http://2011.igem.org/wiki/index.php?title=Special:Contributions/UP_Stefan&feed=atom&limit=50&target=UP_Stefan&year=&month=2011.igem.org - User contributions [en]2024-03-28T17:56:08ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Potsdam_Bioware/Project/SummaryTeam:Potsdam Bioware/Project/Summary2011-10-29T01:56:30Z<p>UP Stefan: /* Summary */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Summary ==<br />
<br />
===Modification, Selection and Production of Cyclic Peptides for Therapy===<br />
One key task of biopharmaceuticals is the binding and blocking of deregulated proteins. Picking the right lead structure for biopharmaceuticals is very important for success. This year, we developed a novel system for the modification, selection and optimization of peptides showing potential for protease inhibition. These promising inhibitors are found in cyanobacteria and are called Microviridins. They belong to a class of peptides characterized by unusual ω-ester and ω-amide bonds between the amino-acid side chains, which are also referred to as depsipeptides. These modifications are introduced post translationally by a set of enzymes and result in an extraordinary tricyclic cage structure. <br />
<br><br />
<br><br />
Proteases are a large enzyme family and comprise about 647 human gene products. Therefore they are important drug targets. In addition, many harmful bacteria, viruses and fungi use proteases in their reproduction cycle and growth. The ability to block these proteases is highly relevant for therapy. Prominent examples of targets are the angiotensin-converting enzyme (ACE) and HIV proteases.<br />
<br><br>We chose a 6.5 kb Microviridin gene cluster named ''mdnABCDE'' from ''Mycrocystis aeruginosa'' NIES843. The <i>mdn</i> gene cluster comprises (i) a gene encoding a precursor peptide named MdnA, which is then modified to form the Microviridin, (ii) two genes encoding ATP-grasp-type ligases named ''mdnB'' and ''mdnC'', (iii) an ABC- transporter encoding gene named ''mdnE'' as well as (iv) one gene encoding an N- acetyltransferase of the GNAT family named ''mdnD'' (Ziemert et al., 2008). Our major aim was to modify the MdnA precursor peptide and optimize its protease inhibiting properties. Towards this goal, we synthesized semi-rational ''mdnA'' gene libraries with partially randomized oligonucleotides. One library was cloned, verified by sequencing, and successfully used for selection.<br />
<br><br />
<br><br />
To identify the best candidates inhibiting various given proteases, we established two different selection systems: Phage Display and a new developed ''in-vivo'' selection assay. Phage display is a frequently used technique in laboratories, yet to our knowledge, phage display of cellularly cyclized peptides is new. We constructed a fusion between the surface protein geneIII of the phage and the ''mdnA'' gene within the ''mdnA'' gene cluster. This system was verified by western blotting, Phage-ELISA and controlled phage panning experiments.<br />
<br><br />
<br><br />
We also devised and constructed a recombinant ''in-vivo'' selection system linking protease degradation to antibiotic resistance. This system is divided into three parts: first a protease activity detector device, second a protease generator device, and third a protease blocking device. For the protease activity detector device we fused various protease cleavage sites between a signal sequence and the antibiotic resistance conferring enzyme β-lactamase. β-lactamase provides resistance when its transferred into the periplasm. Thus, when the protease cleaves the signal sequence from the β-lactamase antibiotic resistance is abolished and cells die under selective pressure. However, when our Microviridin variants inhibit the protease, cells survive, allowing for an easy and efficient selection of protease inhibitors. We demonstrated a wide dynamic range of the system between expressed and non-expressed protease in the presence of increasing ampicillin concentrations. Importantly, after co-transformation with our ''mdnA''-libary we were able to isolate several clones, which are currently being characterized.<br />
<br><br />
<br><br />
In addition to the practical work, we established a mathematical model of the ''in-vivo'' selection system. This model, which we were able to fit to experimental data, helped us to understand the selection process. We modeled the reaction kinetics with ordinary differential equations and simulated and fitted data with matlab.<br />
<br><br />
<br><br />
Last but not least, we had several human practice projects. We send a survey to all members of the German parliament, visited a member of the parliament, held seminars on ethics and invited children to the lab.<br />
<br><br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br><br><br />
<br />
== Highlights ==<br />
<br />
<br />
=== Microviridin ===<br />
[[File:UP_HPLC_mvd_01.jpg|left|300px|thumb|'''Figure 1:''' HPLC chromatogram of mdnA]] <br />
<br><br />
The microviridin sub-project aimed for modifications at the genetic level such that protease inhibiting activities of the resulting peptides are enhanced. We used random mutagenesis or focused randomization of oligonucleotides for the creation of gene libraries, which can be screened for <i>mdnA</i>-variants with therapeutically promising mutations. For further experiments, we also fused mdnA to a myc-tag.<br><br />
In addition, all coding genes of the <i>mdn</i>-cluster were converted to BioBricks. The microviridin from <i>mdnA</i> was purified and characterized by HPLC and cyclization was verified by mass spectrometry.<br><br />
For the quick expression of several library clones, we also built auxiliary plasmid backbones with inducible promoters according to a proposed extension of the iGEM cloning standard.<br />
'''[[:Team:Potsdam_Bioware/Project/Details_Microviridin|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br />
=== Phage Display ===<br />
[[File:UP_Phage_Display_02.jpg|left|290px|thumb|'''Figure 2: Phage Display of microviridin.''' Enrichment of MdnA carrying phages upon panning towards a protease was demonstrated.]]<br />
<br><br />
Phage display is a powerful tool for selecting peptides or proteins that bind and regulate the function of target proteins. Phages robustly link the genotype to the phenotype of the presented peptide allowing for selection of protease binders under controlled ''in vitro'' conditions. We established genetics and panning of therapeutically interesting ''mdnA''-libraries towards a panel of several purified proteases. First, the general suitability of phage display for this purpose was shown. An appropriate phagemid vector harboring an <i>mdnA-myc-geneIII</i> fusion gene, was constructed. The expression of the MdnA-myc-geneIII protein in ''E. coli'' was shown by western blot. Production of phage particles carrying MdnA were analyzed by Phage-ELISA. Finally, phage display procedures were optimized and an enrichment of MdnA modified phages over a control was shown. '''[[:Team:Potsdam_Bioware/Project/Details_Phage|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== In Vivo Selection ===<br />
[[Image:UP_SurvTEV_onepla.jpg|left|290px|thumb|'''Figure 3: Ampicillin resistance test.'''<br />
Survival screen, without induced (blue) and with induced (magenta) protease. From left to right increasing ampicillin concentrations are shown.]]<br />
<br />
A proper designed ''in vivo'' selection is very powerful, quick and entails an inherent selection against general toxicity. We designed and employed a multi-component system enabling inexpensive and time-saving selection of microviridin libraries blocking various proteases. Our genetic circuit links with high sensitivity and wide dynamic range a protease generator, a protease detector and a protease inhibitor device. This is achieved by coupling the export of the enzyme ß-lactamase to a signal sequence via an interspersed modularly cloned protease cleavage site. Inducible lactamase and protease generators are united on a single plasmid while the <i>mdnA</i> library is provided in trans. Functionality of the system was characterized under a multitude of conditions for different proteases. Ultimately, a one step selection of a genetic <i>mdnA</i> library against the protease TEV with increasing concentrations of the antibiotic ampicillin yielded inhibiting clones. '''[[:Team:Potsdam_Bioware/Project/Details_Selection|[more]]]'''<br />
<br><br><br />
<br><br><br />
<br><br><br> <br><br />
<br />
=== Modeling ===<br />
[[File: UP_3Dplot_Lact_06.png|left|300px|thumb|'''Figure 4:''' Plot predicting lactamase concentrations in the periplasm (and thus the cell fittness) in dependence of the enzyme inhibition reaction coefficient K<sub>D</sub>.]]<br />
<br><br />
There is no synthetic biology without modeling, of course. We focused on systems modeling of our ''in vivo'' selection system in which reaction kinetics are analyzed and outcomes are predicted or fitted to experimental data. On one hand, predictions based on known parameters enable the rational choice of optimized conditions (e.g. induction levels of the protease generator). On the other hand, fitting to biological readouts (e.g. survival) enables deciphering of intrinsic parameters (e.g. interactions constants).<br><br><br />
Reactions were coded as ordinary differential equations under consideration of sequential induction time points. Substance concentrations were numerically propagated through time. A useful dynamic range and also a certain robustness of our system were confirmed. We learned about correct time-scales for triggering and we predicted cell-division rates as a reference for the lab work. In a final step, we fitted our model to wet-lab measurements. '''[[:Team:Potsdam_Bioware/Project/Details_Modeling|[more]]]'''<br><br><br><br><br><br />
<br />
=== Ethics ===<br />
<br />
For information about an ethics seminar and a survey among politicians see '''[[:Team:Potsdam_Bioware/Safety_Ethics|[here]]]'''.<br />
<br />
=== Software ===<br />
<br />
Have a look at the features and screenshots of our BioLog app on this '''[[:Team:Potsdam_Bioware/Software|[page]]]'''.</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/SummaryTeam:Potsdam Bioware/Project/Summary2011-10-29T01:47:24Z<p>UP Stefan: /* Modification, Selection and Production of Cyclic Peptides for Therapy */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Summary ==<br />
<br />
===Modification, Selection and Production of Cyclic Peptides for Therapy===<br />
One key task of biopharmaceuticals is the binding and blocking of deregulated proteins. Picking the right lead structure for biopharmaceuticals is very important for success. This year, we developed a novel system for the modification, selection and optimization of peptides showing potential for protease inhibition. These promising inhibitors are found in cyanobacteria and are called Microviridins. They belong to a class of peptides characterized by unusual ω-ester and ω-amide bonds between the amino-acid side chains, which are also referred to as depsipeptides. These modifications are introduced post translationally by a set of enzymes and result in an extraordinary tricyclic cage structure. <br />
<br><br />
<br><br />
Proteases are a large enzyme family and comprise about 647 human gene products. Therefore they are important drug targets. In addition, many harmful bacteria, viruses and fungi use proteases in their reproduction cycle and growth. The ability to block these proteases is highly relevant for therapy. Prominent examples of targets are the angiotensin-converting enzyme (ACE) and HIV proteases.<br />
<br><br>We chose a 6.5 kb Microviridin gene cluster named ''mdnABCDE'' from ''Mycrocystis aeruginosa'' NIES843. The <i>mdn</i> gene cluster comprises (i) a gene encoding a precursor peptide named MdnA, which is then modified to form the Microviridin, (ii) two genes encoding ATP-grasp-type ligases named ''mdnB'' and ''mdnC'', (iii) an ABC- transporter encoding gene named ''mdnE'' as well as (iv) one gene encoding an N- acetyltransferase of the GNAT family named ''mdnD'' (Ziemert et al., 2008). Our major aim was to modify the MdnA precursor peptide and optimize its protease inhibiting properties. Towards this goal, we synthesized semi-rational ''mdnA'' gene libraries with partially randomized oligonucleotides. One library was cloned, verified by sequencing, and successfully used for selection.<br />
<br><br />
<br><br />
To identify the best candidates inhibiting various given proteases, we established two different selection systems: Phage Display and a new developed ''in-vivo'' selection assay. Phage display is a frequently used technique in laboratories, yet to our knowledge, phage display of cellularly cyclized peptides is new. We constructed a fusion between the surface protein geneIII of the phage and the ''mdnA'' gene within the ''mdnA'' gene cluster. This system was verified by western blotting, Phage-ELISA and controlled phage panning experiments.<br />
<br><br />
<br><br />
We also devised and constructed a recombinant ''in-vivo'' selection system linking protease degradation to antibiotic resistance. This system is divided into three parts: first a protease activity detector device, second a protease generator device, and third a protease blocking device. For the protease activity detector device we fused various protease cleavage sites between a signal sequence and the antibiotic resistance conferring enzyme β-lactamase. β-lactamase confers only resistance when transferred to the periplasm of ''E. coli''. Thus, when the protease cleaves the signal sequence from the β-lactamase antibiotic resistance is abolished and cells die under selective pressure. However, when our Microviridin variants inhibit the protease, cells survive, allowing for an easy and efficient selection of protease inhibitors. We demonstrated a wide dynamic range of the system between expressed and non-expressed protease in the presence of increasing ampicillin concentrations. Importantly, after co-transformation with our ''mdnA''-libary we were able to isolate several clones, which are currently being characterized.<br />
<br><br />
<br><br />
In addition to the practical work, we established a mathematical model of the ''in-vivo'' selection system. This model, which we were able to fit to experimental data, helped us to understand the selection process. We modeled the reaction kinetics with ordinary differential equations and simulated and fitted data with matlab.<br />
<br><br />
<br><br />
Last but not least, we had several human practice projects. We send a survey to all members of the German parliament, visited a member of the parliament, held seminars on ethics and invited children to the lab.<br />
<br><br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br><br><br />
<br />
== Highlights ==<br />
<br />
<br />
=== Microviridin ===<br />
[[File:UP_HPLC_mvd_01.jpg|left|300px|thumb|'''Figure 1:''' HPLC chromatogram of mdnA]] <br />
<br><br />
The microviridin sub-project aimed for modifications at the genetic level such that protease inhibiting activities of the resulting peptides are enhanced. We used random mutagenesis or focused randomization of oligonucleotides for the creation of gene libraries, which can be screened for <i>mdnA</i>-variants with therapeutically promising mutations. For further experiments, we also fused mdnA to a myc-tag.<br><br />
In addition, all coding genes of the <i>mdn</i>-cluster were converted to BioBricks. The microviridin from <i>mdnA</i> was purified and characterized by HPLC and cyclization was verified by mass spectrometry.<br><br />
For the quick expression of several library clones, we also built auxiliary plasmid backbones with inducible promoters according to a proposed extension of the iGEM cloning standard.<br />
'''[[:Team:Potsdam_Bioware/Project/Details_Microviridin|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br />
=== Phage Display ===<br />
[[File:UP_Phage_Display_02.jpg|left|290px|thumb|'''Figure 2: Phage Display of microviridin.''' Enrichment of MdnA carrying phages upon panning towards a protease was demonstrated.]]<br />
<br><br />
Phage display is a powerful tool for selecting peptides or proteins that bind and regulate the function of target proteins. Phages robustly link the genotype to the phenotype of the presented peptide allowing for selection of protease binders under controlled ''in vitro'' conditions. We established genetics and panning of therapeutically interesting ''mdnA''-libraries towards a panel of several purified proteases. First, the general suitability of phage display for this purpose was shown. An appropriate phagemid vector harboring an <i>mdnA-myc-geneIII</i> fusion gene, was constructed. The expression of the MdnA-myc-geneIII protein in ''E. coli'' was shown by western blot. Production of phage particles carrying MdnA were analyzed by Phage-ELISA. Finally, phage display procedures were optimized and an enrichment of MdnA modified phages over a control was shown. '''[[:Team:Potsdam_Bioware/Project/Details_Phage|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== In Vivo Selection ===<br />
[[Image:UP_SurvTEV_onepla.jpg|left|290px|thumb|'''Figure 3: Ampicillin resistance test.'''<br />
Survival screen, without induced (blue) and with induced (magenta) protease. From left to right increasing ampicillin concentrations are shown.]]<br />
<br />
A proper designed ''in vivo'' selection is very powerful, quick and entails an inherent selection against general toxicity. We designed and employed a multi-component system enabling inexpensive and time-saving selection of microviridin libraries blocking various proteases. Our genetic circuit links with high sensitivity and wide dynamic range a protease generator, a protease detector and a protease inhibitor device. This is achieved by coupling the export of the enzyme ß-lactamase to a signal sequence via an interspersed modularly cloned protease cleavage site. Inducible lactamase and protease generators are united on a single plasmid while the <i>mdnA</i> library is provided in trans. Functionality of the system was characterized under a multitude of conditions for different proteases. Ultimately, a one step selection of a genetic <i>mdnA</i> library against the protease TEV with increasing concentrations of the antibiotic ampicillin yielded inhibiting clones. '''[[:Team:Potsdam_Bioware/Project/Details_Selection|[more]]]'''<br />
<br><br><br />
<br><br><br />
<br><br><br> <br><br />
<br />
=== Modeling ===<br />
[[File: UP_3Dplot_Lact_06.png|left|300px|thumb|'''Figure 4:''' Plot predicting lactamase concentrations in the periplasm (and thus the cell fittness) in dependence of the enzyme inhibition reaction coefficient K<sub>D</sub>.]]<br />
<br><br />
There is no synthetic biology without modeling, of course. We focused on systems modeling of our ''in vivo'' selection system in which reaction kinetics are analyzed and outcomes are predicted or fitted to experimental data. On one hand, predictions based on known parameters enable the rational choice of optimized conditions (e.g. induction levels of the protease generator). On the other hand, fitting to biological readouts (e.g. survival) enables deciphering of intrinsic parameters (e.g. interactions constants).<br><br><br />
Reactions were coded as ordinary differential equations under consideration of sequential induction time points. Substance concentrations were numerically propagated through time. A useful dynamic range and also a certain robustness of our system were confirmed. We learned about correct time-scales for triggering and we predicted cell-division rates as a reference for the lab work. In a final step, we fitted our model to wet-lab measurements. '''[[:Team:Potsdam_Bioware/Project/Details_Modeling|[more]]]'''<br><br><br><br><br><br />
<br />
=== Ethics ===<br />
<br />
For information about an ethics seminar and a survey among politicians see '''[[:Team:Potsdam_Bioware/Safety_Ethics|[here]]]'''.<br />
<br />
=== Software ===<br />
<br />
Have a look at the features and screenshots of our BioLog app on this '''[[:Team:Potsdam_Bioware/Software|[page]]]'''.</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_ModelingTeam:Potsdam Bioware/Project/Details Modeling2011-10-29T01:45:34Z<p>UP Stefan: /* Results */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
<br />
<br />
<br />
== Modeling ==<br />
<br />
There is no synthetic biology without modeling, of course. In principle there is structure modeling and system modeling. In structure modeling the conformation and structure of proteins is examined and steric consequences for reactions or the whole system can be estimated. We focused on the second sort of modeling: The system modeling in which the reaction kinetics of the whole system is analyzed, outcomes are predicted and parameters correlated to measurements. Thus, a synthetic biology approach can be chosen because a better understanding of the system is achieved and further changes can be planed - just like in engineering.<br />
<br />
=== Model ===<br />
<br />
We built a model of our <i>in vivo</i> selection system to be able to engineer our system effectively. The following schema shows the major reactions taking place in our cell system. The Romanic numbers indicate the system relevant (partially triggered) reactions that were written down as equations and then numerically propagated through time.<br />
<br> <br />
[[File:UP_schema_modeling2v.png|center|650px|thumb|'''Figure 1:'''Simplified schema of our cell system including labels and markers for the chemical reactions in it. Marked reactions are considered in our system modeling.]]<br />
<br />
The schema shows the triggered expression of mircoviridin (our inhibitor), the protease (that needs to be inhibited for medical reasons) and the β-lactamase (that protects the cell from an antibiotic). The protease cleaves at a specific recognition site in the linker peptide. This will abolish the β-lactamase export and in consequnece the cell will die. <br />
<br><br />
There are three important trigger activation times:<br> <br />
* (t0) - start (microviridin added already)<br> <br />
* t1 - start expression of protease<br> <br />
* t2 - start expression of β-lactamase<br> <br />
* t3 - ampicillin added into medium<br> <br />
* (t4) - end of the experiment: cell cultures survive or die.<br><br />
Keeping these times in mind, the reactions can be written down in seven chemical reaction equations of different sort and order.<br />
<br><br />
[[File:UP_reaction_equations.png|center|700px|thumb|'''Figure 2:''' Reaction equations between relevant molecules in the microviridin-inhibitor-concept including indications about the triggered time period (t1,t2,t3). The Romanic numbers correspond to the reactions marked in the above schema.]]<br />
<br />
=== Concentration equations ===<br />
<br />
From the above reaction equations differential equations can be derived that describe the change of substance concentrations. Three concentrations are fixed, however:<br />
<br><br />
[[File: UP_given_concentrations.png|center|550px|thumb|'''Figure 3:''' Given substance concentrations for differential equations. Those concentrations are added to the system and will not increase over time.]]<br />
<br />
<br />
To calculate each concentration at a certain time, several parameters (also refered to as constants or factors) have to be estimated. A literature research was not able to provide us with exact values for our system but a typical range for comparable systems could be assigned to all values. Most of the parameter units had to be transformed. The robustnes of the system towards changes of the most important constants was examined (see results) and some parameters could be estimated by fitting the system to lab measurements (see parameter estimation).<br />
<br />
All not-constant concentrations can be represented in form of differential equations. Between time t1 and t2 four parameters are introduced: <br><br />
* k+1 in (1/s*molecules)- parameter for the association of MdnA (microviridin) and Prot (protease)<br><br />
* k-1 in (1/s) - parameter for the dissociation of the inhibited protease<br><br />
* kexpr.prot in (molecules/s) - parameter for the expression of protease<br><br />
* kdeg1 in (1/s) - parameter for the degradation of protease<br />
<br><br />
[[File: UP_ode_t1onwards.png|center|550px|thumb|'''Figure 4:''' Differential equations for substance concentrations from t1 until t2. At t1 the expression of protease begins.]]<br />
<br />
<br />
Between time t2 and t3 six additional parameters are introduced: <br><br />
* k+2 in (1/s*molecules)- factor for association of Prot (protease) and TorABla (β-lactamase)<br><br />
* k-2 in (1/s) - parameter for the dissociation of protease and substrate<br><br />
* kcat in (1/s) – parameter for the catalytic enzyme reaction that cleaves the signale TorA sequence from the β-lactamase inactivating the export<br><br />
* kexpr.Tor in (molecules/s) - parameter for the the expression of β-lactamase<br><br />
* kdeg2 in (1/s) – parameter for the degradation of β-lactamase<br><br />
* ktransTor (1/s) – parameter by which β-lactamase in the cytoplasm is able to pass the membrane and get into the periplasm<br />
<br><br />
[[File: UP_ode_t2onwards.png|center|550px|thumb|'''Figure 5:''' Differential equations for substance concentrations from t1 until t2. At t2 the expression of lactamase begins.]]<br />
<br />
<br />
After t3 there are only two more factors that we should introduce: <br><br />
* ktransAmp (1/s) – parameter by which the added ampicillin in the medium is able to pass the outer membrane and get into the periplasm<br><br />
* kcat2 in (1/s*molecules) – parameter for the catalytic enzyme reaction of ampicillin deactivation<br />
<br><br />
[[File: UP_ode_t3onwards.png|center|700px|thumb|'''Figure 6:''' Differential equations for substance concentrations from t1 until t2. At t1 ampicillin is added.]]<br />
<br />
=== Results ===<br />
<br />
The equations above were solved using MATLAB. WARNING: These equations are moderately to very stiff! A solution can only be obtained using the functions ''ode23t'' or ''ode23s''!<br><br />
Using the Avogadro constant and the volume of a ''E.coli'' cell (9*10^-16 L) and a periplasm-volume of (3*10^-17 L), following constants were calculated and compared with similar literature values:<br><br />
* k+1 = 8.e-6 (1/molecules*s)<br><br />
* k-1 = 4.e-4 (1/s)<br><br />
* kexpr.Prot = 0.6 (molecules/s)<br><br />
* kdegr1 = 17.e-4 (1/s)<br><br />
* k+2 = 4.e-5 (1/molecules*s)<br><br />
* k-2 = 2.e-4 (1/s)<br><br />
* kcat = 8 (1/s)<br><br />
* kexpr.Tor = 1 (1/s)<br><br />
* kdegr2 = 2.e-3 (1/s)<br><br />
* ktransTor = 1.e-3 (1/s)<br><br />
* ktransAmp = 5.e-3 (1/s)<br><br />
* Kcat2 = 0.001 (1/molecules*s)<br><br />
* mdnA(0) = 3000 (molecules)<br><br />
* Amp(0 Med) = 8.e+2 (molecules)<br />
* alpha = 0.3163 (forumlar parameter)<br />
<br />
Now the concentration equations could be visualized in all three time segments:<br />
<br><br />
[[File: UP_odet_solution_all_times.png|center|700px|thumb|'''Figure 7a:''' MATLAB numerical solution to our system of differential equations for substance concentrations (above) over all time segments.]]<br />
<br />
It can be seen that there is only a negligible amount of ampicillin in the periplasm if the expression works as indicated. This amount of ampicillin has only a small effect on the growth rate of the cells. There is also a wide tolerance left for suboptimal conditions. The trigger time t1, t2 and t3 shall be about one hour after one another.<br />
<br><br />
In the first section the effect of the inhibition can clearly be seen: Even though the continuous expression of protease, only a very small number remains active in the cytoplasm. In the second section β-lactamase is expressed. Most of the β-lactamase in the cytoplasm is destroyed by the protease, however the number is by far large enough to steadily release molecules into the periplasm where it cannot be affected by the protease any more. Because the volume of the periplasm is very small, the concentration there is even higher than the absolute amount of molecules in the graph suggests compared to the amount of molecules in the cytoplasm. The third section shows that the concentrations remain very steady and the cells are ampicillin resistant.<br />
<br />
<br><br />
In case microviridin is not expressed the simulation indicates following substance concentrations that were correlated to our lab data of the same system (see parameter estimation). Under those conditions cells die quickly.<br />
<br><br />
[[File: UP_odet_solution_all_times_without_mdnA.png|center|700px|thumb|'''Figure 7b:''' MATLAB numerical solution to our system of differential equations for substance concentrations under absence of microviridin.]]<br />
<br />
In case the protease is also not expressed the simulation indicates following substance concentrations. They were correlated to measurements as well. Protected by β-lactamase cells survive well on ampicillin plates.<br />
<br><br />
[[File: UP_odet_solution_all_times_without_protease.png|center|700px|thumb|'''Figure 7c:''' MATLAB numerical solution to our system of differential equations for substance concentrations under absence of Protease.]]<br />
<br />
Cells in which all parts of our system work are very ampicillin resistant and grow fast: They double their cell volume about every 20 minutes. If an error would appear and the ampicillin concentration inside the periplasma would increase over 2 µg/mL, the growth rate would slow down drastically and the cells might die. This way defect cell cultures can easily be separated from the good ones.<br />
<br><br />
<br />
We constructed a formula for the ratio of the growth rate (cells double during this timespan) to the ampicillin concentration in ''n µg/mL'' out of data that we gained out of the lab.<br />
The cell doubling time can approximately be calculated by: ''T(growth) = 20min+10*2^(2*n-1)''.<br />
Analogous to this the cell surviving percentage can be estimated by: ''surviving cells in % = 25/(20min+10*2^(alpha*n-1))''.<br />
<br><br />
<br />
Here we display one example of how the change of a simulation constant impacts the growth of the cells and that our system is robust: The parameter K<sub>D</sub> (Dissociation of microviridin and protease divided by the association) is central to our model and the β-lactamase concentration in the periplasm an indicator for the cell survival. To see the dependence of β-lactamase on this constant, following plot (figure 8) was created:<br />
<br><br />
[[File: UP_3Dplot_Lact_06.png|center|600px|thumb|'''Figure 8:''' Starting at t3: β-lactamase concentration inside the periplasm. A significant change of the simulation factor K<sub>D</sub> (Dissociation of microviridin and protease divided by the association) can result in a lower β-lactamase concentration and thus less fitness of the cell (because the β-lactamase protects the cell from the antibiotic ampicillin).]]<br />
<br />
Figure 9 shows the relating cell doubling time that can be measured in the lab.<br />
<br><br />
[[File: UP_3Dplot_cells_06.png|center|600px|thumb|'''Figure 9:''' Starting at t3: Growth (double) rate of cells dependent on the ampicillin concentration in the periplasm. A significant change of the simulation factor K<sub>D</sub> (Dissociation of microviridin and protease divided by the association) can result in less fitness of the cell and a reduction of its growth rate.]]<br />
<br />
The β-lactamase concentration in the periplasm can be very small. Thus for this concentration a semi-stochastig model was produced by handling this value as integer. Such graphs are less smooth (see figures 10 and 11) (parameters were insignificantly changed).<br />
<br />
[[File: UP_3Dplot_Lact_07.png|center|600px|thumb|'''Figure 10:''' Starting at t3: Semi-stochastic β-lactamase concentration inside the periplasm.]]<br />
<br />
[[File: UP_3Dplot_cells_07.png|center|600px|thumb|'''Figure 11:''' Starting at t3: Semi-stochastic growth (double) rate of cells dependent on the ampicillin concentration in the periplasm.]]<br />
<br />
We were able to see that our system works very well in theory. We learned about correct time-scales for our triggering and we were able to identify expected cell-division rates as reference for lab work.<br />
<br />
=== Parameter estimation ===<br />
<br />
As mentioned before most model parameters could only be estimated by looking at the order of magnitude of substances in similar systems and lab experiences combined with unit transformation. To make our model more reliable we improved critical parts of our model by relating it to lab measurements of cell survival in different variations of our system (see section <i>in vivo</i> selection). Especially two experiments were used: The percentage of surviving cells on different ampicillin concentration mediums with induced β-lactamase expression and a system with induced β-lactamse and also induced protease expression.<br />
<br />
We implemented a routine for parameter estimation in MATLAB with the help of which the two parameter values could be specified (in direct relation to lab measurements) that we were least certain about before: alpha (the parameter in the formula (see above) connecting the ampicillin concentration in the periplasm and the percentage of surviving cells) and k(+2) (the parameter of association of the protease and the β-lactamase - here the aspect plays a role that the β-lactamase must first be folded before it can be transported into the periplasm, so in total the chance for association is higher due to the longer retention time). Alpha was specified to 0.3163 and k(+2) was specified to 2.0253*(1.e-5).<br />
<br />
As can be seen on following figures 12 and 13, the model responses reliably to the measurements (see <i>in vivo</i> selection for more information). Thus predictions of the model concerning robustness (see results) and trigger times can be trusted.<br />
<br><br />
[[File: UP_comparison_lactamase.png|center|500px|thumb|'''Figure 12:''' Comparison of model and measurement at t4 > 3h: Percentage of surviving cells dependent on the ampicillin concentration added to the medium. The lactamase expression is induced.]]<br />
<br />
[[File: UP_comparison_lactamase_and_protease.png|center|500px|thumb|'''Figure 13:''' Comparison of model and measurement at t4 > 3h: Percentage of surviving cells dependent on the ampicillin concentration added to the medium. Lactamase and protease expression is induced.]]<br />
<br />
=== MATLAB code ===<br />
<br />
[[Media:conzt1.m]]<br><br />
[[Media:conzt2.m]]<br><br />
[[Media:conzt3.m]]<br><br />
[[Media:UP_plottfuntionendreiaufeinmal2.m]]<br><br />
[[Media:UP_celldivision2.m]]<br><br />
[[Media:UP_lactamase2.m]]<br><br />
[[Media:UP_parameter_estimation_1.m]]<br><br />
[[Media:UP_parameter_estimation_2.m]]<br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/SummaryTeam:Potsdam Bioware/Project/Summary2011-10-29T01:41:36Z<p>UP Stefan: /* In Vivo Selection */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Summary ==<br />
<br />
===Modification, Selection and Production of Cyclic Peptides for Therapy===<br />
One key task of biopharmaceuticals is the binding and blocking of deregulated proteins. Picking the right lead structure for biopharmaceuticals is very important for success. This year, we developed a novel system for the modification, selection and optimization of peptides showing potential for protease inhibition. These promising inhibitors are found in cyanobacteria and are called Microviridins. They belong to a class of peptides characterized by unusual ω-ester and ω-amide bonds between the amino-acid side chains, which are also referred to as depsipeptides. These modifications are introduced post translationally by a set of enzymes and result in an extraordinary tricyclic cage structure. <br />
<br><br />
<br><br />
Proteases are a large enzyme family and comprise about 647 human gene products. Therefore they are important drug targets. In addition, many harmful bacteria, viruses and fungi use proteases in their reproduction cycle and growth. The ability to block these proteases is highly relevant for therapy. Prominent examples of targets are the angiotensin-converting enzyme (ACE) and HIV proteases.<br />
<br><br>We chose a 6.5 kb Microviridin gene cluster named ''mdnABCDE'' from ''Mycrocystis aeruginosa'' NIES843. The mdn gene cluster comprises (i) a gene encoding a precursor peptide named MdnA, which is then modified to form the Microviridin, (ii) two genes encoding ATP-grasp-type ligases named ''mdnB'' and ''mdnC'', (iii) an ABC- transporter encoding gene named ''mdnE'' as well as (iv) one gene encoding an N- acetyltransferase of the GNAT family named ''mdnD'' (Ziemert et al., 2008). Our major aim was to modify the MdnA precursor peptide and optimize its protease inhibiting properties. Towards this goal, we synthesized semi-rational ''mdnA'' gene libraries with partially randomized oligonucleotides. One library was cloned, verified by sequencing, and successfully used for selection.<br />
<br><br />
<br><br />
To identify the best candidates inhibiting various given proteases, we established two different selection systems: Phage Display and a new developed ''in-vivo'' selection assay. Phage display is a frequently used technique in laboratories, yet to our knowledge, phage display of cellularly cyclized peptides is new. We constructed a fusion between the surface protein geneIII of the phage and the ''mdnA'' gene within the ''mdnA'' gene cluster. This system was verified by western blotting, Phage-ELISA and controlled phage panning experiments.<br />
<br><br />
<br><br />
We also devised and constructed a recombinant ''in-vivo'' selection system linking protease degradation to antibiotic resistance. This system is divided into three parts: first a protease activity detector device, second a protease generator device, and third a protease blocking device. For the protease activity detector device we fused various protease cleavage sites between a signal sequence and the antibiotic resistance conferring enzyme β-lactamase. β-lactamase confers only resistance when transferred to the periplasm of ''E. coli''. Thus, when the protease cleaves the signal sequence from the β-lactamase antibiotic resistance is abolished and cells die under selective pressure. However, when our Microviridin variants inhibit the protease, cells survive, allowing for an easy and efficient selection of protease inhibitors. We demonstrated a wide dynamic range of the system between expressed and non-expressed protease in the presence of increasing ampicillin concentrations. Importantly, after co-transformation with our ''mdnA''-libary we were able to isolate several clones, which are currently being characterized.<br />
<br><br />
<br><br />
In addition to the practical work, we established a mathematical model of the ''in-vivo'' selection system. This model, which we were able to fit to experimental data, helped us to understand the selection process. We modeled the reaction kinetics with ordinary differential equations and simulated and fitted data with matlab.<br />
<br><br />
<br><br />
Last but not least, we had several human practice projects. We send a survey to all members of the German parliament, visited a member of the parliament, held seminars on ethics and invited children to the lab.<br />
<br><br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br><br><br />
<br />
== Highlights ==<br />
<br />
<br />
=== Microviridin ===<br />
[[File:UP_HPLC_mvd_01.jpg|left|300px|thumb|'''Figure 1:''' HPLC chromatogram of mdnA]] <br />
<br><br />
The microviridin sub-project aimed for modifications at the genetic level such that protease inhibiting activities of the resulting peptides are enhanced. We used random mutagenesis or focused randomization of oligonucleotides for the creation of gene libraries, which can be screened for <i>mdnA</i>-variants with therapeutically promising mutations. For further experiments, we also fused mdnA to a myc-tag.<br><br />
In addition, all coding genes of the <i>mdn</i>-cluster were converted to BioBricks. The microviridin from <i>mdnA</i> was purified and characterized by HPLC and cyclization was verified by mass spectrometry.<br><br />
For the quick expression of several library clones, we also built auxiliary plasmid backbones with inducible promoters according to a proposed extension of the iGEM cloning standard.<br />
'''[[:Team:Potsdam_Bioware/Project/Details_Microviridin|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br />
=== Phage Display ===<br />
[[File:UP_Phage_Display_02.jpg|left|290px|thumb|'''Figure 2: Phage Display of microviridin.''' Enrichment of MdnA carrying phages upon panning towards a protease was demonstrated.]]<br />
<br><br />
Phage display is a powerful tool for selecting peptides or proteins that bind and regulate the function of target proteins. Phages robustly link the genotype to the phenotype of the presented peptide allowing for selection of protease binders under controlled ''in vitro'' conditions. We established genetics and panning of therapeutically interesting ''mdnA''-libraries towards a panel of several purified proteases. First, the general suitability of phage display for this purpose was shown. An appropriate phagemid vector harboring an <i>mdnA-myc-geneIII</i> fusion gene, was constructed. The expression of the MdnA-myc-geneIII protein in ''E. coli'' was shown by western blot. Production of phage particles carrying MdnA were analyzed by Phage-ELISA. Finally, phage display procedures were optimized and an enrichment of MdnA modified phages over a control was shown. '''[[:Team:Potsdam_Bioware/Project/Details_Phage|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== In Vivo Selection ===<br />
[[Image:UP_SurvTEV_onepla.jpg|left|290px|thumb|'''Figure 3: Ampicillin resistance test.'''<br />
Survival screen, without induced (blue) and with induced (magenta) protease. From left to right increasing ampicillin concentrations are shown.]]<br />
<br />
A proper designed ''in vivo'' selection is very powerful, quick and entails an inherent selection against general toxicity. We designed and employed a multi-component system enabling inexpensive and time-saving selection of microviridin libraries blocking various proteases. Our genetic circuit links with high sensitivity and wide dynamic range a protease generator, a protease detector and a protease inhibitor device. This is achieved by coupling the export of the enzyme ß-lactamase to a signal sequence via an interspersed modularly cloned protease cleavage site. Inducible lactamase and protease generators are united on a single plasmid while the <i>mdnA</i> library is provided in trans. Functionality of the system was characterized under a multitude of conditions for different proteases. Ultimately, a one step selection of a genetic <i>mdnA</i> library against the protease TEV with increasing concentrations of the antibiotic ampicillin yielded inhibiting clones. '''[[:Team:Potsdam_Bioware/Project/Details_Selection|[more]]]'''<br />
<br><br><br />
<br><br><br />
<br><br><br> <br><br />
<br />
=== Modeling ===<br />
[[File: UP_3Dplot_Lact_06.png|left|300px|thumb|'''Figure 4:''' Plot predicting lactamase concentrations in the periplasm (and thus the cell fittness) in dependence of the enzyme inhibition reaction coefficient K<sub>D</sub>.]]<br />
<br><br />
There is no synthetic biology without modeling, of course. We focused on systems modeling of our ''in vivo'' selection system in which reaction kinetics are analyzed and outcomes are predicted or fitted to experimental data. On one hand, predictions based on known parameters enable the rational choice of optimized conditions (e.g. induction levels of the protease generator). On the other hand, fitting to biological readouts (e.g. survival) enables deciphering of intrinsic parameters (e.g. interactions constants).<br><br><br />
Reactions were coded as ordinary differential equations under consideration of sequential induction time points. Substance concentrations were numerically propagated through time. A useful dynamic range and also a certain robustness of our system were confirmed. We learned about correct time-scales for triggering and we predicted cell-division rates as a reference for the lab work. In a final step, we fitted our model to wet-lab measurements. '''[[:Team:Potsdam_Bioware/Project/Details_Modeling|[more]]]'''<br><br><br><br><br><br />
<br />
=== Ethics ===<br />
<br />
For information about an ethics seminar and a survey among politicians see '''[[:Team:Potsdam_Bioware/Safety_Ethics|[here]]]'''.<br />
<br />
=== Software ===<br />
<br />
Have a look at the features and screenshots of our BioLog app on this '''[[:Team:Potsdam_Bioware/Software|[page]]]'''.</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/SummaryTeam:Potsdam Bioware/Project/Summary2011-10-29T01:39:06Z<p>UP Stefan: /* Phage Display */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Summary ==<br />
<br />
===Modification, Selection and Production of Cyclic Peptides for Therapy===<br />
One key task of biopharmaceuticals is the binding and blocking of deregulated proteins. Picking the right lead structure for biopharmaceuticals is very important for success. This year, we developed a novel system for the modification, selection and optimization of peptides showing potential for protease inhibition. These promising inhibitors are found in cyanobacteria and are called Microviridins. They belong to a class of peptides characterized by unusual ω-ester and ω-amide bonds between the amino-acid side chains, which are also referred to as depsipeptides. These modifications are introduced post translationally by a set of enzymes and result in an extraordinary tricyclic cage structure. <br />
<br><br />
<br><br />
Proteases are a large enzyme family and comprise about 647 human gene products. Therefore they are important drug targets. In addition, many harmful bacteria, viruses and fungi use proteases in their reproduction cycle and growth. The ability to block these proteases is highly relevant for therapy. Prominent examples of targets are the angiotensin-converting enzyme (ACE) and HIV proteases.<br />
<br><br>We chose a 6.5 kb Microviridin gene cluster named ''mdnABCDE'' from ''Mycrocystis aeruginosa'' NIES843. The mdn gene cluster comprises (i) a gene encoding a precursor peptide named MdnA, which is then modified to form the Microviridin, (ii) two genes encoding ATP-grasp-type ligases named ''mdnB'' and ''mdnC'', (iii) an ABC- transporter encoding gene named ''mdnE'' as well as (iv) one gene encoding an N- acetyltransferase of the GNAT family named ''mdnD'' (Ziemert et al., 2008). Our major aim was to modify the MdnA precursor peptide and optimize its protease inhibiting properties. Towards this goal, we synthesized semi-rational ''mdnA'' gene libraries with partially randomized oligonucleotides. One library was cloned, verified by sequencing, and successfully used for selection.<br />
<br><br />
<br><br />
To identify the best candidates inhibiting various given proteases, we established two different selection systems: Phage Display and a new developed ''in-vivo'' selection assay. Phage display is a frequently used technique in laboratories, yet to our knowledge, phage display of cellularly cyclized peptides is new. We constructed a fusion between the surface protein geneIII of the phage and the ''mdnA'' gene within the ''mdnA'' gene cluster. This system was verified by western blotting, Phage-ELISA and controlled phage panning experiments.<br />
<br><br />
<br><br />
We also devised and constructed a recombinant ''in-vivo'' selection system linking protease degradation to antibiotic resistance. This system is divided into three parts: first a protease activity detector device, second a protease generator device, and third a protease blocking device. For the protease activity detector device we fused various protease cleavage sites between a signal sequence and the antibiotic resistance conferring enzyme β-lactamase. β-lactamase confers only resistance when transferred to the periplasm of ''E. coli''. Thus, when the protease cleaves the signal sequence from the β-lactamase antibiotic resistance is abolished and cells die under selective pressure. However, when our Microviridin variants inhibit the protease, cells survive, allowing for an easy and efficient selection of protease inhibitors. We demonstrated a wide dynamic range of the system between expressed and non-expressed protease in the presence of increasing ampicillin concentrations. Importantly, after co-transformation with our ''mdnA''-libary we were able to isolate several clones, which are currently being characterized.<br />
<br><br />
<br><br />
In addition to the practical work, we established a mathematical model of the ''in-vivo'' selection system. This model, which we were able to fit to experimental data, helped us to understand the selection process. We modeled the reaction kinetics with ordinary differential equations and simulated and fitted data with matlab.<br />
<br><br />
<br><br />
Last but not least, we had several human practice projects. We send a survey to all members of the German parliament, visited a member of the parliament, held seminars on ethics and invited children to the lab.<br />
<br><br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br><br><br />
<br />
== Highlights ==<br />
<br />
<br />
=== Microviridin ===<br />
[[File:UP_HPLC_mvd_01.jpg|left|300px|thumb|'''Figure 1:''' HPLC chromatogram of mdnA]] <br />
<br><br />
The microviridin sub-project aimed for modifications at the genetic level such that protease inhibiting activities of the resulting peptides are enhanced. We used random mutagenesis or focused randomization of oligonucleotides for the creation of gene libraries, which can be screened for <i>mdnA</i>-variants with therapeutically promising mutations. For further experiments, we also fused mdnA to a myc-tag.<br><br />
In addition, all coding genes of the <i>mdn</i>-cluster were converted to BioBricks. The microviridin from <i>mdnA</i> was purified and characterized by HPLC and cyclization was verified by mass spectrometry.<br><br />
For the quick expression of several library clones, we also built auxiliary plasmid backbones with inducible promoters according to a proposed extension of the iGEM cloning standard.<br />
'''[[:Team:Potsdam_Bioware/Project/Details_Microviridin|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br />
=== Phage Display ===<br />
[[File:UP_Phage_Display_02.jpg|left|290px|thumb|'''Figure 2: Phage Display of microviridin.''' Enrichment of MdnA carrying phages upon panning towards a protease was demonstrated.]]<br />
<br><br />
Phage display is a powerful tool for selecting peptides or proteins that bind and regulate the function of target proteins. Phages robustly link the genotype to the phenotype of the presented peptide allowing for selection of protease binders under controlled ''in vitro'' conditions. We established genetics and panning of therapeutically interesting ''mdnA''-libraries towards a panel of several purified proteases. First, the general suitability of phage display for this purpose was shown. An appropriate phagemid vector harboring an <i>mdnA-myc-geneIII</i> fusion gene, was constructed. The expression of the MdnA-myc-geneIII protein in ''E. coli'' was shown by western blot. Production of phage particles carrying MdnA were analyzed by Phage-ELISA. Finally, phage display procedures were optimized and an enrichment of MdnA modified phages over a control was shown. '''[[:Team:Potsdam_Bioware/Project/Details_Phage|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== In Vivo Selection ===<br />
[[Image:UP_SurvTEV_onepla.jpg|left|290px|thumb|'''Figure 3: Ampicillin resistance test.'''<br />
Survival screen, without induced (blue) and with induced (magenta) protease. From left to right increasing ampicillin concentrations are shown.]]<br />
<br />
A proper designed ''in vivo'' selection is very powerful, quick and entails an inherent selection against general toxicity. We designed and employed a multi-component system enabling inexpensive and time-saving selection of microviridin libraries blocking various proteases. Our genetic circuit links with high sensitivity and wide dynamic range a protease generator, a protease detector and a protease inhibitor device. This is achieved by coupling the export of the enzyme ß-lactamase to a signal sequence via an interspersed modularly cloned protease cleavage site. Inducible lactamase and protease generators are united on a single plasmid while the MdnA library is provided in trans. Functionality of the system was characterized under a multitude of conditions for different proteases. Ultimately, a one step selection of a genetic mdnA library against the protease TEV with increasing concentrations of the antibiotic ampicillin yielded inhibiting clones. '''[[:Team:Potsdam_Bioware/Project/Details_Selection|[more]]]'''<br />
<br><br><br />
<br><br><br />
<br><br><br> <br><br />
<br />
=== Modeling ===<br />
[[File: UP_3Dplot_Lact_06.png|left|300px|thumb|'''Figure 4:''' Plot predicting lactamase concentrations in the periplasm (and thus the cell fittness) in dependence of the enzyme inhibition reaction coefficient K<sub>D</sub>.]]<br />
<br><br />
There is no synthetic biology without modeling, of course. We focused on systems modeling of our ''in vivo'' selection system in which reaction kinetics are analyzed and outcomes are predicted or fitted to experimental data. On one hand, predictions based on known parameters enable the rational choice of optimized conditions (e.g. induction levels of the protease generator). On the other hand, fitting to biological readouts (e.g. survival) enables deciphering of intrinsic parameters (e.g. interactions constants).<br><br><br />
Reactions were coded as ordinary differential equations under consideration of sequential induction time points. Substance concentrations were numerically propagated through time. A useful dynamic range and also a certain robustness of our system were confirmed. We learned about correct time-scales for triggering and we predicted cell-division rates as a reference for the lab work. In a final step, we fitted our model to wet-lab measurements. '''[[:Team:Potsdam_Bioware/Project/Details_Modeling|[more]]]'''<br><br><br><br><br><br />
<br />
=== Ethics ===<br />
<br />
For information about an ethics seminar and a survey among politicians see '''[[:Team:Potsdam_Bioware/Safety_Ethics|[here]]]'''.<br />
<br />
=== Software ===<br />
<br />
Have a look at the features and screenshots of our BioLog app on this '''[[:Team:Potsdam_Bioware/Software|[page]]]'''.</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/SummaryTeam:Potsdam Bioware/Project/Summary2011-10-29T01:36:56Z<p>UP Stefan: /* Microviridin */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Summary ==<br />
<br />
===Modification, Selection and Production of Cyclic Peptides for Therapy===<br />
One key task of biopharmaceuticals is the binding and blocking of deregulated proteins. Picking the right lead structure for biopharmaceuticals is very important for success. This year, we developed a novel system for the modification, selection and optimization of peptides showing potential for protease inhibition. These promising inhibitors are found in cyanobacteria and are called Microviridins. They belong to a class of peptides characterized by unusual ω-ester and ω-amide bonds between the amino-acid side chains, which are also referred to as depsipeptides. These modifications are introduced post translationally by a set of enzymes and result in an extraordinary tricyclic cage structure. <br />
<br><br />
<br><br />
Proteases are a large enzyme family and comprise about 647 human gene products. Therefore they are important drug targets. In addition, many harmful bacteria, viruses and fungi use proteases in their reproduction cycle and growth. The ability to block these proteases is highly relevant for therapy. Prominent examples of targets are the angiotensin-converting enzyme (ACE) and HIV proteases.<br />
<br><br>We chose a 6.5 kb Microviridin gene cluster named ''mdnABCDE'' from ''Mycrocystis aeruginosa'' NIES843. The mdn gene cluster comprises (i) a gene encoding a precursor peptide named MdnA, which is then modified to form the Microviridin, (ii) two genes encoding ATP-grasp-type ligases named ''mdnB'' and ''mdnC'', (iii) an ABC- transporter encoding gene named ''mdnE'' as well as (iv) one gene encoding an N- acetyltransferase of the GNAT family named ''mdnD'' (Ziemert et al., 2008). Our major aim was to modify the MdnA precursor peptide and optimize its protease inhibiting properties. Towards this goal, we synthesized semi-rational ''mdnA'' gene libraries with partially randomized oligonucleotides. One library was cloned, verified by sequencing, and successfully used for selection.<br />
<br><br />
<br><br />
To identify the best candidates inhibiting various given proteases, we established two different selection systems: Phage Display and a new developed ''in-vivo'' selection assay. Phage display is a frequently used technique in laboratories, yet to our knowledge, phage display of cellularly cyclized peptides is new. We constructed a fusion between the surface protein geneIII of the phage and the ''mdnA'' gene within the ''mdnA'' gene cluster. This system was verified by western blotting, Phage-ELISA and controlled phage panning experiments.<br />
<br><br />
<br><br />
We also devised and constructed a recombinant ''in-vivo'' selection system linking protease degradation to antibiotic resistance. This system is divided into three parts: first a protease activity detector device, second a protease generator device, and third a protease blocking device. For the protease activity detector device we fused various protease cleavage sites between a signal sequence and the antibiotic resistance conferring enzyme β-lactamase. β-lactamase confers only resistance when transferred to the periplasm of ''E. coli''. Thus, when the protease cleaves the signal sequence from the β-lactamase antibiotic resistance is abolished and cells die under selective pressure. However, when our Microviridin variants inhibit the protease, cells survive, allowing for an easy and efficient selection of protease inhibitors. We demonstrated a wide dynamic range of the system between expressed and non-expressed protease in the presence of increasing ampicillin concentrations. Importantly, after co-transformation with our ''mdnA''-libary we were able to isolate several clones, which are currently being characterized.<br />
<br><br />
<br><br />
In addition to the practical work, we established a mathematical model of the ''in-vivo'' selection system. This model, which we were able to fit to experimental data, helped us to understand the selection process. We modeled the reaction kinetics with ordinary differential equations and simulated and fitted data with matlab.<br />
<br><br />
<br><br />
Last but not least, we had several human practice projects. We send a survey to all members of the German parliament, visited a member of the parliament, held seminars on ethics and invited children to the lab.<br />
<br><br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br><br><br />
<br />
== Highlights ==<br />
<br />
<br />
=== Microviridin ===<br />
[[File:UP_HPLC_mvd_01.jpg|left|300px|thumb|'''Figure 1:''' HPLC chromatogram of mdnA]] <br />
<br><br />
The microviridin sub-project aimed for modifications at the genetic level such that protease inhibiting activities of the resulting peptides are enhanced. We used random mutagenesis or focused randomization of oligonucleotides for the creation of gene libraries, which can be screened for <i>mdnA</i>-variants with therapeutically promising mutations. For further experiments, we also fused mdnA to a myc-tag.<br><br />
In addition, all coding genes of the <i>mdn</i>-cluster were converted to BioBricks. The microviridin from <i>mdnA</i> was purified and characterized by HPLC and cyclization was verified by mass spectrometry.<br><br />
For the quick expression of several library clones, we also built auxiliary plasmid backbones with inducible promoters according to a proposed extension of the iGEM cloning standard.<br />
'''[[:Team:Potsdam_Bioware/Project/Details_Microviridin|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br />
=== Phage Display ===<br />
[[File:UP_Phage_Display_02.jpg|left|290px|thumb|'''Figure 2: Phage Display of microviridin.''' Enrichment of MdnA carrying phages upon panning towards a protease was demonstrated.]]<br />
<br><br />
Phage display is a powerful tool for selecting peptides or proteins that bind and regulate the function of target proteins. Phages robustly link the genotype to the phenotype of the presented peptide allowing for selection of protease binders under controlled ''in vitro'' conditions. We established genetics and panning of therapeutically interesting ''mdnA''-libraries towards a panel of several purified proteases. First, the general suitability of phage display for this purpose was shown. An appropriate phagemid vector harboring an mdnA-myc-geneIII fusion gene, was constructed. The expression of the mdnA-myc-geneIII protein in ''E. coli'' was shown by western blot. Production of phage particles carrying MdnA was analyzed by Phage-ELISA. Finally, phage display procedures were optimized and enrichment of MdnA modified phages over a control was shown. '''[[:Team:Potsdam_Bioware/Project/Details_Phage|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== In Vivo Selection ===<br />
[[Image:UP_SurvTEV_onepla.jpg|left|290px|thumb|'''Figure 3: Ampicillin resistance test.'''<br />
Survival screen, without induced (blue) and with induced (magenta) protease. From left to right increasing ampicillin concentrations are shown.]]<br />
<br />
A proper designed ''in vivo'' selection is very powerful, quick and entails an inherent selection against general toxicity. We designed and employed a multi-component system enabling inexpensive and time-saving selection of microviridin libraries blocking various proteases. Our genetic circuit links with high sensitivity and wide dynamic range a protease generator, a protease detector and a protease inhibitor device. This is achieved by coupling the export of the enzyme ß-lactamase to a signal sequence via an interspersed modularly cloned protease cleavage site. Inducible lactamase and protease generators are united on a single plasmid while the MdnA library is provided in trans. Functionality of the system was characterized under a multitude of conditions for different proteases. Ultimately, a one step selection of a genetic mdnA library against the protease TEV with increasing concentrations of the antibiotic ampicillin yielded inhibiting clones. '''[[:Team:Potsdam_Bioware/Project/Details_Selection|[more]]]'''<br />
<br><br><br />
<br><br><br />
<br><br><br> <br><br />
<br />
=== Modeling ===<br />
[[File: UP_3Dplot_Lact_06.png|left|300px|thumb|'''Figure 4:''' Plot predicting lactamase concentrations in the periplasm (and thus the cell fittness) in dependence of the enzyme inhibition reaction coefficient K<sub>D</sub>.]]<br />
<br><br />
There is no synthetic biology without modeling, of course. We focused on systems modeling of our ''in vivo'' selection system in which reaction kinetics are analyzed and outcomes are predicted or fitted to experimental data. On one hand, predictions based on known parameters enable the rational choice of optimized conditions (e.g. induction levels of the protease generator). On the other hand, fitting to biological readouts (e.g. survival) enables deciphering of intrinsic parameters (e.g. interactions constants).<br><br><br />
Reactions were coded as ordinary differential equations under consideration of sequential induction time points. Substance concentrations were numerically propagated through time. A useful dynamic range and also a certain robustness of our system were confirmed. We learned about correct time-scales for triggering and we predicted cell-division rates as a reference for the lab work. In a final step, we fitted our model to wet-lab measurements. '''[[:Team:Potsdam_Bioware/Project/Details_Modeling|[more]]]'''<br><br><br><br><br><br />
<br />
=== Ethics ===<br />
<br />
For information about an ethics seminar and a survey among politicians see '''[[:Team:Potsdam_Bioware/Safety_Ethics|[here]]]'''.<br />
<br />
=== Software ===<br />
<br />
Have a look at the features and screenshots of our BioLog app on this '''[[:Team:Potsdam_Bioware/Software|[page]]]'''.</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_MicroviridinTeam:Potsdam Bioware/Project/Details Microviridin2011-10-28T23:37:49Z<p>UP Stefan: /* Expression Backbones */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==Microviridin==<br />
=== Introduction ===<br />
Cyanobacteria are known as the blue-green-algae because their living space is water and their feeding mechanism is photosynthetic. The Cyanobacteria are know for some special and unique metabolites. Some of these metabolites are part of the microviridin family. Special attributes of microviridins are their occurrence as tricyclic depsipeptides, means peptides whose one or more amide-bond is replaced by an ester-bond, unparallel cage-like architecture and their ability to inhibit several proteases. Microviridin B for example is told to function as an elastase inhibitor what would be an therapeutic attempt according to fix the out of control gone function of elastase in lung emphysema. In such a case it is beyond debate how important it is to figure out the biosynthesis of this peptide. The research group of Prof. Dittmann at the University of Potsdam was successful and able to report about the composition of the gene cluster from ''Micocystis aeruginosa'' NIES298 expressing Microviridin B. Moreover they have been convinced that there is existing one unique biosynthetic mechanism for microviridins in <i>Microcystis</i> strains. Unusual for depsipeptides they discovered that they are ribosomal synthesized. The ''mdn'' gene cluster is build up of a gene ''mdnA'', encoding for the putative precursor peptide of the microvirdin, two genes encoding ATP-grasp-type ligases ''mdnB'' and ''mdnC'', an ABC transporter encoding gene ''mdnE'' as well as one encoding an N-acetyltransferase of the GNAT family ''mdnD'' (Ziemert et al., 2008). Moreover in their recent studies they report about the discovery of an existing natural diversity of microviridin precursors genes in set of closely related <i>Mycrocystis</i> laboratory strains. This furthermore would mean and lead to a heterologous production of novel microviridins in ''E. coli''. To give evidence of their discovery they identified and characterized a new Microviridin L from the strain ''Mycrocystis aeruginosa'' NIES843 (Ziemert et al., 2010).<br />
All in all the microviridins, the tricyclic depsipeptide are build up through a gene cluster of five mdn-components each fulfill one function to make the hole cluster functional. This showed the team of Prof. Dr. Dittmann as the peptide is only correctly processed in ''E. coli'' if the entire cluster ''mdnABCDE'' is expressed. In addition the new discovery is promising a small flexibility of the microviridin ligases as an open window for the size of the natural microviridin library and thus microviridin functions and hence for example therapeutic benefit that needs to be discovered. The following illustration is going to give you a better overview (Fig. 1).<br />
[[File:UP_MV_introduction_mdn.jpg|center|525px|thumb|'''Figure 1:''' overview of the mdn gene cluster (GNAT: GCN5-related N-acetyltransferase)]]<br />
<br><br><br />
<br />
=== Modularization of the ''mdn'' gene cluster===<br />
Using two vectors (pARW071 and pARW089, respectively) containing the ''mdn'' biosynthetic gene cluster, we designed PCR primers for the amplification of the ''mdn genes''. Using these primers, we obtained the gene fragments of ''mdnA'', ''mdnB'', ''mdnC'', ''mdnD'', ''mdnE'' and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our BioBricks comply with the BioBrick standards. <br />
<br />
The following image (Fig. 2) shows the generic construction of the BioBrick carrying the'' mdnA gene''.<br />
<br />
[[File:UP_modularization_mdnA_cloning.png|center|500px|thumb|'''Figure 2:''' Cloning Scheme of mdnA-BioBrick (''Cm'' - gene for chloramphenicol resistance)]]<br />
<br />
Microviridin production in ''E. coli'' cells expressing the'' mdn genes'' was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3). <br />
[[File:UP_modularization_HPLC.png|center|500px|thumb|'''Figure 3:''' HPLC chromatogram of MdnA]]<br />
All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.<br />
<br />
Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.<br />
<br />
[[File:UP_modularization_MSII.gif|center|300px|thumb|'''Figure 4:''' Mass Spectrometry Data]]<br />
<br><br><br />
<br />
=== Generating ''mdnA'' gene libraries ===<br />
In searching for optimized and novel microviridin peptides, libraries of mutated microviridins were established. Therefore a number of sites in the amino acid sequence of the precursor peptide (MdnA) were chosen.<br />
In <i>Microcystis</i>, microviridins are synthesized from a ribosomal precursor peptide (MdnA). Therefore the microviridin gene cluster is necessary. This 6.5 kb biosynthesis gene cluster includes two genes (''mdnB'' and ''mdnC''), which encodes ATP-grasp-type ligases and further the ''mdnE'' gene encoding an ABC transporter as well as'' mdnD'' gene encoding a N-acetyltransferase (Ziemert et al., 2008). In consequence the biosynthesis gene cluster genes ''mdnB'', ''mdnC'', ''mdnD'' and ''mdnE'' cannot be mutated. Otherwise only misprocessed and non-function variants of microviridin occur (Ziemert et al., 2008). Thus merely the modification of the ''mdnA'' gene can lead to effective changes in the microviridin structure and function. <br><br />
[[File:UP_lib2_fig.png|center|500px|thumb|'''Figure 5:''' Molecular organization of the ribsosomal precursor peptide (MdnA) of microviridin. MdnA consists of a leader and a core peptide. The N-terminal leader peptide contains highly conserved double glycine motifs. Whereas in the C-terminal 14 amino acid sized core peptide sequence shows variations. Modified from Ziemert et al. (2008) and Ziemert et al. (2010).]]<br />
The precursor peptide MdnA consists of a leader peptide and a core peptide (Fig. 5). Ziemert et al. (2008) has shown that the N-terminal leader peptide contains highly conserved double glycine motifs (Ziemert et al. 2008). However remarkable variations were found in region, which encodes the 14-amino acid sequence of the C-terminal microviridin core peptide (Ziemert et al. 2010). Consequently, mutations in the MdnA core peptide seem to be the most promising option for modification of microviridin. For cyclization, the amino acid side chains of the core peptide form omega-ester and omega-amino bonds. One loop is formed between threonine and aspartic acid, the second loop between lysine and aspartic acid and the third loop between serine and glutamic acid (Fig. 5). The sequence of this loop forming amino acids constitute an exception while mutation of the ''mdnA'' gene (Ziemert et al., 2008 and 2010).<br />
<br />
To find optimized and novel microviridin variants, several libraries carrying modifications in the sequence of the MdnA core peptide were established. These libraries were generated by randomized oligonucleotide synthesis. Therefore a forward oligonucleotide displaying a part of conserved leader peptide sequence was built. Further a reverse oligonucleotide was created. The first part of this oligonucleotide is 20 bases, which overlap with the forward oligonucleotide and which is necessary for hybridization of both, forward and reverse, oligonucleotides. In the middle of the sequence modified bases were inserted. For cloning, the forward oligonucleotide starts with the blunt restricted site of <i>Sfo</i>I. This results therein that the oligonucleotide can ligate in the <i>Sfo</i>I restricted vector without previous digest of the oligonucleotide. The last part of the reverse oligonucleotides displays an <i>Aat</i>II restriction site for cloning. Digestion with <i>Aat</i>II results in sticky ends, thus wrong way round insertion of the oligonucleotide is impossible. Forward and reverse oligonucleotides were combined by performing a fill-in reaction. <br />
Because we designed several libraries with different diversity rates, the modified bases, which are inserted in the middle of the reverse primer, vary. All of our generated libraries contain mutations of the sequence of the MdnA core peptide at several sites, but not at the loop forming sites. Thus we are thinking that cyclization of microviridin happens accurately. A library with a diversity of 45,360 (focused library 1) and a second focused library showing a minor diversity of 810 (focused library 2) were designed. The included sequence modifications plus the diversity of our libraries are shown in figure 6. In our further studies we worked on focused library 2 primarily. In this library the nucleotide sequences encoding glycine residues were changed in GVK in all cases (Figure 6). GVK stands for guanine at the first position, adenine, cytosine or guanine at the second position and further thymine at the third position of the codon. These three possible codons encode for the amino acids alanine, aspartic acid and glycine. The nucleotide sequence N-terminal tyrosine residue was changed in THT describing thymine (first position), adenine, cytosine or thymine at the second position and thymine (third position). These codons encode isoleucine, leucine and phenylalanine. Additionally, the sequence of phenylalanine in the core peptide sequence was shift to NNK. NKK stands for one of the four nucleotides at the first position, and guanine or thymine at the second and third position. Consequently, this codon encodes the amino acids arginine, glycine, tryptophan, methionine, leucine, valine, serine, cysteine, isoleucine and phenylalanine. <br />
[[File:UP_lib2_fig2.png|center|500px|thumb|'''Figure 6:''' Design of libraries based on the nucleotide sequence of the ribosomal precursor peptide (MdnA). To different libraries, focused library 1 and focused library 2 were generated by randomised oligonucleotide synthesis. The modifications compared to the wild type sequence are highlighted. The definition of the used code is also shown. The first libray (focused library 1) displays a diversity of 45360 and the second, focused library 2 a diversity of 810. In the further studies focused library 2 was used to find optimized microviridin variants. ]]<br />
To construct this library a fill-in reaction using the designed forward and reverse oligonucleotides was performed. Subsequently resulting randomized oligonucleotide was digested with the restriction enzyme <i>Aat</i>II. In addition, the vector pUP089 was digested with the restriction enzymes <i>Aat</i>II and <i>Sfo</i>I. After ligation of fragments, randomized oligonucleotide and vector, the focused library 2 (ligation product) was transformed in chemocompetent ''E. coli'' XL1-Blue cells. This procedure was done several times and a library size of 1233 colonies was reached. <br />
[[File:UP_lib2_fig3.png|center|500px|thumb|'''Figure 7:''' Confirmation of focused library 2. Different clones of the generated focused library 2 were sequenced. Alignments with the wild type sequence (reference sequence) were performed. As shown (for a selection of clones) in all of our sequenced samples the requested modifications were established.]]<br />
For confirmation of the focused library 2, sequencing analysis of a number of clones was performed. In all of our sequenced samples the requested modifications were established (figure 7). A sequence logo was generated for the 14 amino acid sized core peptide of MdnA. This sequence logo, which is illustrated in figure 8, indicates that the desired sites stay conserved. The others are modified. <br />
[[File:UP_lib2_fig4.png|center|500px|thumb|'''Figure 8:''' Sequence logo plot of focused library 2. The 14 amino acid sized core peptide of MdnA was sequenced. The sequence of several clones was used to generate a sequence logo. This indicates that the desired sites stay conserved. The others nucleotides and resulting amino acids are modified. ]]<br />
The generated and modified focused library 2 should be used as basis for <i>in vivo</i> selection and phage display screening. Furthermore we purpose the objective to search for optimized and novel microviridins by using this library.<br />
<br><br><br />
<br />
=== Expression Backbones ===<br />
In a subtask of our work we wanted to construct auxiliary expression backbones with inducible promotors. In contrast to constitutive systems inducible systems only express the protein of interest after adding an inducer, which can be for example AHL or arabinose.<br />
We decided to construct IPTG- and arabinose-inducible systems. Therefore we amplified the promotor region and fused it to a reporter gene, which is constituted by YFP. Using this we have the option to verify the presence of the inducible promotor and also the function of the induction process by fluorescence. Our idea was to use the reporter gene as a placeholder. Via restriction enzyme digestion using the iGEM restriction enzyme sites you will be able to replace the reporter gene with your gene of interest. As vector backbone we made use of the pSB1A3, which has ampicillin resistance (Fig. 9). The constructs using pSB1K3 (kanamycin resistance) and pSB1C3 (chloramphenicol resistance) are still on the anvil. <br />
[[File:UP_expression_backbones_cloning.png|center|500px|thumb|'''Figure 9:''' Cloning Scheme of Expression Backbones. ''amp'' - gene for ampicillin resistance]]<br />
<br />
We successfully tested the IPTG- and arabinose-inducible system (Fig 10). Using fluorescence microscopy we were able to detect the YFP-expression after an induction time of approximately 1.5 hrs. To prove the hypothesis we opposed an induced sample with a non-induced control for each promotor. By use of brightfield combined with differential interference contrast microscopy we traced the ''E. coli'' cells and switched then to the YFP detecting channel to investigate the fluorescence of these cells.<br />
<br />
[[File:UP_Expression_Backbones_Fluorescence.png|center|800px|thumb|'''Figure 10:''' Fluorescence Microscopy. control - not induced; induced - induction with IPTG or Arabinose, respectively]]<br />
<br />
We also tested the inducible systems by using fluorescence spectroscopy. For this experiment we induced both systems at a time when the cultures were located in phase of exponential growth. After inducing, the cultures were analyzed by fluorescence spectroscopy. In this analyze the cells were excited by 500 nm. The resultant emission was measured in a spectrum between 510 and 580 nm. (Fig 11;12)<br />
<table><td>[[File:UP_psB1A3_Ara_YFPII.png|left|410px|thumb|'''Figure 11:''' Emission (526nm) of pSB1A3_Ara_YFP and control – '' E.coli'' cells (including pSB1A3_Ara_YFP) were induced and measured at certain moments.]] </td> <td>[[File:UP_psB1A3_IPTG_YFPII.png|right|410px|thumb|'''Figure 12:''' Emission (526nm) of induced pSB1A3_lac_YFP and control – ''E.coli'' cells (including pSB1A3_lac_YFP) were induced and measured at certain moments.]]</td><br />
</table>In contrast to the control of the arabinose inducible system the Lac-control also shows fluorescence. This demonstrates the leaky <i>Lac</i>-promotor, what is also a scientifically proven fact. A leaky promotor means that the promotor is not regulated in a leakproof manner. Even in repressed conditions the transcript can arise.<br />
<br />
The lacking of the <i>LacI</i> gene on the vector is the reason for expression of YFP in both controls. For further research in this field it is necessary to clone the <i>LacI</i> gene inside the constructed vector.<br />
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<br />
===References===<br />
Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74<br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/TeamTeam:Potsdam Bioware/Team2011-10-28T23:35:52Z<p>UP Stefan: /* Contribution */</p>
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<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Team ==<br />
<br><br />
[[File:UP_team_amsterdam.jpg|center|500px]]<br />
<br><br />
[[File:UP_iGEM_team_roof.jpg|center|500px]]<br />
<br />
== Members ==<br />
<br />
=== Instructors ===<br />
<table><tr><br />
<br />
<td width="25%">[[File:UP_Katja.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td width="25%"><br />
'''Katja Arndt'''<br><br />
<br />
Head of group Molecular<br><br />
Biotechnology<br><br />
</td><br />
<br />
<td width="25%">[[File:UP_Kristian.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td width="25%"><br />
'''Kristian Müller'''<br><br />
<br />
Head of group Synthetic<br><br />
Biosystems<br><br />
</td> <br />
<br />
</tr><br />
</table><br />
<br />
<br><br />
<br />
=== Students ===<br />
<br />
=== Microviridin ===<br />
<br />
<table><tr><br />
<br />
<td>[[File:UP_Nicole.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Nicole Albrecht'''<br><br />
<br />
Major: Biotechnology,<br><br />
Molecularbiology, Biochemistry <br><br />
<br />
Semester: 8th <br><br />
</td><br />
<br />
<td>[[File:UP_Katharina.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Katharina Berger'''<br><br />
<br />
Major: Biological Science,<br><br />
Molecularbiology, Biochemistry <br><br />
<br />
Semester: 8th<br><br />
</td> <br />
<br />
</tr><br />
<br />
<tr><br />
<br />
<td>[[File:UP_Nadja.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Nadja Bjelopoljak'''<br><br />
<br />
Major: Biological Science,<br><br />
Biochemistry, Molecularbiology <br><br />
<br />
Semester: 8th <br><br />
</td><br />
<br />
<td>[[File:UP_Nadine.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Nadine Boehmer'''<br><br />
<br />
Major: Biological Science,<br><br />
Biochemistry, Molecularbiology <br><br />
<br />
Semester: 8th <br><br />
</td><br />
</tr><br />
<br />
<tr><br />
<br />
<td>[[File:UP_VanessaB.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Vanessa Boehmer'''<br><br />
<br />
Major: Biological Science,<br><br />
Molecularbiology <br><br />
<br />
Semester: 4th<br><br />
</td><br />
<br />
<td>[[File:UP_Jessica.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Jessica Eger'''<br><br />
<br />
Major: Biological Science,<br><br />
Molecularbiology, Biochemistry<br><br />
<br />
Semester: 8th<br><br />
</td><br />
<br />
</tr><br />
<br />
<tr><br />
<br />
<td>[[File:UP_Steffi.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Steffi Sempert'''<br><br />
<br />
Major: Molecularbiotechnology,<br><br />
Molecularbiology, Biochemistry <br><br />
<br />
Semester: 10th<br><br />
</td><br />
<br />
<td>[[File:UP_Niels.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Niels Weisbach'''<br><br />
<br />
Major: Biology,<br><br />
Molecularbiology, Biochemistry<br><br />
<br />
Semester: 9th<br><br />
</td><br />
<br />
</tr></table><br />
<br />
<br><br />
<br />
=== In vivo Selection ===<br />
<br />
<table><br />
<tr><br />
<td><br />
[[File:UP_Sebastian.jpg|left|200px]]<br />
</td><br />
<td><br />
'''Sebastian Hanke'''<br><br />
<br />
Major: Biological Science, <br><br />
Molecularbiology, Biochemistry <br><br />
<br />
Semester: 8th<br><br />
</td><br />
<td><br />
[[File:UP_Paul.jpg|left|200px]]<br />
</td><br />
<td><br />
'''Paul Kaufmann'''<br><br />
<br />
Major: Biological Science,<br><br />
Molecularbiology, Biochemistry <br><br />
<br />
Semester: 8th<br><br />
</td><br />
</tr><tr><br />
<br />
<td>[[File:UP_SaschaR.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Sascha Ramm'''<br><br />
<br />
Major: Biological Science,<br><br />
Molecularbiology, Biochemistry <br> <br />
<br />
Semester: 8th<br><br />
</td><br />
<br />
<br />
<br />
<td>[[File:UP_Stefan.jpg|left|200px]]<br />
</td><br />
<br />
<td><br />
'''Stefan Wahlefeld'''<br><br />
<br />
Major: Biotechnology,<br><br />
Molecularbiology, Biochemistry <br><br />
<br />
Semester: 8th<br><br />
</td><br />
<br />
</tr></table><br />
<br />
<br><br />
<br />
=== Phage Display ===<br />
<br />
<table><tr><br />
<br />
<td>[[File:UP_Sandrina.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Sandrina Heyde'''<br><br />
<br />
Major: Biological Science,<br><br />
Molecularbiology, Biochemistry <br><br />
<br />
Semester: 8th<br><br />
</td><br />
<br />
<td>[[File:UP_Sabine.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Sabine Meyer'''<br><br />
<br />
Major: Biological Science,<br><br />
Molecularbiology, Biochemistry <br><br />
<br />
Semester: 8th<br><br />
</td><br />
<br />
</tr></table><br />
<br />
<br><br />
<br />
=== Modelling and Software ===<br />
<br />
<table><tr><br />
<br />
<td>[[File:UP_Niklas.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
'''Niklas Laasch'''<br><br />
<br />
Major: Biological Science,<br><br />
Biochemistry <br><br />
<br />
Semester: 4th<br><br />
<br />
<br />
</td><br />
<td>[[File:UP_Oliver.jpg|left|200px]]<br />
</td><br />
<br />
<td><br />
'''Oliver Zimmer'''<br><br />
<br />
Major: Telematics<br><br />
<br />
Semester: 4th<br><br />
<br><br />
<br><br />
<br><br />
</td><br />
<br />
</tr><tr><br />
<br />
<td>[[File:UP_TobiasW.jpg|left|200px]]<br />
<br />
</td><br />
<br />
<td><br />
<br />
'''Tobias Wenzel'''<br><br />
<br />
Major: Physics<br><br />
<br />
Semester: 6th<br><br />
<br />
</td><br />
<br />
</tr></table><br />
<br />
<br><br />
<br />
== Contribution ==<br />
<br />
The chance to be part of this competition, of this incredible project incorporating organization and communication skills, teamwork, scientific know-how, time management, budget management, independent working, enthusiasm for synthetic biology would never have been come true without the endorsement on time and valuable advice of our supervisors Prof. Katja Arndt and Dr. Kristian Müller. We cordially owe our deepest gratitude to them.<br />
<br><br>We are also extremely thankful to Prof. Elke Dittmann and her team providing us the microviridin gene cluster and their precious advices and support throughout the journey right from the beginning.<!-- Tolkien lässt grüßen--><br />
<br><br>We would like to thank Gunter Stier for the TEV protease.<br />
<br><br>Among the crews at the University of Potsdam we want to express our gratitude to the group of Prof. Steup and Dr. Fettke for the support with MS analysis and last but not least the team of Prof. Walz giving us the opportunity to use their fluorescence microscope. Additionally, are we very thankful to the groups of Prof. Dittmann, Prof. Ignatova and Prof. Seckler for their greatness to afford us to use their autoclaves. We would also like to thank Prof. Huisinga for the interesting feedback to our model as well as the financial support.<br />
<br />
Ultimately, we are greatly thankful to Tim, Tobi, Sven, and Vijay, PhD. students of Katja and Kristian, for their encouragement. We know, it wasn't always easy with us, and hereby we formally apologize. For your patience with us throughout the summer, we also want to say a huge Thank you!<br />
<br><br><br />
The contribution of our team is listed in detail in the lab journal provided.</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_PhageTeam:Potsdam Bioware/Project/Details Phage2011-10-28T23:20:11Z<p>UP Stefan: /* Cloning strategy */</p>
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== Phage Display ==<br />
===Introduction===<br />
<br />
Phage Display is an efficient tool for selecting protein or peptides with specific binding properties from a large recombinant library. This proteins are represented on the surface of bacteriophages. This enables the coupling of phenotype and stable packaged genotype because the proteins which form the phage including the proteins of interest are coded in its genome. To test the suitability of phage display system as an appropriate screening method for recombinant <i>mdnA</i> libraries a vector containing a <i>mdnA-myc-geneIII</i>-fusion gene was generated. This vector contains a plasmid origin of replication, so it can be amplified like plasmids. Additionally it contains a f1 ori which enables the packaging of single strand DNA into phages. The vector also contains the whole <i>mdn</i>-cluster which is needed to produce the MdnA peptide. Between <i>mdnA</i> and <i>gene III</i> is a <i>myc</i>-tag located, which is used for an easy detection. The successful expression of the MdnA-myc-geneIII-fusion protein on the surface of the phage was determined by ELISA using anti-myc antibody 9E10 after transforming ''E. coli'' cells and purifying the produced phages. The next step was to perform a phage display on a know target of the MdnA. To test the fundamental suitability of this screening method, phages representing MdnA on their surface and phages not representing <i>MdnA</i> in a ratio of one to one were incubated with immobilized trypsin which is known as a target of MdnA. After the first panning round a marked concentrating of phages carrying MdnA was recognized.<br><br />
<br />
===Cloning strategy===<br />
<br />
The phage display vector pPDV089 was derived from the plasmid pARW089 which carries the whole <i>mdn</i>-cluster. This plasmid contains a plasmid origin of replication and additionally a f1 ori which enables the packaging of single strand DNA into phages. For selective amplification ampcillin and kanamycin resistance genes are included. To create the phagemid pPDV089 standard cloning procedure were performed. <br />
First <i>mdnA</i> was deleted by excising using the restriction enzymes <i>Sfo</i>I and <i>Aat</i>II. The next step comprised the introduction of a <i>mdnA-geneIII</i>-fusion gene. Therefore <i>gene III</i> was amplified from pak100blaKDIR and <i>mdnA</i> from pARW089 by PCR. The primers were designed to enable the introduction of iGEM and other restriction sites required for further cloning steps. The purified PCR product <i>geneIII</i> was digested by <i>NgoM</i>IV and <i>Aat</i>II whereas the PCR product <i>mdnA</i> was digested by <i>Sfo</i>I and <i>Age</i>I. Subsequent the three fragment ligation of <i>mdnA</i> and <i>geneIII</i> into the digested vector has been conducted. Thus a <i>mdnA-geneIII</i>-fusion part according to RFC25 was generated whereby <i>Age</i>I and <i>NgoM</i>IV overhangs are compatible and placed in frame with the protein sequence. The ligation of <i>Age</i>I and <i>NgoM</i>IV overhangs resulted in a scar coding for the threonine and glycine. Because the introduction of restriction sites before <i>mdnA</i> leaded to a great distance between ribosome binding site (RBS) and <i>mdnA</i>. A second RBS was inserted among <i>Sfo</i>I and <i>Xba</i>I recognition sites to ensure a sufficiently expression rate of the <i>mdnA-geneIII</i>-fusion gene. The ''myc'' sequence located between <i>mdnA</i> and <i>gene III</i> allows the detection of the expression of the <i>mdnA-geneIII</i>-fusion protein on the surface of the phage using western blot or ELISA. In the last step the kanamycin resistance gene was disturbed because for phage display the selection of cells carriyng both helper phages and the phagemid is beneficial and the helper phages have a kanamycin resistance, too. Therefore a 300 bp fragment of the kanamycin resistance gene was deleted using the restriction enzyme <i>Nsi</i>I which had two recognition sites in the kanamycin gene.<br />
<br />
<br />
<br />
{| border="0" cellspacing="0" cellpadding="2" <br />
<br />
| [[File:UP cloning strategy.png|center|450px|thumb|'''Figure 1: Cloning strategy for creating a plasmid which can be used for phage display with <i>mdnA</i>. '''<i>MdnA</i> was cut out of the vector and a <i>mdnA-myc-geneIII</i> fusion gene was created and ligated with the vector pARW089 containing the <i>mdn</i>-cluster without <i>mdnA</i>.]] || [[File:UP pPDV089.png|center|350px|thumb|'''Figure 2: Designed vector pPDV089 carrying the <i>mdnA-myc-gene III</i> fusion gene.''' Therefore the <i>mdnA</i> sequence was cut out of the vector pARW089 and the ligated <i>mdnA-myc-geneIII</i> gene was inserted]] <br />
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<br />
===Control of expression of ''mdnA''-''myc''-''geneIII'' in ''E. coli''===<br />
<br />
The expression of the ''mdnA''-''myc''-''geneIII'' fusion gene was analyzed by western blotting. ''E. coli'' cells transformed with the phagemid pPDV089 were harvested and lysated. The proteins were electrophoretically separated and transferred to a membrane. The ''mdnA''-''myc''-''geneIII''-fusion proteins were detected using specific anti-''myc''-antibodies and horseradish peroxidase (HRP)-linked antibodies as secondary antibodies. Enhanced chemiluminescence (ECL) was used to visualize the protein. ECL is based on the emission of light during the HRP-catalyzed oxidation of luminol, which was captured by a camera. The western blot analysis resulted in a band of a size just below the 30 kDa mark representing the ''mdnA''-''myc''-''geneIII''-fusion protein (24 kDa).<br />
<br />
[[File:UP_coomassie+western blot.png|center|400px|thumb|'''Figure 3: Control of expression of ''mdnA''-''myc''-''geneIII'' in ''E. coli'' by western blotting.''' For detection anti-''myc''-antibodies and secondary HRP-linked antibodies were used. The resulting band represents the ''mdnA''-''myc''-''geneIII''-fusion protein.]]<br />
<br><br />
<br><br />
<br><br />
<br />
===Detection of phages carrying ''mdnA'' on their surface by ELISA===<br />
<br />
The next step was the detection of the expression of the ''mdnA''-''myc''-''geneIII''-fusion gene on the surface of the phage. So ''E. coli'' cells strain XL1-Blue were first transformed with the phagemid pPDV089 before they were infected with helper phages. ''E. coli'' cells containing both plasmids were selected. An ELISA test was performed to determine whether these cells are able to produce phage particles carrying the MdnA peptide on their surface. To perform this test anti-''c-myc''-antibodies were immobilized on ELISA plates and incubated with purified phages. For detection a second antibody coupled with horse radish peroxidase (HRP) was used which binds the gene VIII protein of the phages. The HRP substrate o-phenyldiamine (OPD) was added and in case of binding a color reaction was expected. The color shift from achromatic to yellow in wells incubated with phages produced in XL1-Blue cells showed the successful expression of ''mdnA''-c-''myc''-''geneIII''-fusion protein on the phages.<br><br />
For more precise results the absorption at 492 nm was measured. The data were presented in a bar plot. As a negative control helper phages were added instead of produced phages. Furthermore two wells were prepared were the secondary antibody was not added.<br />
The graphic shows clearly the much higher absorption measured in wells, which were incubated with phage particles of interest produced in XL1-Blue cells. As has already pointed out this shows the succeeded expression of ''mdnA''-c-''myc''-''geneIII''-fusion protein on the surface of the phages.<br />
<br />
<br />
<div align="center"><br />
[[File:UP_ELISA3_mit_fehlerindikator.png|center|400px|thumb| '''Figure 4:''' Detection of phages carrying ''mdnA'' on their surface by ELISA. The bar plot shows the absorption at 492 nm. Anti-''myc''-antibodies were immobilized. For detection a second antibody coupled with horse radish peroxidase (HRP) was used which binds the gene VIII coat protein of the phages. The left bar shows the absorption of the wells containing helper phages (negative control), the right bar shows the absorption of wells containing ''mdnA'' carrying phages]]</div><br><br />
<br><br />
<br />
===Testing phage display with unmodified ''mdnA'' to examine its suitability as screening method===<br />
<br />
To test the fundamental suitability of this screening method, phages representing unmodified ''mdnA'' on their surface and phages not representing ''mdnA'' (helper phages) in a ratio of one to one were incubated with immobilized trypsin which is known as a target of ''mdnA''. The display was conducted in ELISA plates. The bound phages were eluted using a buffer with low pH value and neutralized afterwards. To check how many phages interacted with trypsin, ''E. coli'' cells XL1-Blue were re-infected with eluted phages and plated on agar with different antibiotics. Cells infected with phages carrying ''mdnA'' are able to grow on agar with ampicillin whereas cells infected with helper phages are able to grow on agar with kanamycin. To control the success of the panning round additionally ''E. coli'' cells were infected with phage mix before panning and plated on agar. Subsequent the number of clones grew on ampicillin and kanamycin before and after panning was compared. During the running of this step it was noticed that much more cells were infected with helper phages than with phages carrying ''mdnA'' despite of the engaged 1:1 ratio. This was surprising and indicated that ''mdnA'' on the surface of the phages may inhibit their infectivity. After controlling the plates an infection ratio of phages carrying ''mdnA'' to helper phages of 1:400 was calculated. This fact should be analyzed in further experiments.<br><br />
The results of the first phage display are plotted in the figure below. After one panning round an enrichment of phages carrying ''mdnA'' was expected. This is attributable to the fact that phage particles carrying ''mdnA''-c-''myc'''-gene III-fusion protein on their surface are expected to bind specifically to the immobilized trypsin. Unfortunately this was not observed in this experiment. The ratio of cells growing on kanamycin agar before (4000) to cells growing on kanamycin agar (cells containing helper phages) after panning (750) was determined as 5:1. The ratio of cells growing on ampicillin agar before (12) to cells growing on ampicillin agar (cells containing ''mdnA'' carrying phages) after panning (2) was nearly equal. Thus no enrichment of ''mdnA'' carrying phages occurred in the first experiment. So it was decided to repeat this experiment under improved conditions. Therefor the number of washing steps during the described experimental procedure was increased. Here the ratio of cells growing on kanamycin agar before (3000) to cells growing on kanamycin agar (cells containing helper phages) after panning (29) was determined as 103:1. The ratio of cells growing on ampicillin agar before (26) to cells growing on ampicillin agar (cells containing ''mdnA'' carrying phages) after panning (2) was determined as 13:1. Thus an enrichment factor of eight was reached for the phages displaying ''mdnA'' on their surface. <br><br />
These results indicate that the unmodified ''mdnA'' expressed on the phages binds specifically to the immobilized trypsin. Therefore it can be deduced that ''mdnA'' is presented in a functional 3D structure. These findings suggest that phage display in general is an appropriate method for screening a recombinant ''mdnA'' library. Further experiments are required to optimize this system.<br />
<br />
<br />
[[File:UP panning 3.png|center|400px|thumb| '''Figure 5: Optimization of phage display.''' After optimized conditions (right) a clear concentration of phages carrying ''mdnA'' after one panning round was noted. ''E. coli'' cells were infected with phage mix (helper phages and phages carrying ''mdnA'') before and after panning and plated on agar containing kanamycin or ampicillin. The ratio of cells growing on ampicillin or kanamycin agar before panning to cells growing on ampicillin or kanamycin agar after was calculated. Helper phages which acted as negative control have a kanamycine resistance whereby phages carrying ''mdnA'' have an ampicillin resistance.]]<br />
<br />
===Testing phage display with unmodified mdnA against further proteases===<br />
<br />
Furthermore the interaction of unmodified mdnA with other proteases was determined. From the literature (Ziemert, 2010) the high inhibitory activity of microviridin L, besides trypsin, against chymotrypsin and elastase is also known. So a phage display with these enzymes was performed. Additionally papain, proteinase K, mycolysin and pepsin were tested for which no interaction was shown yet. For all enzymes an equal amount of ''E. coli'' cells and phages were used. In agreement with data from the literature interaction of microviridin with chymotrypsin and elastase was confirmed. Additionally an interaction with papain was noted. All other proteases were not bound by microviridin. <br />
<br />
[[File:UP_test of different enzymes.png|center|400px|thumb|'''Figure 6: Phage display against different proteases.''' After panning the number of clones was counted. In agreement with data from the literature interaction of microviridin with chymotrypsin and elastase was confirmed. Additionally an interaction with papain was noted.]]<br />
<br><br><br />
<br />
===References===<br />
*Fuh G., Sidhu S.S. (2000). Efficient phage display of polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein. FEBS Lett. 480(2-3):231-4<br />
<br />
*Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R., Plückthun, A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Meth. 201(1):35-55 <br />
<br />
*Rakonjac J., Feng J., Model P. (1999). Filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of pIII. J Mol Biol. 289(5):1253-65<br />
*Smith, G.P. (1985). Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virus surface. Science 228: 1315-17<br />
<br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
*Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74 <br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_MicroviridinTeam:Potsdam Bioware/Project/Details Microviridin2011-10-28T23:19:09Z<p>UP Stefan: /* Introduction */</p>
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==Microviridin==<br />
=== Introduction ===<br />
Cyanobacteria are known as the blue-green-algae because their living space is water and their feeding mechanism is photosynthetic. The Cyanobacteria are know for some special and unique metabolites. Some of these metabolites are part of the microviridin family. Special attributes of microviridins are their occurrence as tricyclic depsipeptides, means peptides whose one or more amide-bond is replaced by an ester-bond, unparallel cage-like architecture and their ability to inhibit several proteases. Microviridin B for example is told to function as an elastase inhibitor what would be an therapeutic attempt according to fix the out of control gone function of elastase in lung emphysema. In such a case it is beyond debate how important it is to figger out the biosynthesis of this peptide. The research group of Prof. Dittmann at the University of Potsdam was successful and able to report about the composition of the gene cluster from ''Micocystis aeruginosa'' NIES298 expressing Microviridin B. Moreover they have been convinced that there is existing one unique biosynthetic mechanism for microviridins in <i>Microcystis</i> strains. Unusual for depsipeptides they discovered that they are ribosomal synthesized. The ''mdn'' gene cluster is build up of a gene ''mdnA'', encoding for the putative precursor peptide of the microvirdin, two genes encoding ATP-grasp-type ligases ''mdnB'' and ''mdnC'', an ABC transporter encoding gene ''mdnE'' as well as one encoding an N- acetyltransferase of the GNAT family ''mdnD'' (Ziemert et al., 2008). Moreover in their recent studies they on the contrary report about the discovery of an existing natural diversity of microviridin precursors genes in set of closely related <i>Mycrocystis</i> laboratory strains. This furthermore would mean and lead to a heterologous production of novel microviridins in ''E. coli''. To give evidence of their discovery they identified and characterized a new Microviridin L from the strain ''Mycrocystis aeruginosa'' NIES843 (Ziemert et al., 2010).<br />
All in all the microviridins, the tricyclic depsipeptide are build up through a gene cluster of five mdn-components each fulfill one function to make the hole cluster functional. This showed the team of Prof. Dr. Dittmann as the peptide is only correctly processed in ''E. coli'' if the entire cluster ''mdnABCDE'' is expressed. In addition the new discovery is promising a small flexibility of the microviridin ligases as an open window for the size of the natural microviridin library and thus microviridin functions and hence for example therapeutic benefit that needs to be discovered. The following illustration is going to give you a better overview (Fig. 1).<br />
[[File:UP_MV_introduction_mdn.jpg|center|525px|thumb|'''Figure 1:''' overview of the mdn gene cluster (GNAT: GCN5-related N-acetyltransferase)]]<br />
<br><br><br />
<br />
=== Modularization of the ''mdn'' gene cluster===<br />
Using two vectors (pARW071 and pARW089, respectively) containing the ''mdn'' biosynthetic gene cluster, we designed PCR primers for the amplification of the ''mdn genes''. Using these primers, we obtained the gene fragments of ''mdnA'', ''mdnB'', ''mdnC'', ''mdnD'', ''mdnE'' and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our BioBricks comply with the BioBrick standards. <br />
<br />
The following image (Fig. 2) shows the generic construction of the BioBrick carrying the'' mdnA gene''.<br />
<br />
[[File:UP_modularization_mdnA_cloning.png|center|500px|thumb|'''Figure 2:''' Cloning Scheme of mdnA-BioBrick (''Cm'' - gene for chloramphenicol resistance)]]<br />
<br />
Microviridin production in ''E. coli'' cells expressing the'' mdn genes'' was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3). <br />
[[File:UP_modularization_HPLC.png|center|500px|thumb|'''Figure 3:''' HPLC chromatogram of MdnA]]<br />
All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.<br />
<br />
Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.<br />
<br />
[[File:UP_modularization_MSII.gif|center|300px|thumb|'''Figure 4:''' Mass Spectrometry Data]]<br />
<br><br><br />
<br />
=== Generating ''mdnA'' gene libraries ===<br />
In searching for optimized and novel microviridin peptides, libraries of mutated microviridins were established. Therefore a number of sites in the amino acid sequence of the precursor peptide (MdnA) were chosen.<br />
In <i>Microcystis</i>, microviridins are synthesized from a ribosomal precursor peptide (MdnA). Therefore the microviridin gene cluster is necessary. This 6.5 kb biosynthesis gene cluster includes two genes (''mdnB'' and ''mdnC''), which encodes ATP-grasp-type ligases and further the ''mdnE'' gene encoding an ABC transporter as well as'' mdnD'' gene encoding a N-acetyltransferase (Ziemert et al., 2008). In consequence the biosynthesis gene cluster genes ''mdnB'', ''mdnC'', ''mdnD'' and ''mdnE'' cannot be mutated. Otherwise only misprocessed and non-function variants of microviridin occur (Ziemert et al., 2008). Thus merely the modification of the ''mdnA'' gene can lead to effective changes in the microviridin structure and function. <br><br />
[[File:UP_lib2_fig.png|center|500px|thumb|'''Figure 5:''' Molecular organization of the ribsosomal precursor peptide (MdnA) of microviridin. MdnA consists of a leader and a core peptide. The N-terminal leader peptide contains highly conserved double glycine motifs. Whereas in the C-terminal 14 amino acid sized core peptide sequence shows variations. Modified from Ziemert et al. (2008) and Ziemert et al. (2010).]]<br />
The precursor peptide MdnA consists of a leader peptide and a core peptide (Fig. 5). Ziemert et al. (2008) has shown that the N-terminal leader peptide contains highly conserved double glycine motifs (Ziemert et al. 2008). However remarkable variations were found in region, which encodes the 14-amino acid sequence of the C-terminal microviridin core peptide (Ziemert et al. 2010). Consequently, mutations in the MdnA core peptide seem to be the most promising option for modification of microviridin. For cyclization, the amino acid side chains of the core peptide form omega-ester and omega-amino bonds. One loop is formed between threonine and aspartic acid, the second loop between lysine and aspartic acid and the third loop between serine and glutamic acid (Fig. 5). The sequence of this loop forming amino acids constitute an exception while mutation of the ''mdnA'' gene (Ziemert et al., 2008 and 2010).<br />
<br />
To find optimized and novel microviridin variants, several libraries carrying modifications in the sequence of the MdnA core peptide were established. These libraries were generated by randomized oligonucleotide synthesis. Therefore a forward oligonucleotide displaying a part of conserved leader peptide sequence was built. Further a reverse oligonucleotide was created. The first part of this oligonucleotide is 20 bases, which overlap with the forward oligonucleotide and which is necessary for hybridization of both, forward and reverse, oligonucleotides. In the middle modified bases are inserted. For cloning the forward oligonucleotide starts with the blunt restricted recognition site of <i>Sfo</i>I. This results therein that the oligonucleotide can ligate in the <i>Sfo</i>I restricted vector without previous restriction of the oligonucleotide. The last part of the reverse oligonucleotides displays an <i>Aat</i>II restriction site for cloning. Digestion with <i>Aat</i>II results in sticky end, thus wrong way round insertion of the oligonucleotide is impossible. Forward and reverse oligonucleotides were combined by performing a fill-in reaction. <br />
Because we designed several libraries with different diversity rates, the modified bases, which are inserted in the middle of the reverse primer, vary. All of our generated libraries contain mutations of the sequence of the MdnA core peptide at several sites, but not at the loop forming sites. Thus we are thinking that cyclization of microviridin happens accurately. A library with a diversity of 45,360 (focused library 1) and a second focused library showing a minor diversity of 810 (focused library 2) were designed. The included sequence modifications plus the diversity of our libraries are shown in figure 6. In our further studies we worked on focused library 2 primarily. In this library the nucleotide sequences encoding glycine residues were changed in GVK in all cases (Figure 6). GVK stands for guanine at the first position, adenine, cytosine or guanine at the second position and further thymine at the third position of the codon. These three possible codons encode for the amino acids alanine, aspartic acid and glycine. The nucleotide sequence N-terminal tyrosine residue was changed in THT describing thymine (first position), adenine, cytosine or thymine at the second position and thymine (third position). These codons encode isoleucine, leucine and phenylalanine. Additionally, the sequence of phenylalanine in the core peptide sequence was shift to NNK. NKK stands for one of the four nucleotides at the first position, and guanine or thymine at the second and third position. Consequently, this codon encodes the amino acids arginine, glycine, tryptophan, methionine, leucine, valine, serine, cysteine, isoleucine and phenylalanine. <br />
[[File:UP_lib2_fig2.png|center|500px|thumb|'''Figure 6:''' Design of libraries based on the nucleotide sequence of the ribosomal precursor peptide (MdnA). To different libraries, focused library 1 and focused library 2 were generated by randomised oligonucleotide synthesis. The modifications compared to the wild type sequence are highlighted. The definition of the used code is also shown. The first libray (focused library 1) displays a diversity of 45360 and the second, focused library 2 a diversity of 810. In the further studies focused library 2 was used to find optimized microviridin variants. ]]<br />
To construct this library a fill-in reaction using the designed forward and reverse oligonucleotides was performed. Subsequently resulting randomized oligonucleotide was digested with the restriction enzyme <i>Aat</i>II. In addition, the vector pUP089 was digested with the restriction enzymes <i>Aat</i>II and <i>Sfo</i>I. After ligation of fragments, randomized oligonucleotide and vector, the focused library 2 (ligation product) was transformed in chemocompetent ''E. coli'' XL1-Blue cells. This procedure was done several times and a library size of 1233 colonies was reached. <br />
[[File:UP_lib2_fig3.png|center|500px|thumb|'''Figure 7:''' Confirmation of focused library 2. Different clones of the generated focused library 2 were sequenced. Alignments with the wild type sequence (reference sequence) were performed. As shown (for a selection of clones) in all of our sequenced samples the requested modifications were established.]]<br />
For confirmation of the focused library 2, sequencing analysis of a number of clones was performed. In all of our sequenced samples the requested modifications were established (figure 7). A sequence logo was generated for the 14 amino acid sized core peptide of MdnA. This sequence logo, which is illustrated in figure 8, indicates that the desired sites stay conserved. The others are modified. <br />
[[File:UP_lib2_fig4.png|center|500px|thumb|'''Figure 8:''' Sequence logo plot of focused library 2. The 14 amino acid sized core peptide of MdnA was sequenced. The sequence of several clones was used to generate a sequence logo. This indicates that the desired sites stay conserved. The others nucleotides and resulting amino acids are modified. ]]<br />
The generated and modified focused library 2 should be used as basis for <i>in vivo</i> selection and phage display screening. Furthermore we purpose the objective to search for optimized and novel microviridins by using this library.<br />
<br><br><br />
<br />
=== Expression Backbones ===<br />
In a subtask of our work we wanted to construct auxiliary expression backbones with inducible promotors. In contrast to constitutive systems inducible systems only express the protein of interest after adding an inducer, which can be for example AHL or arabinose.<br />
We decided to construct IPTG- and arabinose-inducible systems. Therefore we amplified the promotor region and fused it to a reporter gene, which is constituted by YFP. Using this we have the option to verify the presence of the inducible promotor and also the function of the induction process by fluorescence. Our idea was to use the reporter gene as a placeholder. Via restriction enzyme digestion using the iGEM restriction enzyme sites you will be able to replace the reporter gene with your gene of interest. As vector backbone we made use of the pSB1A3, which has ampicillin resistance (Fig. 9). The constructs using pSB1K3 (kanamycin resistance) and pSB1C3 (chloramphenicol resistance) are still on the anvil. <br />
[[File:UP_expression_backbones_cloning.png|center|500px|thumb|'''Figure 9:''' Cloning Scheme of Expression Backbones. ''amp'' - gene for ampicillin resistance]]<br />
<br />
We successfully tested the IPTG- and arabinose-inducible system (Fig 10). Using fluorescence microscopy we were able to detect the YFP-expression after an induction time of approximately 1.5 hrs. To prove the hypothesis we opposed an induced sample with a non-induced control for each promotor. By use of brightfield combined with differential interference contrast microscopy we traced the ''E. coli'' cells and switched then to the YFP detecting channel to investigate the fluorescence of these cells.<br />
<br />
[[File:UP_Expression_Backbones_Fluorescence.png|center|800px|thumb|'''Figure 10:''' Fluorescence Microscopy. control - not induced; induced - induction with IPTG or Arabinose, respectively]]<br />
<br />
We also tested the inducible systems by using fluorescence spectroscopy. For this experiment we induced both systems at a time when the cultures were located in phase of exponential growth. After inducing, the cultures were analyzed by fluorescence spectroscopy. In this analyze the cells were excited by 500 nm. The resultant emission was measured in a spectrum between 510 and 580 nm. (Fig 11;12)<br />
<table><td>[[File:UP_psB1A3_Ara_YFPII.png|left|410px|thumb|'''Figure 11:''' Emission (526nm) of pSB1A3_Ara_YFP and control – '' E.coli'' cells (including pSB1A3_Ara_YFP) were induced and measured at certain moments.]] </td> <td>[[File:UP_psB1A3_IPTG_YFPII.png|right|410px|thumb|'''Figure 12:''' Emission (526nm) of induced pSB1A3_lac_YFP and control – ''E.coli'' cells (including pSB1A3_lac_YFP) were induced and measured at certain moments.]]</td><br />
</table>In contrast to the control of the arabinose inducible system the Lac-control also shows fluorescence. This demonstrates the leaky Lac-promotor, what is also a scientifically proven fact. A leaky promotor means that the promotor is not regulated in a leakproof manner. Even in repressed conditions the transcript can arise.<br />
<br />
The lacking of the Lac-I gene on the vector is the reason for expression of YFP in both controls. For further research in this field it is necessary to clone the Lac-I gene inside the constructed vector.<br />
<br><br><br />
<br />
===References===<br />
Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74<br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_MicroviridinTeam:Potsdam Bioware/Project/Details Microviridin2011-10-28T23:16:09Z<p>UP Stefan: /* Introduction */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==Microviridin==<br />
=== Introduction ===<br />
Cyanobacteria are known as the blue-green-algae because their living space is water and their feeding mechanism is photosynthetic. The Cyanobacteria are know for some special and unique metabolites. One of these metabolites are the microviridin family. Special attributes of microviridins are their occurrence as tricyclic depsipeptides, means peptides whose one or more amide-bond is replaced by an ester-bond, unparallel cage-like architecture and their ability to inhibit several proteases. Microviridin B for example is told to function as an elastase inhibitor what would be an therapeutic attempt according to fix the out of control gone function of elastase in lung emphysema. In such a case it is beyond debate how important it is to figger out the biosynthesis of this peptide. The research group of Prof. Dittmann at the University of Potsdam was successful and able to report about the composition of the gene cluster from ''Micocystis aeruginosa'' NIES298 expressing Microviridin B. Moreover they have been convinced that there is existing one unique biosynthetic mechanism for microviridins in <i>Microcystis</i> strains. Unusual for depsipeptides they discovered that they are ribosomal synthesized. The ''mdn'' gene cluster is build up of a gene ''mdnA'', encoding for the putative precursor peptide of the microvirdin, two genes encoding ATP-grasp-type ligases ''mdnB'' and ''mdnC'', an ABC transporter encoding gene ''mdnE'' as well as one encoding an N- acetyltransferase of the GNAT family ''mdnD'' (Ziemert et al., 2008). Moreover in their recent studies they on the contrary report about the discovery of an existing natural diversity of microviridin precursors genes in set of closely related Mycrocystis laboratory strains. This furthermore would mean and lead to a heterologous production of novel microviridins in ''E. coli''. To give evidence of their discovery they identified and characterized a new Microviridin L from the strain ''Mycrocystis aeruginosa'' NIES843 (Ziemert et al., 2010).<br />
All in all the microviridins, the tricyclic depsipeptide are build up through a gene cluster of five mdn-components each fulfill one function to make the hole cluster functional. This showed the team of Prof. Dr. Dittmann as the peptide is only correctly processed in ''E. coli'' if the entire cluster ''mdnABCDE'' is expressed. In addition the new discovery is promising a small flexibility of the microviridin ligases in Mycrocystis as an open window for the size of the natural microviridin library and thus microviridin functions and hence for example therapeutic benefit that needs to be discovered. The following illustration is going to give you a better overview (Fig. 1).<br />
[[File:UP_MV_introduction_mdn.jpg|center|525px|thumb|'''Figure 1:''' overview of the mdn gene cluster (GNAT: GCN5-related N-acetyltransferase)]]<br />
<br><br><br />
<br />
=== Modularization of the ''mdn'' gene cluster===<br />
Using two vectors (pARW071 and pARW089, respectively) containing the ''mdn'' biosynthetic gene cluster, we designed PCR primers for the amplification of the ''mdn genes''. Using these primers, we obtained the gene fragments of ''mdnA'', ''mdnB'', ''mdnC'', ''mdnD'', ''mdnE'' and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our BioBricks comply with the BioBrick standards. <br />
<br />
The following image (Fig. 2) shows the generic construction of the BioBrick carrying the'' mdnA gene''.<br />
<br />
[[File:UP_modularization_mdnA_cloning.png|center|500px|thumb|'''Figure 2:''' Cloning Scheme of mdnA-BioBrick (''Cm'' - gene for chloramphenicol resistance)]]<br />
<br />
Microviridin production in ''E. coli'' cells expressing the'' mdn genes'' was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3). <br />
[[File:UP_modularization_HPLC.png|center|500px|thumb|'''Figure 3:''' HPLC chromatogram of MdnA]]<br />
All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.<br />
<br />
Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.<br />
<br />
[[File:UP_modularization_MSII.gif|center|300px|thumb|'''Figure 4:''' Mass Spectrometry Data]]<br />
<br><br><br />
<br />
=== Generating ''mdnA'' gene libraries ===<br />
In searching for optimized and novel microviridin peptides, libraries of mutated microviridins were established. Therefore a number of sites in the amino acid sequence of the precursor peptide (MdnA) were chosen.<br />
In <i>Microcystis</i>, microviridins are synthesized from a ribosomal precursor peptide (MdnA). Therefore the microviridin gene cluster is necessary. This 6.5 kb biosynthesis gene cluster includes two genes (''mdnB'' and ''mdnC''), which encodes ATP-grasp-type ligases and further the ''mdnE'' gene encoding an ABC transporter as well as'' mdnD'' gene encoding a N-acetyltransferase (Ziemert et al., 2008). In consequence the biosynthesis gene cluster genes ''mdnB'', ''mdnC'', ''mdnD'' and ''mdnE'' cannot be mutated. Otherwise only misprocessed and non-function variants of microviridin occur (Ziemert et al., 2008). Thus merely the modification of the ''mdnA'' gene can lead to effective changes in the microviridin structure and function. <br><br />
[[File:UP_lib2_fig.png|center|500px|thumb|'''Figure 5:''' Molecular organization of the ribsosomal precursor peptide (MdnA) of microviridin. MdnA consists of a leader and a core peptide. The N-terminal leader peptide contains highly conserved double glycine motifs. Whereas in the C-terminal 14 amino acid sized core peptide sequence shows variations. Modified from Ziemert et al. (2008) and Ziemert et al. (2010).]]<br />
The precursor peptide MdnA consists of a leader peptide and a core peptide (Fig. 5). Ziemert et al. (2008) has shown that the N-terminal leader peptide contains highly conserved double glycine motifs (Ziemert et al. 2008). However remarkable variations were found in region, which encodes the 14-amino acid sequence of the C-terminal microviridin core peptide (Ziemert et al. 2010). Consequently, mutations in the MdnA core peptide seem to be the most promising option for modification of microviridin. For cyclization, the amino acid side chains of the core peptide form omega-ester and omega-amino bonds. One loop is formed between threonine and aspartic acid, the second loop between lysine and aspartic acid and the third loop between serine and glutamic acid (Fig. 5). The sequence of this loop forming amino acids constitute an exception while mutation of the ''mdnA'' gene (Ziemert et al., 2008 and 2010).<br />
<br />
To find optimized and novel microviridin variants, several libraries carrying modifications in the sequence of the MdnA core peptide were established. These libraries were generated by randomized oligonucleotide synthesis. Therefore a forward oligonucleotide displaying a part of conserved leader peptide sequence was built. Further a reverse oligonucleotide was created. The first part of this oligonucleotide is 20 bases, which overlap with the forward oligonucleotide and which is necessary for hybridization of both, forward and reverse, oligonucleotides. In the middle modified bases are inserted. For cloning the forward oligonucleotide starts with the blunt restricted recognition site of <i>Sfo</i>I. This results therein that the oligonucleotide can ligate in the <i>Sfo</i>I restricted vector without previous restriction of the oligonucleotide. The last part of the reverse oligonucleotides displays an <i>Aat</i>II restriction site for cloning. Digestion with <i>Aat</i>II results in sticky end, thus wrong way round insertion of the oligonucleotide is impossible. Forward and reverse oligonucleotides were combined by performing a fill-in reaction. <br />
Because we designed several libraries with different diversity rates, the modified bases, which are inserted in the middle of the reverse primer, vary. All of our generated libraries contain mutations of the sequence of the MdnA core peptide at several sites, but not at the loop forming sites. Thus we are thinking that cyclization of microviridin happens accurately. A library with a diversity of 45,360 (focused library 1) and a second focused library showing a minor diversity of 810 (focused library 2) were designed. The included sequence modifications plus the diversity of our libraries are shown in figure 6. In our further studies we worked on focused library 2 primarily. In this library the nucleotide sequences encoding glycine residues were changed in GVK in all cases (Figure 6). GVK stands for guanine at the first position, adenine, cytosine or guanine at the second position and further thymine at the third position of the codon. These three possible codons encode for the amino acids alanine, aspartic acid and glycine. The nucleotide sequence N-terminal tyrosine residue was changed in THT describing thymine (first position), adenine, cytosine or thymine at the second position and thymine (third position). These codons encode isoleucine, leucine and phenylalanine. Additionally, the sequence of phenylalanine in the core peptide sequence was shift to NNK. NKK stands for one of the four nucleotides at the first position, and guanine or thymine at the second and third position. Consequently, this codon encodes the amino acids arginine, glycine, tryptophan, methionine, leucine, valine, serine, cysteine, isoleucine and phenylalanine. <br />
[[File:UP_lib2_fig2.png|center|500px|thumb|'''Figure 6:''' Design of libraries based on the nucleotide sequence of the ribosomal precursor peptide (MdnA). To different libraries, focused library 1 and focused library 2 were generated by randomised oligonucleotide synthesis. The modifications compared to the wild type sequence are highlighted. The definition of the used code is also shown. The first libray (focused library 1) displays a diversity of 45360 and the second, focused library 2 a diversity of 810. In the further studies focused library 2 was used to find optimized microviridin variants. ]]<br />
To construct this library a fill-in reaction using the designed forward and reverse oligonucleotides was performed. Subsequently resulting randomized oligonucleotide was digested with the restriction enzyme <i>Aat</i>II. In addition, the vector pUP089 was digested with the restriction enzymes <i>Aat</i>II and <i>Sfo</i>I. After ligation of fragments, randomized oligonucleotide and vector, the focused library 2 (ligation product) was transformed in chemocompetent ''E. coli'' XL1-Blue cells. This procedure was done several times and a library size of 1233 colonies was reached. <br />
[[File:UP_lib2_fig3.png|center|500px|thumb|'''Figure 7:''' Confirmation of focused library 2. Different clones of the generated focused library 2 were sequenced. Alignments with the wild type sequence (reference sequence) were performed. As shown (for a selection of clones) in all of our sequenced samples the requested modifications were established.]]<br />
For confirmation of the focused library 2, sequencing analysis of a number of clones was performed. In all of our sequenced samples the requested modifications were established (figure 7). A sequence logo was generated for the 14 amino acid sized core peptide of MdnA. This sequence logo, which is illustrated in figure 8, indicates that the desired sites stay conserved. The others are modified. <br />
[[File:UP_lib2_fig4.png|center|500px|thumb|'''Figure 8:''' Sequence logo plot of focused library 2. The 14 amino acid sized core peptide of MdnA was sequenced. The sequence of several clones was used to generate a sequence logo. This indicates that the desired sites stay conserved. The others nucleotides and resulting amino acids are modified. ]]<br />
The generated and modified focused library 2 should be used as basis for <i>in vivo</i> selection and phage display screening. Furthermore we purpose the objective to search for optimized and novel microviridins by using this library.<br />
<br><br><br />
<br />
=== Expression Backbones ===<br />
In a subtask of our work we wanted to construct auxiliary expression backbones with inducible promotors. In contrast to constitutive systems inducible systems only express the protein of interest after adding an inducer, which can be for example AHL or arabinose.<br />
We decided to construct IPTG- and arabinose-inducible systems. Therefore we amplified the promotor region and fused it to a reporter gene, which is constituted by YFP. Using this we have the option to verify the presence of the inducible promotor and also the function of the induction process by fluorescence. Our idea was to use the reporter gene as a placeholder. Via restriction enzyme digestion using the iGEM restriction enzyme sites you will be able to replace the reporter gene with your gene of interest. As vector backbone we made use of the pSB1A3, which has ampicillin resistance (Fig. 9). The constructs using pSB1K3 (kanamycin resistance) and pSB1C3 (chloramphenicol resistance) are still on the anvil. <br />
[[File:UP_expression_backbones_cloning.png|center|500px|thumb|'''Figure 9:''' Cloning Scheme of Expression Backbones. ''amp'' - gene for ampicillin resistance]]<br />
<br />
We successfully tested the IPTG- and arabinose-inducible system (Fig 10). Using fluorescence microscopy we were able to detect the YFP-expression after an induction time of approximately 1.5 hrs. To prove the hypothesis we opposed an induced sample with a non-induced control for each promotor. By use of brightfield combined with differential interference contrast microscopy we traced the ''E. coli'' cells and switched then to the YFP detecting channel to investigate the fluorescence of these cells.<br />
<br />
[[File:UP_Expression_Backbones_Fluorescence.png|center|800px|thumb|'''Figure 10:''' Fluorescence Microscopy. control - not induced; induced - induction with IPTG or Arabinose, respectively]]<br />
<br />
We also tested the inducible systems by using fluorescence spectroscopy. For this experiment we induced both systems at a time when the cultures were located in phase of exponential growth. After inducing, the cultures were analyzed by fluorescence spectroscopy. In this analyze the cells were excited by 500 nm. The resultant emission was measured in a spectrum between 510 and 580 nm. (Fig 11;12)<br />
<table><td>[[File:UP_psB1A3_Ara_YFPII.png|left|410px|thumb|'''Figure 11:''' Emission (526nm) of pSB1A3_Ara_YFP and control – '' E.coli'' cells (including pSB1A3_Ara_YFP) were induced and measured at certain moments.]] </td> <td>[[File:UP_psB1A3_IPTG_YFPII.png|right|410px|thumb|'''Figure 12:''' Emission (526nm) of induced pSB1A3_lac_YFP and control – ''E.coli'' cells (including pSB1A3_lac_YFP) were induced and measured at certain moments.]]</td><br />
</table>In contrast to the control of the arabinose inducible system the Lac-control also shows fluorescence. This demonstrates the leaky Lac-promotor, what is also a scientifically proven fact. A leaky promotor means that the promotor is not regulated in a leakproof manner. Even in repressed conditions the transcript can arise.<br />
<br />
The lacking of the Lac-I gene on the vector is the reason for expression of YFP in both controls. For further research in this field it is necessary to clone the Lac-I gene inside the constructed vector.<br />
<br><br><br />
<br />
===References===<br />
Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74<br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_MicroviridinTeam:Potsdam Bioware/Project/Details Microviridin2011-10-28T23:14:19Z<p>UP Stefan: /* Modularization of the mdn gene cluster */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==Microviridin==<br />
=== Introduction ===<br />
Cyanobacteria are known as the blue-green-algae because their living space is water and their feeding mechanism is photosynthetic. The Cyanobacteria are know for some special and unique metabolites. One of these metabolites are the microviridin family. Special attributes of microviridins are their occurrence as tricyclic depsipeptides, means peptides whose one or more amide-bond is replaced by an ester-bond, unparallel cage-like architecture and their ability to inhibit several proteases. Microviridin B for example is told to function as an elastase inhibitor what would be an therapeutic attempt according to fix the out of control gone function of elastase in lung emphysema. In such a case it is beyond debate how important it is to figger out the biosynthesis of this peptide. The research group of Prof. Dittmann at the University of Potsdam was successful and able to report about the composition of the gene cluster from ''Micocystis aeruginosa'' NIES298 expressing Microviridin B. Moreover they have been convinced that there is existing one unique biosynthetic mechanism for microviridins in Microcystis. Unusual for depsipeptides they discovered that they are ribosomal synthesized. The ''mdn'' gene cluster is build up of a gene ''mdnA'', encoding for the putative precursor peptide of the microvirdin, two genes encoding ATP-grasp-type ligases ''mdnB'' and ''mdnC'', an ABC transporter encoding gene ''mdnE'' as well as one encoding an N- acetyltransferase of the GNAT family ''mdnD'' (Ziemert et al., 2008). Moreover in their recent studies they on the contrary report about the discovery of an existing natural diversity of microviridin precursors genes in set of closely related Mycrocystis laboratory strains. This furthermore would mean and lead to a heterologous production of novel microviridins in ''E. coli''. To give evidence of their discovery they identified and characterized a new Microviridin L from the strain ''Mycrocystis aeruginosa'' NIES843 (Ziemert et al., 2010).<br />
All in all the microviridins, the tricyclic depsipeptide are build up through a gene cluster of five mdn-components each fulfill one function to make the hole cluster functional. This showed the team of Prof. Dr. Dittmann as the peptide is only correctly processed in ''E. coli'' if the entire cluster ''mdnABCDE'' is expressed. In addition the new discovery is promising a small flexibility of the microviridin ligases in Mycrocystis as an open window for the size of the natural microviridin library and thus microviridin functions and hence for example therapeutic benefit that needs to be discovered. The following illustration is going to give you a better overview (Fig. 1).<br />
[[File:UP_MV_introduction_mdn.jpg|center|525px|thumb|'''Figure 1:''' overview of the mdn gene cluster (GNAT: GCN5-related N-acetyltransferase)]]<br />
<br><br><br />
<br />
=== Modularization of the ''mdn'' gene cluster===<br />
Using two vectors (pARW071 and pARW089, respectively) containing the ''mdn'' biosynthetic gene cluster, we designed PCR primers for the amplification of the ''mdn genes''. Using these primers, we obtained the gene fragments of ''mdnA'', ''mdnB'', ''mdnC'', ''mdnD'', ''mdnE'' and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our BioBricks comply with the BioBrick standards. <br />
<br />
The following image (Fig. 2) shows the generic construction of the BioBrick carrying the'' mdnA gene''.<br />
<br />
[[File:UP_modularization_mdnA_cloning.png|center|500px|thumb|'''Figure 2:''' Cloning Scheme of mdnA-BioBrick (''Cm'' - gene for chloramphenicol resistance)]]<br />
<br />
Microviridin production in ''E. coli'' cells expressing the'' mdn genes'' was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3). <br />
[[File:UP_modularization_HPLC.png|center|500px|thumb|'''Figure 3:''' HPLC chromatogram of MdnA]]<br />
All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.<br />
<br />
Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.<br />
<br />
[[File:UP_modularization_MSII.gif|center|300px|thumb|'''Figure 4:''' Mass Spectrometry Data]]<br />
<br><br><br />
<br />
=== Generating ''mdnA'' gene libraries ===<br />
In searching for optimized and novel microviridin peptides, libraries of mutated microviridins were established. Therefore a number of sites in the amino acid sequence of the precursor peptide (MdnA) were chosen.<br />
In <i>Microcystis</i>, microviridins are synthesized from a ribosomal precursor peptide (MdnA). Therefore the microviridin gene cluster is necessary. This 6.5 kb biosynthesis gene cluster includes two genes (''mdnB'' and ''mdnC''), which encodes ATP-grasp-type ligases and further the ''mdnE'' gene encoding an ABC transporter as well as'' mdnD'' gene encoding a N-acetyltransferase (Ziemert et al., 2008). In consequence the biosynthesis gene cluster genes ''mdnB'', ''mdnC'', ''mdnD'' and ''mdnE'' cannot be mutated. Otherwise only misprocessed and non-function variants of microviridin occur (Ziemert et al., 2008). Thus merely the modification of the ''mdnA'' gene can lead to effective changes in the microviridin structure and function. <br><br />
[[File:UP_lib2_fig.png|center|500px|thumb|'''Figure 5:''' Molecular organization of the ribsosomal precursor peptide (MdnA) of microviridin. MdnA consists of a leader and a core peptide. The N-terminal leader peptide contains highly conserved double glycine motifs. Whereas in the C-terminal 14 amino acid sized core peptide sequence shows variations. Modified from Ziemert et al. (2008) and Ziemert et al. (2010).]]<br />
The precursor peptide MdnA consists of a leader peptide and a core peptide (Fig. 5). Ziemert et al. (2008) has shown that the N-terminal leader peptide contains highly conserved double glycine motifs (Ziemert et al. 2008). However remarkable variations were found in region, which encodes the 14-amino acid sequence of the C-terminal microviridin core peptide (Ziemert et al. 2010). Consequently, mutations in the MdnA core peptide seem to be the most promising option for modification of microviridin. For cyclization, the amino acid side chains of the core peptide form omega-ester and omega-amino bonds. One loop is formed between threonine and aspartic acid, the second loop between lysine and aspartic acid and the third loop between serine and glutamic acid (Fig. 5). The sequence of this loop forming amino acids constitute an exception while mutation of the ''mdnA'' gene (Ziemert et al., 2008 and 2010).<br />
<br />
To find optimized and novel microviridin variants, several libraries carrying modifications in the sequence of the MdnA core peptide were established. These libraries were generated by randomized oligonucleotide synthesis. Therefore a forward oligonucleotide displaying a part of conserved leader peptide sequence was built. Further a reverse oligonucleotide was created. The first part of this oligonucleotide is 20 bases, which overlap with the forward oligonucleotide and which is necessary for hybridization of both, forward and reverse, oligonucleotides. In the middle modified bases are inserted. For cloning the forward oligonucleotide starts with the blunt restricted recognition site of <i>Sfo</i>I. This results therein that the oligonucleotide can ligate in the <i>Sfo</i>I restricted vector without previous restriction of the oligonucleotide. The last part of the reverse oligonucleotides displays an <i>Aat</i>II restriction site for cloning. Digestion with <i>Aat</i>II results in sticky end, thus wrong way round insertion of the oligonucleotide is impossible. Forward and reverse oligonucleotides were combined by performing a fill-in reaction. <br />
Because we designed several libraries with different diversity rates, the modified bases, which are inserted in the middle of the reverse primer, vary. All of our generated libraries contain mutations of the sequence of the MdnA core peptide at several sites, but not at the loop forming sites. Thus we are thinking that cyclization of microviridin happens accurately. A library with a diversity of 45,360 (focused library 1) and a second focused library showing a minor diversity of 810 (focused library 2) were designed. The included sequence modifications plus the diversity of our libraries are shown in figure 6. In our further studies we worked on focused library 2 primarily. In this library the nucleotide sequences encoding glycine residues were changed in GVK in all cases (Figure 6). GVK stands for guanine at the first position, adenine, cytosine or guanine at the second position and further thymine at the third position of the codon. These three possible codons encode for the amino acids alanine, aspartic acid and glycine. The nucleotide sequence N-terminal tyrosine residue was changed in THT describing thymine (first position), adenine, cytosine or thymine at the second position and thymine (third position). These codons encode isoleucine, leucine and phenylalanine. Additionally, the sequence of phenylalanine in the core peptide sequence was shift to NNK. NKK stands for one of the four nucleotides at the first position, and guanine or thymine at the second and third position. Consequently, this codon encodes the amino acids arginine, glycine, tryptophan, methionine, leucine, valine, serine, cysteine, isoleucine and phenylalanine. <br />
[[File:UP_lib2_fig2.png|center|500px|thumb|'''Figure 6:''' Design of libraries based on the nucleotide sequence of the ribosomal precursor peptide (MdnA). To different libraries, focused library 1 and focused library 2 were generated by randomised oligonucleotide synthesis. The modifications compared to the wild type sequence are highlighted. The definition of the used code is also shown. The first libray (focused library 1) displays a diversity of 45360 and the second, focused library 2 a diversity of 810. In the further studies focused library 2 was used to find optimized microviridin variants. ]]<br />
To construct this library a fill-in reaction using the designed forward and reverse oligonucleotides was performed. Subsequently resulting randomized oligonucleotide was digested with the restriction enzyme <i>Aat</i>II. In addition, the vector pUP089 was digested with the restriction enzymes <i>Aat</i>II and <i>Sfo</i>I. After ligation of fragments, randomized oligonucleotide and vector, the focused library 2 (ligation product) was transformed in chemocompetent ''E. coli'' XL1-Blue cells. This procedure was done several times and a library size of 1233 colonies was reached. <br />
[[File:UP_lib2_fig3.png|center|500px|thumb|'''Figure 7:''' Confirmation of focused library 2. Different clones of the generated focused library 2 were sequenced. Alignments with the wild type sequence (reference sequence) were performed. As shown (for a selection of clones) in all of our sequenced samples the requested modifications were established.]]<br />
For confirmation of the focused library 2, sequencing analysis of a number of clones was performed. In all of our sequenced samples the requested modifications were established (figure 7). A sequence logo was generated for the 14 amino acid sized core peptide of MdnA. This sequence logo, which is illustrated in figure 8, indicates that the desired sites stay conserved. The others are modified. <br />
[[File:UP_lib2_fig4.png|center|500px|thumb|'''Figure 8:''' Sequence logo plot of focused library 2. The 14 amino acid sized core peptide of MdnA was sequenced. The sequence of several clones was used to generate a sequence logo. This indicates that the desired sites stay conserved. The others nucleotides and resulting amino acids are modified. ]]<br />
The generated and modified focused library 2 should be used as basis for <i>in vivo</i> selection and phage display screening. Furthermore we purpose the objective to search for optimized and novel microviridins by using this library.<br />
<br><br><br />
<br />
=== Expression Backbones ===<br />
In a subtask of our work we wanted to construct auxiliary expression backbones with inducible promotors. In contrast to constitutive systems inducible systems only express the protein of interest after adding an inducer, which can be for example AHL or arabinose.<br />
We decided to construct IPTG- and arabinose-inducible systems. Therefore we amplified the promotor region and fused it to a reporter gene, which is constituted by YFP. Using this we have the option to verify the presence of the inducible promotor and also the function of the induction process by fluorescence. Our idea was to use the reporter gene as a placeholder. Via restriction enzyme digestion using the iGEM restriction enzyme sites you will be able to replace the reporter gene with your gene of interest. As vector backbone we made use of the pSB1A3, which has ampicillin resistance (Fig. 9). The constructs using pSB1K3 (kanamycin resistance) and pSB1C3 (chloramphenicol resistance) are still on the anvil. <br />
[[File:UP_expression_backbones_cloning.png|center|500px|thumb|'''Figure 9:''' Cloning Scheme of Expression Backbones. ''amp'' - gene for ampicillin resistance]]<br />
<br />
We successfully tested the IPTG- and arabinose-inducible system (Fig 10). Using fluorescence microscopy we were able to detect the YFP-expression after an induction time of approximately 1.5 hrs. To prove the hypothesis we opposed an induced sample with a non-induced control for each promotor. By use of brightfield combined with differential interference contrast microscopy we traced the ''E. coli'' cells and switched then to the YFP detecting channel to investigate the fluorescence of these cells.<br />
<br />
[[File:UP_Expression_Backbones_Fluorescence.png|center|800px|thumb|'''Figure 10:''' Fluorescence Microscopy. control - not induced; induced - induction with IPTG or Arabinose, respectively]]<br />
<br />
We also tested the inducible systems by using fluorescence spectroscopy. For this experiment we induced both systems at a time when the cultures were located in phase of exponential growth. After inducing, the cultures were analyzed by fluorescence spectroscopy. In this analyze the cells were excited by 500 nm. The resultant emission was measured in a spectrum between 510 and 580 nm. (Fig 11;12)<br />
<table><td>[[File:UP_psB1A3_Ara_YFPII.png|left|410px|thumb|'''Figure 11:''' Emission (526nm) of pSB1A3_Ara_YFP and control – '' E.coli'' cells (including pSB1A3_Ara_YFP) were induced and measured at certain moments.]] </td> <td>[[File:UP_psB1A3_IPTG_YFPII.png|right|410px|thumb|'''Figure 12:''' Emission (526nm) of induced pSB1A3_lac_YFP and control – ''E.coli'' cells (including pSB1A3_lac_YFP) were induced and measured at certain moments.]]</td><br />
</table>In contrast to the control of the arabinose inducible system the Lac-control also shows fluorescence. This demonstrates the leaky Lac-promotor, what is also a scientifically proven fact. A leaky promotor means that the promotor is not regulated in a leakproof manner. Even in repressed conditions the transcript can arise.<br />
<br />
The lacking of the Lac-I gene on the vector is the reason for expression of YFP in both controls. For further research in this field it is necessary to clone the Lac-I gene inside the constructed vector.<br />
<br><br><br />
<br />
===References===<br />
Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74<br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_MicroviridinTeam:Potsdam Bioware/Project/Details Microviridin2011-10-28T23:13:48Z<p>UP Stefan: /* Modularization of the mdn gene cluster */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==Microviridin==<br />
=== Introduction ===<br />
Cyanobacteria are known as the blue-green-algae because their living space is water and their feeding mechanism is photosynthetic. The Cyanobacteria are know for some special and unique metabolites. One of these metabolites are the microviridin family. Special attributes of microviridins are their occurrence as tricyclic depsipeptides, means peptides whose one or more amide-bond is replaced by an ester-bond, unparallel cage-like architecture and their ability to inhibit several proteases. Microviridin B for example is told to function as an elastase inhibitor what would be an therapeutic attempt according to fix the out of control gone function of elastase in lung emphysema. In such a case it is beyond debate how important it is to figger out the biosynthesis of this peptide. The research group of Prof. Dittmann at the University of Potsdam was successful and able to report about the composition of the gene cluster from ''Micocystis aeruginosa'' NIES298 expressing Microviridin B. Moreover they have been convinced that there is existing one unique biosynthetic mechanism for microviridins in Microcystis. Unusual for depsipeptides they discovered that they are ribosomal synthesized. The ''mdn'' gene cluster is build up of a gene ''mdnA'', encoding for the putative precursor peptide of the microvirdin, two genes encoding ATP-grasp-type ligases ''mdnB'' and ''mdnC'', an ABC transporter encoding gene ''mdnE'' as well as one encoding an N- acetyltransferase of the GNAT family ''mdnD'' (Ziemert et al., 2008). Moreover in their recent studies they on the contrary report about the discovery of an existing natural diversity of microviridin precursors genes in set of closely related Mycrocystis laboratory strains. This furthermore would mean and lead to a heterologous production of novel microviridins in ''E. coli''. To give evidence of their discovery they identified and characterized a new Microviridin L from the strain ''Mycrocystis aeruginosa'' NIES843 (Ziemert et al., 2010).<br />
All in all the microviridins, the tricyclic depsipeptide are build up through a gene cluster of five mdn-components each fulfill one function to make the hole cluster functional. This showed the team of Prof. Dr. Dittmann as the peptide is only correctly processed in ''E. coli'' if the entire cluster ''mdnABCDE'' is expressed. In addition the new discovery is promising a small flexibility of the microviridin ligases in Mycrocystis as an open window for the size of the natural microviridin library and thus microviridin functions and hence for example therapeutic benefit that needs to be discovered. The following illustration is going to give you a better overview (Fig. 1).<br />
[[File:UP_MV_introduction_mdn.jpg|center|525px|thumb|'''Figure 1:''' overview of the mdn gene cluster (GNAT: GCN5-related N-acetyltransferase)]]<br />
<br><br><br />
<br />
=== Modularization of the ''mdn'' gene cluster===<br />
Using two vectors (pARW071 and pARW089, respectively) containing the ''mdn'' biosynthetic gene cluster, we designed PCR primers for the amplification of the ''mdn genes''. Using these primers, we obtained the gene fragments of ''mdnA'', ''mdnB'', ''mdnC'', ''mdnD'', ''mdnE'' and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our BioBricks comply with the BioBrick standards. <br />
<br />
The following image (Fig. 2) shows the generic construction of the BioBrick carrying the'' mdnA gene''.<br />
<br />
[[File:UP_modularization_mdnA_cloning.png|center|500px|thumb|'''Figure 2:''' Cloning Scheme of mdnA-BioBrick (''Cm'' - gene for chloramphenicol resistance)]]<br />
<br />
Microviridin production in ''E. coli'' cells expressing the'' mdn genes'' was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3). <br />
[[File:UP_modularization_HPLC.png|center|500px|thumb|'''Figure 3:''' HPLC chromatogram of MdnA]]<br />
All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.<br />
<br />
Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.<br />
<br />
[[File:UP_modularization_MSII.gif|center|300px|thumb|'''Figure 4:''' Mass Spectrometry Data of microvirdin]]<br />
<br><br><br />
<br />
=== Generating ''mdnA'' gene libraries ===<br />
In searching for optimized and novel microviridin peptides, libraries of mutated microviridins were established. Therefore a number of sites in the amino acid sequence of the precursor peptide (MdnA) were chosen.<br />
In <i>Microcystis</i>, microviridins are synthesized from a ribosomal precursor peptide (MdnA). Therefore the microviridin gene cluster is necessary. This 6.5 kb biosynthesis gene cluster includes two genes (''mdnB'' and ''mdnC''), which encodes ATP-grasp-type ligases and further the ''mdnE'' gene encoding an ABC transporter as well as'' mdnD'' gene encoding a N-acetyltransferase (Ziemert et al., 2008). In consequence the biosynthesis gene cluster genes ''mdnB'', ''mdnC'', ''mdnD'' and ''mdnE'' cannot be mutated. Otherwise only misprocessed and non-function variants of microviridin occur (Ziemert et al., 2008). Thus merely the modification of the ''mdnA'' gene can lead to effective changes in the microviridin structure and function. <br><br />
[[File:UP_lib2_fig.png|center|500px|thumb|'''Figure 5:''' Molecular organization of the ribsosomal precursor peptide (MdnA) of microviridin. MdnA consists of a leader and a core peptide. The N-terminal leader peptide contains highly conserved double glycine motifs. Whereas in the C-terminal 14 amino acid sized core peptide sequence shows variations. Modified from Ziemert et al. (2008) and Ziemert et al. (2010).]]<br />
The precursor peptide MdnA consists of a leader peptide and a core peptide (Fig. 5). Ziemert et al. (2008) has shown that the N-terminal leader peptide contains highly conserved double glycine motifs (Ziemert et al. 2008). However remarkable variations were found in region, which encodes the 14-amino acid sequence of the C-terminal microviridin core peptide (Ziemert et al. 2010). Consequently, mutations in the MdnA core peptide seem to be the most promising option for modification of microviridin. For cyclization, the amino acid side chains of the core peptide form omega-ester and omega-amino bonds. One loop is formed between threonine and aspartic acid, the second loop between lysine and aspartic acid and the third loop between serine and glutamic acid (Fig. 5). The sequence of this loop forming amino acids constitute an exception while mutation of the ''mdnA'' gene (Ziemert et al., 2008 and 2010).<br />
<br />
To find optimized and novel microviridin variants, several libraries carrying modifications in the sequence of the MdnA core peptide were established. These libraries were generated by randomized oligonucleotide synthesis. Therefore a forward oligonucleotide displaying a part of conserved leader peptide sequence was built. Further a reverse oligonucleotide was created. The first part of this oligonucleotide is 20 bases, which overlap with the forward oligonucleotide and which is necessary for hybridization of both, forward and reverse, oligonucleotides. In the middle modified bases are inserted. For cloning the forward oligonucleotide starts with the blunt restricted recognition site of <i>Sfo</i>I. This results therein that the oligonucleotide can ligate in the <i>Sfo</i>I restricted vector without previous restriction of the oligonucleotide. The last part of the reverse oligonucleotides displays an <i>Aat</i>II restriction site for cloning. Digestion with <i>Aat</i>II results in sticky end, thus wrong way round insertion of the oligonucleotide is impossible. Forward and reverse oligonucleotides were combined by performing a fill-in reaction. <br />
Because we designed several libraries with different diversity rates, the modified bases, which are inserted in the middle of the reverse primer, vary. All of our generated libraries contain mutations of the sequence of the MdnA core peptide at several sites, but not at the loop forming sites. Thus we are thinking that cyclization of microviridin happens accurately. A library with a diversity of 45,360 (focused library 1) and a second focused library showing a minor diversity of 810 (focused library 2) were designed. The included sequence modifications plus the diversity of our libraries are shown in figure 6. In our further studies we worked on focused library 2 primarily. In this library the nucleotide sequences encoding glycine residues were changed in GVK in all cases (Figure 6). GVK stands for guanine at the first position, adenine, cytosine or guanine at the second position and further thymine at the third position of the codon. These three possible codons encode for the amino acids alanine, aspartic acid and glycine. The nucleotide sequence N-terminal tyrosine residue was changed in THT describing thymine (first position), adenine, cytosine or thymine at the second position and thymine (third position). These codons encode isoleucine, leucine and phenylalanine. Additionally, the sequence of phenylalanine in the core peptide sequence was shift to NNK. NKK stands for one of the four nucleotides at the first position, and guanine or thymine at the second and third position. Consequently, this codon encodes the amino acids arginine, glycine, tryptophan, methionine, leucine, valine, serine, cysteine, isoleucine and phenylalanine. <br />
[[File:UP_lib2_fig2.png|center|500px|thumb|'''Figure 6:''' Design of libraries based on the nucleotide sequence of the ribosomal precursor peptide (MdnA). To different libraries, focused library 1 and focused library 2 were generated by randomised oligonucleotide synthesis. The modifications compared to the wild type sequence are highlighted. The definition of the used code is also shown. The first libray (focused library 1) displays a diversity of 45360 and the second, focused library 2 a diversity of 810. In the further studies focused library 2 was used to find optimized microviridin variants. ]]<br />
To construct this library a fill-in reaction using the designed forward and reverse oligonucleotides was performed. Subsequently resulting randomized oligonucleotide was digested with the restriction enzyme <i>Aat</i>II. In addition, the vector pUP089 was digested with the restriction enzymes <i>Aat</i>II and <i>Sfo</i>I. After ligation of fragments, randomized oligonucleotide and vector, the focused library 2 (ligation product) was transformed in chemocompetent ''E. coli'' XL1-Blue cells. This procedure was done several times and a library size of 1233 colonies was reached. <br />
[[File:UP_lib2_fig3.png|center|500px|thumb|'''Figure 7:''' Confirmation of focused library 2. Different clones of the generated focused library 2 were sequenced. Alignments with the wild type sequence (reference sequence) were performed. As shown (for a selection of clones) in all of our sequenced samples the requested modifications were established.]]<br />
For confirmation of the focused library 2, sequencing analysis of a number of clones was performed. In all of our sequenced samples the requested modifications were established (figure 7). A sequence logo was generated for the 14 amino acid sized core peptide of MdnA. This sequence logo, which is illustrated in figure 8, indicates that the desired sites stay conserved. The others are modified. <br />
[[File:UP_lib2_fig4.png|center|500px|thumb|'''Figure 8:''' Sequence logo plot of focused library 2. The 14 amino acid sized core peptide of MdnA was sequenced. The sequence of several clones was used to generate a sequence logo. This indicates that the desired sites stay conserved. The others nucleotides and resulting amino acids are modified. ]]<br />
The generated and modified focused library 2 should be used as basis for <i>in vivo</i> selection and phage display screening. Furthermore we purpose the objective to search for optimized and novel microviridins by using this library.<br />
<br><br><br />
<br />
=== Expression Backbones ===<br />
In a subtask of our work we wanted to construct auxiliary expression backbones with inducible promotors. In contrast to constitutive systems inducible systems only express the protein of interest after adding an inducer, which can be for example AHL or arabinose.<br />
We decided to construct IPTG- and arabinose-inducible systems. Therefore we amplified the promotor region and fused it to a reporter gene, which is constituted by YFP. Using this we have the option to verify the presence of the inducible promotor and also the function of the induction process by fluorescence. Our idea was to use the reporter gene as a placeholder. Via restriction enzyme digestion using the iGEM restriction enzyme sites you will be able to replace the reporter gene with your gene of interest. As vector backbone we made use of the pSB1A3, which has ampicillin resistance (Fig. 9). The constructs using pSB1K3 (kanamycin resistance) and pSB1C3 (chloramphenicol resistance) are still on the anvil. <br />
[[File:UP_expression_backbones_cloning.png|center|500px|thumb|'''Figure 9:''' Cloning Scheme of Expression Backbones. ''amp'' - gene for ampicillin resistance]]<br />
<br />
We successfully tested the IPTG- and arabinose-inducible system (Fig 10). Using fluorescence microscopy we were able to detect the YFP-expression after an induction time of approximately 1.5 hrs. To prove the hypothesis we opposed an induced sample with a non-induced control for each promotor. By use of brightfield combined with differential interference contrast microscopy we traced the ''E. coli'' cells and switched then to the YFP detecting channel to investigate the fluorescence of these cells.<br />
<br />
[[File:UP_Expression_Backbones_Fluorescence.png|center|800px|thumb|'''Figure 10:''' Fluorescence Microscopy. control - not induced; induced - induction with IPTG or Arabinose, respectively]]<br />
<br />
We also tested the inducible systems by using fluorescence spectroscopy. For this experiment we induced both systems at a time when the cultures were located in phase of exponential growth. After inducing, the cultures were analyzed by fluorescence spectroscopy. In this analyze the cells were excited by 500 nm. The resultant emission was measured in a spectrum between 510 and 580 nm. (Fig 11;12)<br />
<table><td>[[File:UP_psB1A3_Ara_YFPII.png|left|410px|thumb|'''Figure 11:''' Emission (526nm) of pSB1A3_Ara_YFP and control – '' E.coli'' cells (including pSB1A3_Ara_YFP) were induced and measured at certain moments.]] </td> <td>[[File:UP_psB1A3_IPTG_YFPII.png|right|410px|thumb|'''Figure 12:''' Emission (526nm) of induced pSB1A3_lac_YFP and control – ''E.coli'' cells (including pSB1A3_lac_YFP) were induced and measured at certain moments.]]</td><br />
</table>In contrast to the control of the arabinose inducible system the Lac-control also shows fluorescence. This demonstrates the leaky Lac-promotor, what is also a scientifically proven fact. A leaky promotor means that the promotor is not regulated in a leakproof manner. Even in repressed conditions the transcript can arise.<br />
<br />
The lacking of the Lac-I gene on the vector is the reason for expression of YFP in both controls. For further research in this field it is necessary to clone the Lac-I gene inside the constructed vector.<br />
<br><br><br />
<br />
===References===<br />
Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74<br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_MicroviridinTeam:Potsdam Bioware/Project/Details Microviridin2011-10-28T23:12:30Z<p>UP Stefan: /* Generating mdnA gene libraries */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==Microviridin==<br />
=== Introduction ===<br />
Cyanobacteria are known as the blue-green-algae because their living space is water and their feeding mechanism is photosynthetic. The Cyanobacteria are know for some special and unique metabolites. One of these metabolites are the microviridin family. Special attributes of microviridins are their occurrence as tricyclic depsipeptides, means peptides whose one or more amide-bond is replaced by an ester-bond, unparallel cage-like architecture and their ability to inhibit several proteases. Microviridin B for example is told to function as an elastase inhibitor what would be an therapeutic attempt according to fix the out of control gone function of elastase in lung emphysema. In such a case it is beyond debate how important it is to figger out the biosynthesis of this peptide. The research group of Prof. Dittmann at the University of Potsdam was successful and able to report about the composition of the gene cluster from ''Micocystis aeruginosa'' NIES298 expressing Microviridin B. Moreover they have been convinced that there is existing one unique biosynthetic mechanism for microviridins in Microcystis. Unusual for depsipeptides they discovered that they are ribosomal synthesized. The ''mdn'' gene cluster is build up of a gene ''mdnA'', encoding for the putative precursor peptide of the microvirdin, two genes encoding ATP-grasp-type ligases ''mdnB'' and ''mdnC'', an ABC transporter encoding gene ''mdnE'' as well as one encoding an N- acetyltransferase of the GNAT family ''mdnD'' (Ziemert et al., 2008). Moreover in their recent studies they on the contrary report about the discovery of an existing natural diversity of microviridin precursors genes in set of closely related Mycrocystis laboratory strains. This furthermore would mean and lead to a heterologous production of novel microviridins in ''E. coli''. To give evidence of their discovery they identified and characterized a new Microviridin L from the strain ''Mycrocystis aeruginosa'' NIES843 (Ziemert et al., 2010).<br />
All in all the microviridins, the tricyclic depsipeptide are build up through a gene cluster of five mdn-components each fulfill one function to make the hole cluster functional. This showed the team of Prof. Dr. Dittmann as the peptide is only correctly processed in ''E. coli'' if the entire cluster ''mdnABCDE'' is expressed. In addition the new discovery is promising a small flexibility of the microviridin ligases in Mycrocystis as an open window for the size of the natural microviridin library and thus microviridin functions and hence for example therapeutic benefit that needs to be discovered. The following illustration is going to give you a better overview (Fig. 1).<br />
[[File:UP_MV_introduction_mdn.jpg|center|525px|thumb|'''Figure 1:''' overview of the mdn gene cluster (GNAT: GCN5-related N-acetyltransferase)]]<br />
<br><br><br />
<br />
=== Modularization of the ''mdn'' gene cluster===<br />
Using two vectors (pARW071 and pARW089, respectively) containing the ''mdn'' biosynthetic gene cluster, we designed PCR primers for the amplification of the ''mdn genes''. Using these primers, we obtained the gene fragments of ''mdnA'', ''mdnB'', ''mdnC'', ''mdnD'', ''mdnE'' and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our BioBricks comply with the BioBrick standards. <br />
<br />
The following image (Fig. 2) shows the generic construction of the BioBrick carrying the'' mdnA gene''.<br />
<br />
[[File:UP_modularization_mdnA_cloning.png|center|500px|thumb|'''Figure 2:''' Cloning Scheme of mdnA-BioBrick (''Cm'' - gene for chloramphenicol resistance)]]<br />
<br />
Microviridin production in ''E. coli'' cells expressing the'' mdn genes'' was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3). <br />
[[File:UP_modularization_HPLC.png|center|500px|thumb|'''Figure 3:''' HPLC chromatogram of mdnA]]<br />
All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.<br />
<br />
Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.<br />
<br />
[[File:UP_modularization_MSII.gif|center|300px|thumb|'''Figure 4:''' Mass Spectrometry Data]]<br />
<br><br><br />
<br />
=== Generating ''mdnA'' gene libraries ===<br />
In searching for optimized and novel microviridin peptides, libraries of mutated microviridins were established. Therefore a number of sites in the amino acid sequence of the precursor peptide (MdnA) were chosen.<br />
In <i>Microcystis</i>, microviridins are synthesized from a ribosomal precursor peptide (MdnA). Therefore the microviridin gene cluster is necessary. This 6.5 kb biosynthesis gene cluster includes two genes (''mdnB'' and ''mdnC''), which encodes ATP-grasp-type ligases and further the ''mdnE'' gene encoding an ABC transporter as well as'' mdnD'' gene encoding a N-acetyltransferase (Ziemert et al., 2008). In consequence the biosynthesis gene cluster genes ''mdnB'', ''mdnC'', ''mdnD'' and ''mdnE'' cannot be mutated. Otherwise only misprocessed and non-function variants of microviridin occur (Ziemert et al., 2008). Thus merely the modification of the ''mdnA'' gene can lead to effective changes in the microviridin structure and function. <br><br />
[[File:UP_lib2_fig.png|center|500px|thumb|'''Figure 5:''' Molecular organization of the ribsosomal precursor peptide (MdnA) of microviridin. MdnA consists of a leader and a core peptide. The N-terminal leader peptide contains highly conserved double glycine motifs. Whereas in the C-terminal 14 amino acid sized core peptide sequence shows variations. Modified from Ziemert et al. (2008) and Ziemert et al. (2010).]]<br />
The precursor peptide MdnA consists of a leader peptide and a core peptide (Fig. 5). Ziemert et al. (2008) has shown that the N-terminal leader peptide contains highly conserved double glycine motifs (Ziemert et al. 2008). However remarkable variations were found in region, which encodes the 14-amino acid sequence of the C-terminal microviridin core peptide (Ziemert et al. 2010). Consequently, mutations in the MdnA core peptide seem to be the most promising option for modification of microviridin. For cyclization, the amino acid side chains of the core peptide form omega-ester and omega-amino bonds. One loop is formed between threonine and aspartic acid, the second loop between lysine and aspartic acid and the third loop between serine and glutamic acid (Fig. 5). The sequence of this loop forming amino acids constitute an exception while mutation of the ''mdnA'' gene (Ziemert et al., 2008 and 2010).<br />
<br />
To find optimized and novel microviridin variants, several libraries carrying modifications in the sequence of the MdnA core peptide were established. These libraries were generated by randomized oligonucleotide synthesis. Therefore a forward oligonucleotide displaying a part of conserved leader peptide sequence was built. Further a reverse oligonucleotide was created. The first part of this oligonucleotide is 20 bases, which overlap with the forward oligonucleotide and which is necessary for hybridization of both, forward and reverse, oligonucleotides. In the middle modified bases are inserted. For cloning the forward oligonucleotide starts with the blunt restricted recognition site of <i>Sfo</i>I. This results therein that the oligonucleotide can ligate in the <i>Sfo</i>I restricted vector without previous restriction of the oligonucleotide. The last part of the reverse oligonucleotides displays an <i>Aat</i>II restriction site for cloning. Digestion with <i>Aat</i>II results in sticky end, thus wrong way round insertion of the oligonucleotide is impossible. Forward and reverse oligonucleotides were combined by performing a fill-in reaction. <br />
Because we designed several libraries with different diversity rates, the modified bases, which are inserted in the middle of the reverse primer, vary. All of our generated libraries contain mutations of the sequence of the MdnA core peptide at several sites, but not at the loop forming sites. Thus we are thinking that cyclization of microviridin happens accurately. A library with a diversity of 45,360 (focused library 1) and a second focused library showing a minor diversity of 810 (focused library 2) were designed. The included sequence modifications plus the diversity of our libraries are shown in figure 6. In our further studies we worked on focused library 2 primarily. In this library the nucleotide sequences encoding glycine residues were changed in GVK in all cases (Figure 6). GVK stands for guanine at the first position, adenine, cytosine or guanine at the second position and further thymine at the third position of the codon. These three possible codons encode for the amino acids alanine, aspartic acid and glycine. The nucleotide sequence N-terminal tyrosine residue was changed in THT describing thymine (first position), adenine, cytosine or thymine at the second position and thymine (third position). These codons encode isoleucine, leucine and phenylalanine. Additionally, the sequence of phenylalanine in the core peptide sequence was shift to NNK. NKK stands for one of the four nucleotides at the first position, and guanine or thymine at the second and third position. Consequently, this codon encodes the amino acids arginine, glycine, tryptophan, methionine, leucine, valine, serine, cysteine, isoleucine and phenylalanine. <br />
[[File:UP_lib2_fig2.png|center|500px|thumb|'''Figure 6:''' Design of libraries based on the nucleotide sequence of the ribosomal precursor peptide (MdnA). To different libraries, focused library 1 and focused library 2 were generated by randomised oligonucleotide synthesis. The modifications compared to the wild type sequence are highlighted. The definition of the used code is also shown. The first libray (focused library 1) displays a diversity of 45360 and the second, focused library 2 a diversity of 810. In the further studies focused library 2 was used to find optimized microviridin variants. ]]<br />
To construct this library a fill-in reaction using the designed forward and reverse oligonucleotides was performed. Subsequently resulting randomized oligonucleotide was digested with the restriction enzyme <i>Aat</i>II. In addition, the vector pUP089 was digested with the restriction enzymes <i>Aat</i>II and <i>Sfo</i>I. After ligation of fragments, randomized oligonucleotide and vector, the focused library 2 (ligation product) was transformed in chemocompetent ''E. coli'' XL1-Blue cells. This procedure was done several times and a library size of 1233 colonies was reached. <br />
[[File:UP_lib2_fig3.png|center|500px|thumb|'''Figure 7:''' Confirmation of focused library 2. Different clones of the generated focused library 2 were sequenced. Alignments with the wild type sequence (reference sequence) were performed. As shown (for a selection of clones) in all of our sequenced samples the requested modifications were established.]]<br />
For confirmation of the focused library 2, sequencing analysis of a number of clones was performed. In all of our sequenced samples the requested modifications were established (figure 7). A sequence logo was generated for the 14 amino acid sized core peptide of MdnA. This sequence logo, which is illustrated in figure 8, indicates that the desired sites stay conserved. The others are modified. <br />
[[File:UP_lib2_fig4.png|center|500px|thumb|'''Figure 8:''' Sequence logo plot of focused library 2. The 14 amino acid sized core peptide of MdnA was sequenced. The sequence of several clones was used to generate a sequence logo. This indicates that the desired sites stay conserved. The others nucleotides and resulting amino acids are modified. ]]<br />
The generated and modified focused library 2 should be used as basis for <i>in vivo</i> selection and phage display screening. Furthermore we purpose the objective to search for optimized and novel microviridins by using this library.<br />
<br><br><br />
<br />
=== Expression Backbones ===<br />
In a subtask of our work we wanted to construct auxiliary expression backbones with inducible promotors. In contrast to constitutive systems inducible systems only express the protein of interest after adding an inducer, which can be for example AHL or arabinose.<br />
We decided to construct IPTG- and arabinose-inducible systems. Therefore we amplified the promotor region and fused it to a reporter gene, which is constituted by YFP. Using this we have the option to verify the presence of the inducible promotor and also the function of the induction process by fluorescence. Our idea was to use the reporter gene as a placeholder. Via restriction enzyme digestion using the iGEM restriction enzyme sites you will be able to replace the reporter gene with your gene of interest. As vector backbone we made use of the pSB1A3, which has ampicillin resistance (Fig. 9). The constructs using pSB1K3 (kanamycin resistance) and pSB1C3 (chloramphenicol resistance) are still on the anvil. <br />
[[File:UP_expression_backbones_cloning.png|center|500px|thumb|'''Figure 9:''' Cloning Scheme of Expression Backbones. ''amp'' - gene for ampicillin resistance]]<br />
<br />
We successfully tested the IPTG- and arabinose-inducible system (Fig 10). Using fluorescence microscopy we were able to detect the YFP-expression after an induction time of approximately 1.5 hrs. To prove the hypothesis we opposed an induced sample with a non-induced control for each promotor. By use of brightfield combined with differential interference contrast microscopy we traced the ''E. coli'' cells and switched then to the YFP detecting channel to investigate the fluorescence of these cells.<br />
<br />
[[File:UP_Expression_Backbones_Fluorescence.png|center|800px|thumb|'''Figure 10:''' Fluorescence Microscopy. control - not induced; induced - induction with IPTG or Arabinose, respectively]]<br />
<br />
We also tested the inducible systems by using fluorescence spectroscopy. For this experiment we induced both systems at a time when the cultures were located in phase of exponential growth. After inducing, the cultures were analyzed by fluorescence spectroscopy. In this analyze the cells were excited by 500 nm. The resultant emission was measured in a spectrum between 510 and 580 nm. (Fig 11;12)<br />
<table><td>[[File:UP_psB1A3_Ara_YFPII.png|left|410px|thumb|'''Figure 11:''' Emission (526nm) of pSB1A3_Ara_YFP and control – '' E.coli'' cells (including pSB1A3_Ara_YFP) were induced and measured at certain moments.]] </td> <td>[[File:UP_psB1A3_IPTG_YFPII.png|right|410px|thumb|'''Figure 12:''' Emission (526nm) of induced pSB1A3_lac_YFP and control – ''E.coli'' cells (including pSB1A3_lac_YFP) were induced and measured at certain moments.]]</td><br />
</table>In contrast to the control of the arabinose inducible system the Lac-control also shows fluorescence. This demonstrates the leaky Lac-promotor, what is also a scientifically proven fact. A leaky promotor means that the promotor is not regulated in a leakproof manner. Even in repressed conditions the transcript can arise.<br />
<br />
The lacking of the Lac-I gene on the vector is the reason for expression of YFP in both controls. For further research in this field it is necessary to clone the Lac-I gene inside the constructed vector.<br />
<br><br><br />
<br />
===References===<br />
Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74<br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_MicroviridinTeam:Potsdam Bioware/Project/Details Microviridin2011-10-28T23:09:49Z<p>UP Stefan: /* Generating mdnA gene libraries */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==Microviridin==<br />
=== Introduction ===<br />
Cyanobacteria are known as the blue-green-algae because their living space is water and their feeding mechanism is photosynthetic. The Cyanobacteria are know for some special and unique metabolites. One of these metabolites are the microviridin family. Special attributes of microviridins are their occurrence as tricyclic depsipeptides, means peptides whose one or more amide-bond is replaced by an ester-bond, unparallel cage-like architecture and their ability to inhibit several proteases. Microviridin B for example is told to function as an elastase inhibitor what would be an therapeutic attempt according to fix the out of control gone function of elastase in lung emphysema. In such a case it is beyond debate how important it is to figger out the biosynthesis of this peptide. The research group of Prof. Dittmann at the University of Potsdam was successful and able to report about the composition of the gene cluster from ''Micocystis aeruginosa'' NIES298 expressing Microviridin B. Moreover they have been convinced that there is existing one unique biosynthetic mechanism for microviridins in Microcystis. Unusual for depsipeptides they discovered that they are ribosomal synthesized. The ''mdn'' gene cluster is build up of a gene ''mdnA'', encoding for the putative precursor peptide of the microvirdin, two genes encoding ATP-grasp-type ligases ''mdnB'' and ''mdnC'', an ABC transporter encoding gene ''mdnE'' as well as one encoding an N- acetyltransferase of the GNAT family ''mdnD'' (Ziemert et al., 2008). Moreover in their recent studies they on the contrary report about the discovery of an existing natural diversity of microviridin precursors genes in set of closely related Mycrocystis laboratory strains. This furthermore would mean and lead to a heterologous production of novel microviridins in ''E. coli''. To give evidence of their discovery they identified and characterized a new Microviridin L from the strain ''Mycrocystis aeruginosa'' NIES843 (Ziemert et al., 2010).<br />
All in all the microviridins, the tricyclic depsipeptide are build up through a gene cluster of five mdn-components each fulfill one function to make the hole cluster functional. This showed the team of Prof. Dr. Dittmann as the peptide is only correctly processed in ''E. coli'' if the entire cluster ''mdnABCDE'' is expressed. In addition the new discovery is promising a small flexibility of the microviridin ligases in Mycrocystis as an open window for the size of the natural microviridin library and thus microviridin functions and hence for example therapeutic benefit that needs to be discovered. The following illustration is going to give you a better overview (Fig. 1).<br />
[[File:UP_MV_introduction_mdn.jpg|center|525px|thumb|'''Figure 1:''' overview of the mdn gene cluster (GNAT: GCN5-related N-acetyltransferase)]]<br />
<br><br><br />
<br />
=== Modularization of the ''mdn'' gene cluster===<br />
Using two vectors (pARW071 and pARW089, respectively) containing the ''mdn'' biosynthetic gene cluster, we designed PCR primers for the amplification of the ''mdn genes''. Using these primers, we obtained the gene fragments of ''mdnA'', ''mdnB'', ''mdnC'', ''mdnD'', ''mdnE'' and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our BioBricks comply with the BioBrick standards. <br />
<br />
The following image (Fig. 2) shows the generic construction of the BioBrick carrying the'' mdnA gene''.<br />
<br />
[[File:UP_modularization_mdnA_cloning.png|center|500px|thumb|'''Figure 2:''' Cloning Scheme of mdnA-BioBrick (''Cm'' - gene for chloramphenicol resistance)]]<br />
<br />
Microviridin production in ''E. coli'' cells expressing the'' mdn genes'' was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3). <br />
[[File:UP_modularization_HPLC.png|center|500px|thumb|'''Figure 3:''' HPLC chromatogram of mdnA]]<br />
All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.<br />
<br />
Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.<br />
<br />
[[File:UP_modularization_MSII.gif|center|300px|thumb|'''Figure 4:''' Mass Spectrometry Data]]<br />
<br><br><br />
<br />
=== Generating ''mdnA'' gene libraries ===<br />
In searching for optimized and novel microviridin peptides, libraries of mutated microviridins were established. Therefore a number of sites in the amino acid sequence of the precursor peptide (MdnA) were chosen.<br />
In <i>Microcystis</i>, microviridins are synthesized from a ribosomal precursor peptide (MdnA). Therefore the microviridin gene cluster is necessary. This 6.5 kb biosynthesis gene cluster includes two genes (''mdnB'' and ''mdnC''), which encodes ATP-grasp-type ligases and further the ''mdnE'' gene encoding an ABC transporter as well as'' mdnD'' gene encoding a N-acetyltransferase (Ziemert et al., 2008). In consequence the biosynthesis gene cluster genes ''mdnB'', ''mdnC'', ''mdnD'' and ''mdnE'' cannot be mutated. Otherwise only misprocessed and non-function variants of microviridin occur (Ziemert et al., 2008). Thus merely the modification of the ''mdnA'' gene can lead to effective changes in the microviridin structure and function. <br><br />
[[File:UP_lib2_fig.png|center|500px|thumb|'''Figure 5:''' Molecular organization of the ribsosomal precursor peptide (MdnA) of microviridin. MdnA consists of a leader and a core peptide. The N-terminal leader peptide contains highly conserved double glycine motifs. Whereas in the C-terminal 14 amino acid sized core peptide sequence shows variations. Modified from Ziemert et al. (2008) and Ziemert et al. (2010).]]<br />
The precursor peptide MdnA consists of a leader peptide and a core peptide (Fig. 5). Ziemert et al. (2008) has shown that the N-terminal leader peptide contains highly conserved double glycine motifs (Ziemert et al. 2008). However remarkable variations were found in region, which encodes the 14-amino acid sequence of the C-terminal microviridin core peptide (Ziemert et al. 2010). Consequently, mutations in the MdnA core peptide seem to be the most promising option for modification of microviridin. For cyclization, the amino acid side chains of the core peptide form omega-ester and omega-amino bonds. One loop is formed between threonine and aspartic acid, the second loop between lysine and aspartic acid and the third loop between serine and glutamic acid (Fig. 5). The sequence of this loop forming amino acids constitute an exception while mutation of the ''mdnA'' gene (Ziemert et al., 2008 and 2010).<br />
<br />
To find optimized and novel microviridin variants, several libraries carrying modifications in the sequence of the MdnA core peptide were established. These libraries were generated by randomized oligonucleotide synthesis. Therefore a forward oligonucleotide displaying a part of conserved leader peptide sequence was built. Further a reverse oligonucleotide was created. The first part of this oligonucleotide is 20 bases, which overlap with the forward oligonucleotide and which is necessary for hybridization of both, forward and reverse, oligonucleotides. In the middle modified bases are inserted. For cloning the forward oligonucleotide starts with the blunt restricted recognition site of <i>Sfo</i>I. This results therein that the oligonucleotide can ligate in the <i>Sfo</i>I restricted vector without previous restriction of the oligonucleotide. The last part of the reverse oligonucleotides displays an <i>Aat</i>II restriction site for cloning. Digestion with <i>Aat</i>II results in sticky end, thus wrong way round insertion of the oligonucleotide is impossible. Forward and reverse oligonucleotides were combined by performing a fill-in reaction. <br />
Because we designed several libraries with different diversity rates, the modified bases, which are inserted in the middle of the reverse primer, vary. All of our generated libraries contain mutations of the sequence of the MdnA core peptide at several sites, but not at the loop forming sites. Thus we are thinking that cyclization of microviridin happens accurately. A library with a diversity of 45,360 (focused library 1) and a second focused library showing a minor diversity of 810 (focused library 2) were designed. The included sequence modifications plus the diversity of our libraries are shown in figure 6. In our further studies we worked on focused library 2 primarily. In this library the nucleotide sequences encoding glycine residues were changed in GVK in all cases (Figure 6). GVK stands for guanine at the first position, adenine, cytosine or guanine at the second position and further thymine at the third position of the codon. These three possible codons encode for the amino acids alanine, aspartic acid and glycine. The nucleotide sequence N-terminal tyrosine residue was changed in THT describing thymine (first position), adenine, cytosine or thymine at the second position and thymine (third position). These codons encode isoleucine, leucine and phenylalanine. Additionally, the sequence of phenylalanine in the core peptide sequence was shift to NNK. NKK stands for one of the four nucleotides at the first position, and guanine or thymine at the second and third position. Consequently, this codon encodes the amino acids arginine, glycine, tryptophan, methionine, leucine, valine, serine, cysteine, isoleucine and phenylalanine. <br />
[[File:UP_lib2_fig2.png|center|500px|thumb|'''Figure 6:''' Design of libraries based on the nucleotide sequence of the ribosomal precursor peptide (MdnA). To different libraries, focused library 1 and focused library 2 were generated by randomised oligonucleotide synthesis. The modifications compared to the wild type sequence are highlighted. The definition of the used code is also shown. The first libray (focused library 1) displays a diversity of 45360 and the second, focused library 2 a diversity of 810. In the further studies focused library 2 was used to find optimized microviridin variants. ]]<br />
To construct this library a fill-in reaction using the designed forward and reverse oligonucleotides was performed. Subsequently resulting randomized oligonucleotide was digested with the restriction enzyme AatII. In addition, the vector pUP089 was digested with the restriction enzymes AatII and SfoI. After ligation of fragments, randomized oligonucleotide and vector, the focused library 2 (ligation product) was transformed in chemocompetent ''E. coli'' XL1-Blue cells. This procedure was done several times and a library size of 1233 colonies was reached. <br />
[[File:UP_lib2_fig3.png|center|500px|thumb|'''Figure 7:''' Confirmation of focused library 2. Different clones of the generated focused library 2 were sequenced. Alignments with the wild type sequence (reference sequence) were performed. As shown (for a selection of clones) in all of our sequenced samples the requested modifications were established.]]<br />
For confirmation of the focused library 2, sequencing analysis of a number of clones was performed. In all of our sequenced samples the requested modifications were established (figure 7). A sequence logo was generated for the 14 amino acid sized core peptide of MdnA. This sequence logo, which is illustrated in figure 8, indicates that the desired sites stay conserved. The others are modified. <br />
[[File:UP_lib2_fig4.png|center|500px|thumb|'''Figure 8:''' Sequence logo plot of focused library 2. The 14 amino acid sized core peptide of MdnA was sequenced. The sequence of several clones was used to generate a sequence logo. This indicates that the desired sites stay conserved. The others nucleotides and resulting amino acids are modified. ]]<br />
The generated and modified focused library 2 should be used as basis for in-vivo selection and phage-display screening. Furthermore we purpose the objective to search for optimized and novel microviridins by using this library.<br />
<br><br><br />
<br />
=== Expression Backbones ===<br />
In a subtask of our work we wanted to construct auxiliary expression backbones with inducible promotors. In contrast to constitutive systems inducible systems only express the protein of interest after adding an inducer, which can be for example AHL or arabinose.<br />
We decided to construct IPTG- and arabinose-inducible systems. Therefore we amplified the promotor region and fused it to a reporter gene, which is constituted by YFP. Using this we have the option to verify the presence of the inducible promotor and also the function of the induction process by fluorescence. Our idea was to use the reporter gene as a placeholder. Via restriction enzyme digestion using the iGEM restriction enzyme sites you will be able to replace the reporter gene with your gene of interest. As vector backbone we made use of the pSB1A3, which has ampicillin resistance (Fig. 9). The constructs using pSB1K3 (kanamycin resistance) and pSB1C3 (chloramphenicol resistance) are still on the anvil. <br />
[[File:UP_expression_backbones_cloning.png|center|500px|thumb|'''Figure 9:''' Cloning Scheme of Expression Backbones. ''amp'' - gene for ampicillin resistance]]<br />
<br />
We successfully tested the IPTG- and arabinose-inducible system (Fig 10). Using fluorescence microscopy we were able to detect the YFP-expression after an induction time of approximately 1.5 hrs. To prove the hypothesis we opposed an induced sample with a non-induced control for each promotor. By use of brightfield combined with differential interference contrast microscopy we traced the ''E. coli'' cells and switched then to the YFP detecting channel to investigate the fluorescence of these cells.<br />
<br />
[[File:UP_Expression_Backbones_Fluorescence.png|center|800px|thumb|'''Figure 10:''' Fluorescence Microscopy. control - not induced; induced - induction with IPTG or Arabinose, respectively]]<br />
<br />
We also tested the inducible systems by using fluorescence spectroscopy. For this experiment we induced both systems at a time when the cultures were located in phase of exponential growth. After inducing, the cultures were analyzed by fluorescence spectroscopy. In this analyze the cells were excited by 500 nm. The resultant emission was measured in a spectrum between 510 and 580 nm. (Fig 11;12)<br />
<table><td>[[File:UP_psB1A3_Ara_YFPII.png|left|410px|thumb|'''Figure 11:''' Emission (526nm) of pSB1A3_Ara_YFP and control – '' E.coli'' cells (including pSB1A3_Ara_YFP) were induced and measured at certain moments.]] </td> <td>[[File:UP_psB1A3_IPTG_YFPII.png|right|410px|thumb|'''Figure 12:''' Emission (526nm) of induced pSB1A3_lac_YFP and control – ''E.coli'' cells (including pSB1A3_lac_YFP) were induced and measured at certain moments.]]</td><br />
</table>In contrast to the control of the arabinose inducible system the Lac-control also shows fluorescence. This demonstrates the leaky Lac-promotor, what is also a scientifically proven fact. A leaky promotor means that the promotor is not regulated in a leakproof manner. Even in repressed conditions the transcript can arise.<br />
<br />
The lacking of the Lac-I gene on the vector is the reason for expression of YFP in both controls. For further research in this field it is necessary to clone the Lac-I gene inside the constructed vector.<br />
<br><br><br />
<br />
===References===<br />
Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74<br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_MicroviridinTeam:Potsdam Bioware/Project/Details Microviridin2011-10-28T23:07:59Z<p>UP Stefan: /* Generating mdnA gene libraries */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==Microviridin==<br />
=== Introduction ===<br />
Cyanobacteria are known as the blue-green-algae because their living space is water and their feeding mechanism is photosynthetic. The Cyanobacteria are know for some special and unique metabolites. One of these metabolites are the microviridin family. Special attributes of microviridins are their occurrence as tricyclic depsipeptides, means peptides whose one or more amide-bond is replaced by an ester-bond, unparallel cage-like architecture and their ability to inhibit several proteases. Microviridin B for example is told to function as an elastase inhibitor what would be an therapeutic attempt according to fix the out of control gone function of elastase in lung emphysema. In such a case it is beyond debate how important it is to figger out the biosynthesis of this peptide. The research group of Prof. Dittmann at the University of Potsdam was successful and able to report about the composition of the gene cluster from ''Micocystis aeruginosa'' NIES298 expressing Microviridin B. Moreover they have been convinced that there is existing one unique biosynthetic mechanism for microviridins in Microcystis. Unusual for depsipeptides they discovered that they are ribosomal synthesized. The ''mdn'' gene cluster is build up of a gene ''mdnA'', encoding for the putative precursor peptide of the microvirdin, two genes encoding ATP-grasp-type ligases ''mdnB'' and ''mdnC'', an ABC transporter encoding gene ''mdnE'' as well as one encoding an N- acetyltransferase of the GNAT family ''mdnD'' (Ziemert et al., 2008). Moreover in their recent studies they on the contrary report about the discovery of an existing natural diversity of microviridin precursors genes in set of closely related Mycrocystis laboratory strains. This furthermore would mean and lead to a heterologous production of novel microviridins in ''E. coli''. To give evidence of their discovery they identified and characterized a new Microviridin L from the strain ''Mycrocystis aeruginosa'' NIES843 (Ziemert et al., 2010).<br />
All in all the microviridins, the tricyclic depsipeptide are build up through a gene cluster of five mdn-components each fulfill one function to make the hole cluster functional. This showed the team of Prof. Dr. Dittmann as the peptide is only correctly processed in ''E. coli'' if the entire cluster ''mdnABCDE'' is expressed. In addition the new discovery is promising a small flexibility of the microviridin ligases in Mycrocystis as an open window for the size of the natural microviridin library and thus microviridin functions and hence for example therapeutic benefit that needs to be discovered. The following illustration is going to give you a better overview (Fig. 1).<br />
[[File:UP_MV_introduction_mdn.jpg|center|525px|thumb|'''Figure 1:''' overview of the mdn gene cluster (GNAT: GCN5-related N-acetyltransferase)]]<br />
<br><br><br />
<br />
=== Modularization of the ''mdn'' gene cluster===<br />
Using two vectors (pARW071 and pARW089, respectively) containing the ''mdn'' biosynthetic gene cluster, we designed PCR primers for the amplification of the ''mdn genes''. Using these primers, we obtained the gene fragments of ''mdnA'', ''mdnB'', ''mdnC'', ''mdnD'', ''mdnE'' and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our BioBricks comply with the BioBrick standards. <br />
<br />
The following image (Fig. 2) shows the generic construction of the BioBrick carrying the'' mdnA gene''.<br />
<br />
[[File:UP_modularization_mdnA_cloning.png|center|500px|thumb|'''Figure 2:''' Cloning Scheme of mdnA-BioBrick (''Cm'' - gene for chloramphenicol resistance)]]<br />
<br />
Microviridin production in ''E. coli'' cells expressing the'' mdn genes'' was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3). <br />
[[File:UP_modularization_HPLC.png|center|500px|thumb|'''Figure 3:''' HPLC chromatogram of mdnA]]<br />
All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.<br />
<br />
Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.<br />
<br />
[[File:UP_modularization_MSII.gif|center|300px|thumb|'''Figure 4:''' Mass Spectrometry Data]]<br />
<br><br><br />
<br />
=== Generating ''mdnA'' gene libraries ===<br />
In searching for optimized and novel microviridin peptides, libraries of mutated microviridins were established. Therefore a number of sites in the amino acid sequence of the precursor peptide (MdnA) were chosen.<br />
In <i>Microcystis</i>, microviridins are synthesized from a ribosomal precursor peptide (MdnA). Therefore the microviridin gene cluster is necessary. This 6.5 kb biosynthesis gene cluster includes two genes (''mdnB'' and ''mdnC''), which encodes ATP-grasp-type ligases and further the ''mdnE'' gene encoding an ABC transporter as well as'' mdnD'' gene encoding a N-acetyltransferase (Ziemert et al., 2008). In consequence the biosynthesis gene cluster genes ''mdnB'', ''mdnC'', ''mdnD'' and ''mdnE'' cannot be mutated. Otherwise only misprocessed and non-function variants of microviridin occur (Ziemert et al., 2008). Thus merely the modification of the ''mdnA'' gene can lead to effective changes in the microviridin structure and function. <br><br />
[[File:UP_lib2_fig.png|center|500px|thumb|'''Figure 5:''' Molecular organization of the ribsosomal precursor peptide (MdnA) of microviridin. MdnA consists of a leader and a core peptide. The N-terminal leader peptide contains highly conserved double glycine motifs. Whereas in the C-terminal 14 amino acid sized core peptide sequence shows variations. Modified from Ziemert et al. (2008) and Ziemert et al. (2010).]]<br />
The precursor peptide MdnA consists of a leader peptide and a core peptide (Fig. 5). Ziemert et al. (2008) has shown that the N-terminal leader peptide contains highly conserved double glycine motifs (Ziemert et al. 2008). However remarkable variations were found in region, which encodes the 14-amino acid sequence of the C-terminal microviridin core peptide (Ziemert et al. 2010). Consequently, mutations in the MdnA core peptide seem to be the most promising option for modification of microviridin. For cyclization, the amino acid side chains of the core peptide form omega-ester and omega-amino bonds. One loop is formed between threonine and aspartic acid, the second loop between lysine and aspartic acid and the third loop between serine and glutamic acid (Fig. 5). The sequence of this loop forming amino acids constitute an exception while mutation of the ''mdnA'' gene (Ziemert et al., 2008 and 2010).<br />
<br />
To find optimized and novel microviridin variants, several libraries carrying modifications in the sequence of the MdnA core peptide were established. These libraries were generated by randomized oligonucleotide synthesis. Therefore a forward oligonucleotide displaying a part of conserved leader peptide sequence was built. Further a reverse oligonucleotide was created. The first part of this oligonucleotide is 20 bases, which overlap with the forward oligonucleotide and which is necessary for hybridization of both, forward and reverse, oligonucleotides. In the middle modified bases are inserted. For cloning the forward oligonucleotide starts with the blunt restricted recognition site of SfoI. This results therein that the oligonucleotide can ligate in the SfoI restricted vector without previous restriction of the oligonucleotide. The last part of the reverse oligonucleotides displays an AatII restriction site for cloning. Digestion with AatII results in sticky end, thus wrong way round insertion of the oligonucleotide is impossible. Forward and reverse oligonucleotides were combined by performing a fill-in reaction. <br />
Because we designed several libraries with different diversity rates, the modified bases, which are inserted in the middle of the reverse primer, vary. All of our generated libraries contain mutations of the sequence of the MdnA core peptide at several sites, but not at the loop forming sites. Thus we are thinking that cyclization of microviridin happens accurately. A library with a diversity of 45,360 (focused library 1) and a second focused library showing a minor diversity of 810 (focused library 2) were designed. The included sequence modifications plus the diversity of our libraries are shown in figure 6. In our further studies we worked on focused library 2 primarily. In this library the nucleotide sequences encoding glycine residues were changed in GVK in all cases (Figure 6). GVK stands for guanine at the first position, adenine, cytosine or guanine at the second position and further thymine at the third position of the codon. These three possible codons encode for the amino acids alanine, aspartic acid and glycine. The nucleotide sequence N-terminal tyrosine residue was changed in THT describing thymine (first position), adenine, cytosine or thymine at the second position and thymine (third position). These codons encode isoleucine, leucine and phenylalanine. Additionally, the sequence of phenylalanine in the core peptide sequence was shift to NNK. NKK stands for one of the four nucleotides at the first position, and guanine or thymine at the second and third position. Consequently, this codon encodes the amino acids arginine, glycine, tryptophan, methionine, leucine, valine, serine, cysteine, isoleucine and phenylalanine. <br />
[[File:UP_lib2_fig2.png|center|500px|thumb|'''Figure 6:''' Design of libraries based on the nucleotide sequence of the ribosomal precursor peptide (MdnA). To different libraries, focused library 1 and focused library 2 were generated by randomised oligonucleotide synthesis. The modifications compared to the wild type sequence are highlighted. The definition of the used code is also shown. The first libray (focused library 1) displays a diversity of 45360 and the second, focused library 2 a diversity of 810. In the further studies focused library 2 was used to find optimized microviridin variants. ]]<br />
To construct this library a fill-in reaction using the designed forward and reverse oligonucleotides was performed. Subsequently resulting randomized oligonucleotide was digested with the restriction enzyme AatII. In addition, the vector pUP089 was digested with the restriction enzymes AatII and SfoI. After ligation of fragments, randomized oligonucleotide and vector, the focused library 2 (ligation product) was transformed in chemocompetent ''E. coli'' XL1-Blue cells. This procedure was done several times and a library size of 1233 colonies was reached. <br />
[[File:UP_lib2_fig3.png|center|500px|thumb|'''Figure 7:''' Confirmation of focused library 2. Different clones of the generated focused library 2 were sequenced. Alignments with the wild type sequence (reference sequence) were performed. As shown (for a selection of clones) in all of our sequenced samples the requested modifications were established.]]<br />
For confirmation of the focused library 2, sequencing analysis of a number of clones was performed. In all of our sequenced samples the requested modifications were established (figure 7). A sequence logo was generated for the 14 amino acid sized core peptide of MdnA. This sequence logo, which is illustrated in figure 8, indicates that the desired sites stay conserved. The others are modified. <br />
[[File:UP_lib2_fig4.png|center|500px|thumb|'''Figure 8:''' Sequence logo plot of focused library 2. The 14 amino acid sized core peptide of MdnA was sequenced. The sequence of several clones was used to generate a sequence logo. This indicates that the desired sites stay conserved. The others nucleotides and resulting amino acids are modified. ]]<br />
The generated and modified focused library 2 should be used as basis for in-vivo selection and phage-display screening. Furthermore we purpose the objective to search for optimized and novel microviridins by using this library.<br />
<br><br><br />
<br />
=== Expression Backbones ===<br />
In a subtask of our work we wanted to construct auxiliary expression backbones with inducible promotors. In contrast to constitutive systems inducible systems only express the protein of interest after adding an inducer, which can be for example AHL or arabinose.<br />
We decided to construct IPTG- and arabinose-inducible systems. Therefore we amplified the promotor region and fused it to a reporter gene, which is constituted by YFP. Using this we have the option to verify the presence of the inducible promotor and also the function of the induction process by fluorescence. Our idea was to use the reporter gene as a placeholder. Via restriction enzyme digestion using the iGEM restriction enzyme sites you will be able to replace the reporter gene with your gene of interest. As vector backbone we made use of the pSB1A3, which has ampicillin resistance (Fig. 9). The constructs using pSB1K3 (kanamycin resistance) and pSB1C3 (chloramphenicol resistance) are still on the anvil. <br />
[[File:UP_expression_backbones_cloning.png|center|500px|thumb|'''Figure 9:''' Cloning Scheme of Expression Backbones. ''amp'' - gene for ampicillin resistance]]<br />
<br />
We successfully tested the IPTG- and arabinose-inducible system (Fig 10). Using fluorescence microscopy we were able to detect the YFP-expression after an induction time of approximately 1.5 hrs. To prove the hypothesis we opposed an induced sample with a non-induced control for each promotor. By use of brightfield combined with differential interference contrast microscopy we traced the ''E. coli'' cells and switched then to the YFP detecting channel to investigate the fluorescence of these cells.<br />
<br />
[[File:UP_Expression_Backbones_Fluorescence.png|center|800px|thumb|'''Figure 10:''' Fluorescence Microscopy. control - not induced; induced - induction with IPTG or Arabinose, respectively]]<br />
<br />
We also tested the inducible systems by using fluorescence spectroscopy. For this experiment we induced both systems at a time when the cultures were located in phase of exponential growth. After inducing, the cultures were analyzed by fluorescence spectroscopy. In this analyze the cells were excited by 500 nm. The resultant emission was measured in a spectrum between 510 and 580 nm. (Fig 11;12)<br />
<table><td>[[File:UP_psB1A3_Ara_YFPII.png|left|410px|thumb|'''Figure 11:''' Emission (526nm) of pSB1A3_Ara_YFP and control – '' E.coli'' cells (including pSB1A3_Ara_YFP) were induced and measured at certain moments.]] </td> <td>[[File:UP_psB1A3_IPTG_YFPII.png|right|410px|thumb|'''Figure 12:''' Emission (526nm) of induced pSB1A3_lac_YFP and control – ''E.coli'' cells (including pSB1A3_lac_YFP) were induced and measured at certain moments.]]</td><br />
</table>In contrast to the control of the arabinose inducible system the Lac-control also shows fluorescence. This demonstrates the leaky Lac-promotor, what is also a scientifically proven fact. A leaky promotor means that the promotor is not regulated in a leakproof manner. Even in repressed conditions the transcript can arise.<br />
<br />
The lacking of the Lac-I gene on the vector is the reason for expression of YFP in both controls. For further research in this field it is necessary to clone the Lac-I gene inside the constructed vector.<br />
<br><br><br />
<br />
===References===<br />
Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74<br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_MicroviridinTeam:Potsdam Bioware/Project/Details Microviridin2011-10-28T23:07:17Z<p>UP Stefan: /* Expression Backbones */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==Microviridin==<br />
=== Introduction ===<br />
Cyanobacteria are known as the blue-green-algae because their living space is water and their feeding mechanism is photosynthetic. The Cyanobacteria are know for some special and unique metabolites. One of these metabolites are the microviridin family. Special attributes of microviridins are their occurrence as tricyclic depsipeptides, means peptides whose one or more amide-bond is replaced by an ester-bond, unparallel cage-like architecture and their ability to inhibit several proteases. Microviridin B for example is told to function as an elastase inhibitor what would be an therapeutic attempt according to fix the out of control gone function of elastase in lung emphysema. In such a case it is beyond debate how important it is to figger out the biosynthesis of this peptide. The research group of Prof. Dittmann at the University of Potsdam was successful and able to report about the composition of the gene cluster from ''Micocystis aeruginosa'' NIES298 expressing Microviridin B. Moreover they have been convinced that there is existing one unique biosynthetic mechanism for microviridins in Microcystis. Unusual for depsipeptides they discovered that they are ribosomal synthesized. The ''mdn'' gene cluster is build up of a gene ''mdnA'', encoding for the putative precursor peptide of the microvirdin, two genes encoding ATP-grasp-type ligases ''mdnB'' and ''mdnC'', an ABC transporter encoding gene ''mdnE'' as well as one encoding an N- acetyltransferase of the GNAT family ''mdnD'' (Ziemert et al., 2008). Moreover in their recent studies they on the contrary report about the discovery of an existing natural diversity of microviridin precursors genes in set of closely related Mycrocystis laboratory strains. This furthermore would mean and lead to a heterologous production of novel microviridins in ''E. coli''. To give evidence of their discovery they identified and characterized a new Microviridin L from the strain ''Mycrocystis aeruginosa'' NIES843 (Ziemert et al., 2010).<br />
All in all the microviridins, the tricyclic depsipeptide are build up through a gene cluster of five mdn-components each fulfill one function to make the hole cluster functional. This showed the team of Prof. Dr. Dittmann as the peptide is only correctly processed in ''E. coli'' if the entire cluster ''mdnABCDE'' is expressed. In addition the new discovery is promising a small flexibility of the microviridin ligases in Mycrocystis as an open window for the size of the natural microviridin library and thus microviridin functions and hence for example therapeutic benefit that needs to be discovered. The following illustration is going to give you a better overview (Fig. 1).<br />
[[File:UP_MV_introduction_mdn.jpg|center|525px|thumb|'''Figure 1:''' overview of the mdn gene cluster (GNAT: GCN5-related N-acetyltransferase)]]<br />
<br><br><br />
<br />
=== Modularization of the ''mdn'' gene cluster===<br />
Using two vectors (pARW071 and pARW089, respectively) containing the ''mdn'' biosynthetic gene cluster, we designed PCR primers for the amplification of the ''mdn genes''. Using these primers, we obtained the gene fragments of ''mdnA'', ''mdnB'', ''mdnC'', ''mdnD'', ''mdnE'' and for the whole cluster, respectively. Due to the sophisticated incorporation of the sequences of the iGEM restriction enzymes into the primer sequence our BioBricks comply with the BioBrick standards. <br />
<br />
The following image (Fig. 2) shows the generic construction of the BioBrick carrying the'' mdnA gene''.<br />
<br />
[[File:UP_modularization_mdnA_cloning.png|center|500px|thumb|'''Figure 2:''' Cloning Scheme of mdnA-BioBrick (''Cm'' - gene for chloramphenicol resistance)]]<br />
<br />
Microviridin production in ''E. coli'' cells expressing the'' mdn genes'' was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3). <br />
[[File:UP_modularization_HPLC.png|center|500px|thumb|'''Figure 3:''' HPLC chromatogram of mdnA]]<br />
All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.<br />
<br />
Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.<br />
<br />
[[File:UP_modularization_MSII.gif|center|300px|thumb|'''Figure 4:''' Mass Spectrometry Data]]<br />
<br><br><br />
<br />
=== Generating ''mdnA'' gene libraries ===<br />
In searching for optimized and novel microviridin peptides, libraries of mutated microviridins were established. Therefore a number of sites in the amino acid sequence of the precursor peptide (MdnA) were chosen.<br />
In Microcystis, microviridins are synthesized from a ribosomal precursor peptide (MdnA). Therefore the microviridin gene cluster is necessary. This 6.5 kb biosynthesis gene cluster includes two genes (''mdnB'' and ''mdnC''), which encodes ATP-grasp-type ligases and further the ''mdnE'' gene encoding an ABC transporter as well as'' mdnD'' gene encoding a N-acetyltransferase (Ziemert et al., 2008). In consequence the biosynthesis gene cluster genes ''mdnB'', ''mdnC'', ''mdnD'' and ''mdnE'' cannot be mutated. Otherwise only misprocessed and non-function variants of microviridin occur (Ziemert et al., 2008). Thus merely the modification of the ''mdnA'' gene can lead to effective changes in the microviridin structure and function. <br><br />
[[File:UP_lib2_fig.png|center|500px|thumb|'''Figure 5:''' Molecular organization of the ribsosomal precursor peptide (MdnA) of microviridin. MdnA consists of a leader and a core peptide. The N-terminal leader peptide contains highly conserved double glycine motifs. Whereas in the C-terminal 14 amino acid sized core peptide sequence shows variations. Modified from Ziemert et al. (2008) and Ziemert et al. (2010).]]<br />
The precursor peptide MdnA consists of a leader peptide and a core peptide (Fig. 5). Ziemert et al. (2008) has shown that the N-terminal leader peptide contains highly conserved double glycine motifs (Ziemert et al. 2008). However remarkable variations were found in region, which encodes the 14-amino acid sequence of the C-terminal microviridin core peptide (Ziemert et al. 2010). Consequently, mutations in the MdnA core peptide seem to be the most promising option for modification of microviridin. For cyclization, the amino acid side chains of the core peptide form omega-ester and omega-amino bonds. One loop is formed between threonine and aspartic acid, the second loop between lysine and aspartic acid and the third loop between serine and glutamic acid (Fig. 5). The sequence of this loop forming amino acids constitute an exception while mutation of the ''mdnA'' gene (Ziemert et al., 2008 and 2010).<br />
<br />
To find optimized and novel microviridin variants, several libraries carrying modifications in the sequence of the MdnA core peptide were established. These libraries were generated by randomized oligonucleotide synthesis. Therefore a forward oligonucleotide displaying a part of conserved leader peptide sequence was built. Further a reverse oligonucleotide was created. The first part of this oligonucleotide is 20 bases, which overlap with the forward oligonucleotide and which is necessary for hybridization of both, forward and reverse, oligonucleotides. In the middle modified bases are inserted. For cloning the forward oligonucleotide starts with the blunt restricted recognition site of SfoI. This results therein that the oligonucleotide can ligate in the SfoI restricted vector without previous restriction of the oligonucleotide. The last part of the reverse oligonucleotides displays an AatII restriction site for cloning. Digestion with AatII results in sticky end, thus wrong way round insertion of the oligonucleotide is impossible. Forward and reverse oligonucleotides were combined by performing a fill-in reaction. <br />
Because we designed several libraries with different diversity rates, the modified bases, which are inserted in the middle of the reverse primer, vary. All of our generated libraries contain mutations of the sequence of the MdnA core peptide at several sites, but not at the loop forming sites. Thus we are thinking that cyclization of microviridin happens accurately. A library with a diversity of 45,360 (focused library 1) and a second focused library showing a minor diversity of 810 (focused library 2) were designed. The included sequence modifications plus the diversity of our libraries are shown in figure 6. In our further studies we worked on focused library 2 primarily. In this library the nucleotide sequences encoding glycine residues were changed in GVK in all cases (Figure 6). GVK stands for guanine at the first position, adenine, cytosine or guanine at the second position and further thymine at the third position of the codon. These three possible codons encode for the amino acids alanine, aspartic acid and glycine. The nucleotide sequence N-terminal tyrosine residue was changed in THT describing thymine (first position), adenine, cytosine or thymine at the second position and thymine (third position). These codons encode isoleucine, leucine and phenylalanine. Additionally, the sequence of phenylalanine in the core peptide sequence was shift to NNK. NKK stands for one of the four nucleotides at the first position, and guanine or thymine at the second and third position. Consequently, this codon encodes the amino acids arginine, glycine, tryptophan, methionine, leucine, valine, serine, cysteine, isoleucine and phenylalanine. <br />
[[File:UP_lib2_fig2.png|center|500px|thumb|'''Figure 6:''' Design of libraries based on the nucleotide sequence of the ribosomal precursor peptide (MdnA). To different libraries, focused library 1 and focused library 2 were generated by randomised oligonucleotide synthesis. The modifications compared to the wild type sequence are highlighted. The definition of the used code is also shown. The first libray (focused library 1) displays a diversity of 45360 and the second, focused library 2 a diversity of 810. In the further studies focused library 2 was used to find optimized microviridin variants. ]]<br />
To construct this library a fill-in reaction using the designed forward and reverse oligonucleotides was performed. Subsequently resulting randomized oligonucleotide was digested with the restriction enzyme AatII. In addition, the vector pUP089 was digested with the restriction enzymes AatII and SfoI. After ligation of fragments, randomized oligonucleotide and vector, the focused library 2 (ligation product) was transformed in chemocompetent ''E. coli'' XL1-Blue cells. This procedure was done several times and a library size of 1233 colonies was reached. <br />
[[File:UP_lib2_fig3.png|center|500px|thumb|'''Figure 7:''' Confirmation of focused library 2. Different clones of the generated focused library 2 were sequenced. Alignments with the wild type sequence (reference sequence) were performed. As shown (for a selection of clones) in all of our sequenced samples the requested modifications were established.]]<br />
For confirmation of the focused library 2, sequencing analysis of a number of clones was performed. In all of our sequenced samples the requested modifications were established (figure 7). A sequence logo was generated for the 14 amino acid sized core peptide of MdnA. This sequence logo, which is illustrated in figure 8, indicates that the desired sites stay conserved. The others are modified. <br />
[[File:UP_lib2_fig4.png|center|500px|thumb|'''Figure 8:''' Sequence logo plot of focused library 2. The 14 amino acid sized core peptide of MdnA was sequenced. The sequence of several clones was used to generate a sequence logo. This indicates that the desired sites stay conserved. The others nucleotides and resulting amino acids are modified. ]]<br />
The generated and modified focused library 2 should be used as basis for in-vivo selection and phage-display screening. Furthermore we purpose the objective to search for optimized and novel microviridins by using this library.<br />
<br><br><br />
<br />
=== Expression Backbones ===<br />
In a subtask of our work we wanted to construct auxiliary expression backbones with inducible promotors. In contrast to constitutive systems inducible systems only express the protein of interest after adding an inducer, which can be for example AHL or arabinose.<br />
We decided to construct IPTG- and arabinose-inducible systems. Therefore we amplified the promotor region and fused it to a reporter gene, which is constituted by YFP. Using this we have the option to verify the presence of the inducible promotor and also the function of the induction process by fluorescence. Our idea was to use the reporter gene as a placeholder. Via restriction enzyme digestion using the iGEM restriction enzyme sites you will be able to replace the reporter gene with your gene of interest. As vector backbone we made use of the pSB1A3, which has ampicillin resistance (Fig. 9). The constructs using pSB1K3 (kanamycin resistance) and pSB1C3 (chloramphenicol resistance) are still on the anvil. <br />
[[File:UP_expression_backbones_cloning.png|center|500px|thumb|'''Figure 9:''' Cloning Scheme of Expression Backbones. ''amp'' - gene for ampicillin resistance]]<br />
<br />
We successfully tested the IPTG- and arabinose-inducible system (Fig 10). Using fluorescence microscopy we were able to detect the YFP-expression after an induction time of approximately 1.5 hrs. To prove the hypothesis we opposed an induced sample with a non-induced control for each promotor. By use of brightfield combined with differential interference contrast microscopy we traced the ''E. coli'' cells and switched then to the YFP detecting channel to investigate the fluorescence of these cells.<br />
<br />
[[File:UP_Expression_Backbones_Fluorescence.png|center|800px|thumb|'''Figure 10:''' Fluorescence Microscopy. control - not induced; induced - induction with IPTG or Arabinose, respectively]]<br />
<br />
We also tested the inducible systems by using fluorescence spectroscopy. For this experiment we induced both systems at a time when the cultures were located in phase of exponential growth. After inducing, the cultures were analyzed by fluorescence spectroscopy. In this analyze the cells were excited by 500 nm. The resultant emission was measured in a spectrum between 510 and 580 nm. (Fig 11;12)<br />
<table><td>[[File:UP_psB1A3_Ara_YFPII.png|left|410px|thumb|'''Figure 11:''' Emission (526nm) of pSB1A3_Ara_YFP and control – '' E.coli'' cells (including pSB1A3_Ara_YFP) were induced and measured at certain moments.]] </td> <td>[[File:UP_psB1A3_IPTG_YFPII.png|right|410px|thumb|'''Figure 12:''' Emission (526nm) of induced pSB1A3_lac_YFP and control – ''E.coli'' cells (including pSB1A3_lac_YFP) were induced and measured at certain moments.]]</td><br />
</table>In contrast to the control of the arabinose inducible system the Lac-control also shows fluorescence. This demonstrates the leaky Lac-promotor, what is also a scientifically proven fact. A leaky promotor means that the promotor is not regulated in a leakproof manner. Even in repressed conditions the transcript can arise.<br />
<br />
The lacking of the Lac-I gene on the vector is the reason for expression of YFP in both controls. For further research in this field it is necessary to clone the Lac-I gene inside the constructed vector.<br />
<br><br><br />
<br />
===References===<br />
Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74<br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/SummaryTeam:Potsdam Bioware/Project/Summary2011-10-28T22:43:44Z<p>UP Stefan: /* Modification, Selection and Production of Cyclic Peptides for Therapy */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Summary ==<br />
<br />
===Modification, Selection and Production of Cyclic Peptides for Therapy===<br />
One key task of biopharmaceuticals is the binding and blocking of deregulated proteins. Picking the right lead structure for biopharmaceuticals is very important for success. This year, we developed a novel system for the modification, selection and optimization of peptides showing potential for protease inhibition. These promising inhibitors are found in cyanobacteria and are called Microviridins. They belong to the class peptides characterized by unusual ω-ester and ω-amide bonds between the amino-acid side chains, which are also referred to as depsipeptides. These modifications are introduced post translationally by a set of enzymes and result in an extraordinary tricyclic cage structure. <br />
<br><br>Proteases are a large enzyme family and comprise about 647 human gene products. Therefore they are important drug targets. Additional many harmful bacteria, viruses and fungi use proteasess in their reproduction cycle and growth. The ability of blocking these proteases is a highly relevant therapeutic application. Prominent examples of targets are the angiotensin-converting enzyme (ACE) and HIV proteases.<br />
<br><br>We chose a 6.5 kb Microviridin gene cluster named ''mdnABCDE'' from ''Mycrocystis aeruginosa'' NIES843. The mdn gene cluster comprises a gene encoding a precursor peptide named MdnA, which is then modified to form the Microviridin, two genes encoding ATP-grasp-type ligases named ''mdnB'' and ''mdnC'', an ABC- transporter encoding gene named ''mdnE'' as well as one gene encoding an N- acetyltransferase of the GNAT family named ''mdnD'' (Ziemert et al., 2008). Our major aim was to modify the MdnA precursor peptide and optimize its protease inhibiting properties. Towards this goal, we synthesized semi-rational ''mdnA'' gene libraries with partially randomized oligonucleotides. The library was successfully cloned and verified by mass spectrometry.<br />
<br><br>To identify the best candidates inhibiting various given proteases, we established two different selection systems: Phage Display and a new developed ''in-vivo'' selection assay. Phage display is a frequently used technique in laboratories, yet to our knowledge, phage display of cellularly cyclized peptides is new. We constructed a fusion between the surface protein geneIII of the phage and the ''mdnA'' gene within the ''mdnA'' gene cluster. This was verified by western blotting, an ELISA test and a trypsin assay.<br />
<br><br>We also constructed a recombinant ''in-vivo'' selection system linking protease degradation to antibiotic resistance. The designed device is divided into two parts: first a protease activity detector device and second a protease generator device. For the protease activity detector device we fused various protease cleavage sites between a signal sequence and the antibiotic resistance conferring enzyme β-lactamse. β-lactamse confers only resistance when transferred to the periplasm of ''E. coli''. Thus, when the protease cleaves the signal sequence from the lactamase, antibiotic resistance is abolished and cells die under selective pressure. However, when our Microviridin variants inhibit the protease, cells survive, allowing an easy and efficient inhibitor selection. We achieved different growth rates with increasing ampicillin concentrations. In addition we show cell death when the protease is activated. After cotransformation with the ''mdnA''-libary we were able to isolate several clones, which will be sequenced.<br />
<br><br />
<br><br />
In addition to the practical work we established a mathematical model of the ''in-vivo'' selection system, which helped us to understand the selection process. We modeled the reaction kinetics by ordinary differential equations and coded them in matlab, to predict, later based on our experimental data, several constants like dissoziation and assoziation constants of the interaction between microviridin and the protease.<br><br />
<br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br><br><br />
<br />
== Highlights ==<br />
<br />
<br />
=== Microviridin ===<br />
[[File:UP_HPLC_mvd_01.jpg|left|300px|thumb|'''Figure 1:''' HPLC chromatogram of mdnA]] <br />
<br />
The major aim of the microviridin group was to modify the mdnA such that the protease inhibiting activity is enhanced. Therefore we used random mutagenesis as well as focused oligonucleotids for creating a library, which is ready for being screened for mdnA with a therapeutically promising set of mutations. For further experiments we also fused the mdnA to a myc-tag. So in the future we will be able to purify and isolate the mdnA.<br><br />
Due to the applicability of the whole mdn-cluster the creation of several BioBricks was possible. The construction was done using a given template vector containing the mdn-genes and sophisticated design of primers. Characterization of the BioBricks was done via HPLC analysis, mass spectrometry and western blot.<br><br />
In a subproject we also built auxiliary expression backbones with inducible promoters for easy cloning via the iGEM restriction enzyme sites.<br />
'''[[:Team:Potsdam_Bioware/Project/Details_Microviridin|[more]]]'''<br />
<br><br><br />
<br><br />
<br><br />
<br />
=== Phage Display ===<br />
[[File:UP_Phage_Display_02.jpg|left|290px|thumb|'''Figure 2: Optimization of phage display.''' After optimized conditions (right) a clear concentration of phages carrying mdnA after one panning round was noted.]]<br />
Phage display is a powerful tool for selecting peptides or proteins that bind and regulate the function of target proteins. It is defined as a system in which the protein and its encoding gene are covalently linked. Because of the therapeutic interest of microviridins as protease inhibitors a selection system for screening recombinant <i>mdnA</i>-libraries is of great importance. In our project the fundamental suitability of phage display for this purpose was shown. Therefore an appropriate phagemid, carrying an mdnA-myc-geneIII fusion gene, was constructed. The expression of the mdnA-myc-geneIII protein in the cells was shown by western blotting. Furthermore the production of phage particles carrying MdnA was determined by ELISA and phage display. '''[[:Team:Potsdam_Bioware/Project/Details_Phage|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== In Vivo Selection ===<br />
[[Image:UP_SurvTEV_onepla.jpg|left|290px|thumb|'''Figure 3: Ampicillin resistance test.''' Survival Screen, without induced (blue) and with induced (magenta) protease. From left to right increasing Ampicillin concentrations are shown.]]<br />
In addition to the Phage Display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an in vitro screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for a random protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator.'''[[:Team:Potsdam_Bioware/Project/Details_Selection|[more]]]'''<br />
<br><br><br />
<br><br><br />
<br><br><br />
<br><br><br />
<br><br><br> <br><br />
<br />
=== Modeling ===<br />
[[File: UP_3Dplot_Lact_06.png|left|300px|thumb|'''Figure 4:''' Plot that shows the lactamase concentration inside the periplasm (and thus the cell fittness) in dependence of the Enzyme inhibition reaction coefficient K_D.]]<br />
There is no synthetic biology without modeling, of course. We focused on systems modeling of our invivo selection system in which the reaction kinetics are analyzed and outcomes are predicted. Thus a synthetic biology approach can be chosen because a better understanding of the system is achieved and further changes can be planed - just like in engineering.<br> The reactions in our system were written down as equations under consideration of their induction at different times and the substance concentrations were numerically propagated through time. Using our concentration calculations we were able to see that our system works very well in theory - it is robust against changes of the most important system parameters. We learned about correct time-scales for our triggering and we were able to identify expected cell-division rates as a reference for the lab work. In a final step we were able to fit our model to wet-lab measurements so that predictions are more reliable. '''[[:Team:Potsdam_Bioware/Project/Details_Modeling|[more]]]'''<br><br><br><br><br><br><br />
<br />
=== Ethics ===<br />
<br />
For information about an ethics seminar and a survey among politicians see '''[[:Team:Potsdam_Bioware/Safety_Ethics|[here]]]'''.<br />
<br />
=== Software ===<br />
<br />
Have a look at the features and screenshots of our BioLog app on this '''[[:Team:Potsdam_Bioware/Software|[page]]]'''.</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/SummaryTeam:Potsdam Bioware/Project/Summary2011-10-28T22:42:34Z<p>UP Stefan: /* Modification, Selection and Production of Cyclic Peptides for Therapy */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Summary ==<br />
<br />
===Modification, Selection and Production of Cyclic Peptides for Therapy===<br />
One key task of biopharmaceuticals is the binding and blocking of deregulated proteins. Picking the right lead structure for biopharmaceuticals is very important for success. This year, we developed a novel system for the modification, selection and optimization of peptides showing potential for protease inhibition. These promising inhibitors are found in cyanobacteria and are called Microviridins. They belong to the class peptides characterized by unusual ω-ester and ω-amide bonds between the amino-acid side chains, which are also referred to as depsipeptides. These modifications are introduced post translationally by a set of enzymes and result in an extraordinary tricyclic cage structure. <br />
<br><br>Proteases are a large enzyme family and comprise about 647 human gene products. Therefore they are important drug targets. Additional many harmful bacteria, viruses and fungi use proteasess in their reproduction cycle and growth. The ability of blocking these proteases is a highly relevant therapeutic application. Prominent examples of targets are the angiotensin-converting enzyme (ACE) and HIV proteases.<br />
<br><br>We chose a 6.5 kb Microviridin gene cluster named ''mdnABCDE'' from ''Mycrocystis aeruginosa'' NIES843. The mdn gene cluster comprises a gene encoding a precursor peptide named MdnA, which is then modified to form the Microviridin, two genes encoding ATP-grasp-type ligases named ''mdnB'' and ''mdnC'', an ABC- transporter encoding gene named ''mdnE'' as well as one gene encoding an N- acetyltransferase of the GNAT family named ''mdnD'' (Ziemert et al., 2008). Our major aim was to modify the MdnA precursor peptide and optimize its protease inhibiting properties. Towards this goal, we synthesized semi-rational ''mdnA'' gene libraries with partially randomized oligonucleotides. The library was successfully cloned and verified by mass spectrometry.<br />
<br><br>To identify the best candidates inhibiting various given proteases, we established two different selection systems: Phage Display and a new developed ''in-vivo'' selection assay. Phage display is a frequently used technique in laboratories, yet to our knowledge, phage display of cellularly cyclized peptides is new. We constructed a fusion between the surface protein geneIII of the phage and the ''mdnA'' gene within the ''mdnA'' gene cluster. This was verified by western blotting, an ELISA test and a trypsin assay.<br />
<br><br>We also constructed a recombinant ''in-vivo'' selection system linking protease degradation to antibiotic resistance. The designed device is divided into two parts: first a protease activity detector device and second a protease generator device. For the protease activity detector device we fused various protease cleavage sites between a signal sequence and the antibiotic resistance conferring enzyme β-lactamse. β-lactamse confers only resistance when transferred to the periplasm of ''E. coli''. Thus, when the protease cleaves the signal sequence from the lactamase, antibiotic resistance is abolished and cells die under selective pressure. However, when our Microviridin variants inhibit the protease, cells survive, allowing an easy and efficient inhibitor selection. We achieved different growth rates with increasing ampicillin concentrations. And were able to show cell death when the protease is activated. After cotransformation with the ''mdnA''-libary we were able to isolate several clones, which will be sequenced.<br />
<br><br />
<br><br />
In addition to the practical work we established a mathematical model of the ''in-vivo'' selection system, which helped us to understand the selection process. We modeled the reaction kinetics by ordinary differential equations and coded them in matlab, to predict, later based on our experimental data, several constants like dissoziation and assoziation constants of the interaction between microviridin and the protease.<br><br />
<br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br><br><br />
<br />
== Highlights ==<br />
<br />
<br />
=== Microviridin ===<br />
[[File:UP_HPLC_mvd_01.jpg|left|300px|thumb|'''Figure 1:''' HPLC chromatogram of mdnA]] <br />
<br />
The major aim of the microviridin group was to modify the mdnA such that the protease inhibiting activity is enhanced. Therefore we used random mutagenesis as well as focused oligonucleotids for creating a library, which is ready for being screened for mdnA with a therapeutically promising set of mutations. For further experiments we also fused the mdnA to a myc-tag. So in the future we will be able to purify and isolate the mdnA.<br><br />
Due to the applicability of the whole mdn-cluster the creation of several BioBricks was possible. The construction was done using a given template vector containing the mdn-genes and sophisticated design of primers. Characterization of the BioBricks was done via HPLC analysis, mass spectrometry and western blot.<br><br />
In a subproject we also built auxiliary expression backbones with inducible promoters for easy cloning via the iGEM restriction enzyme sites.<br />
'''[[:Team:Potsdam_Bioware/Project/Details_Microviridin|[more]]]'''<br />
<br><br><br />
<br><br />
<br><br />
<br />
=== Phage Display ===<br />
[[File:UP_Phage_Display_02.jpg|left|290px|thumb|'''Figure 2: Optimization of phage display.''' After optimized conditions (right) a clear concentration of phages carrying mdnA after one panning round was noted.]]<br />
Phage display is a powerful tool for selecting peptides or proteins that bind and regulate the function of target proteins. It is defined as a system in which the protein and its encoding gene are covalently linked. Because of the therapeutic interest of microviridins as protease inhibitors a selection system for screening recombinant <i>mdnA</i>-libraries is of great importance. In our project the fundamental suitability of phage display for this purpose was shown. Therefore an appropriate phagemid, carrying an mdnA-myc-geneIII fusion gene, was constructed. The expression of the mdnA-myc-geneIII protein in the cells was shown by western blotting. Furthermore the production of phage particles carrying MdnA was determined by ELISA and phage display. '''[[:Team:Potsdam_Bioware/Project/Details_Phage|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== In Vivo Selection ===<br />
[[Image:UP_SurvTEV_onepla.jpg|left|290px|thumb|'''Figure 3: Ampicillin resistance test.''' Survival Screen, without induced (blue) and with induced (magenta) protease. From left to right increasing Ampicillin concentrations are shown.]]<br />
In addition to the Phage Display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an in vitro screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for a random protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator.'''[[:Team:Potsdam_Bioware/Project/Details_Selection|[more]]]'''<br />
<br><br><br />
<br><br><br />
<br><br><br />
<br><br><br />
<br><br><br> <br><br />
<br />
=== Modeling ===<br />
[[File: UP_3Dplot_Lact_06.png|left|300px|thumb|'''Figure 4:''' Plot that shows the lactamase concentration inside the periplasm (and thus the cell fittness) in dependence of the Enzyme inhibition reaction coefficient K_D.]]<br />
There is no synthetic biology without modeling, of course. We focused on systems modeling of our invivo selection system in which the reaction kinetics are analyzed and outcomes are predicted. Thus a synthetic biology approach can be chosen because a better understanding of the system is achieved and further changes can be planed - just like in engineering.<br> The reactions in our system were written down as equations under consideration of their induction at different times and the substance concentrations were numerically propagated through time. Using our concentration calculations we were able to see that our system works very well in theory - it is robust against changes of the most important system parameters. We learned about correct time-scales for our triggering and we were able to identify expected cell-division rates as a reference for the lab work. In a final step we were able to fit our model to wet-lab measurements so that predictions are more reliable. '''[[:Team:Potsdam_Bioware/Project/Details_Modeling|[more]]]'''<br><br><br><br><br><br><br />
<br />
=== Ethics ===<br />
<br />
For information about an ethics seminar and a survey among politicians see '''[[:Team:Potsdam_Bioware/Safety_Ethics|[here]]]'''.<br />
<br />
=== Software ===<br />
<br />
Have a look at the features and screenshots of our BioLog app on this '''[[:Team:Potsdam_Bioware/Software|[page]]]'''.</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T22:40:11Z<p>UP Stefan: /* Children, the scientists of tomorrow/Meeting the young minds? */</p>
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<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Roespel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
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[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or biotechnology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|420px|thumb| Our Team and Mr. Roespel at the right]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|420px|thumb|Our team in front of the German "Bundestag".]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html<br />
<br><br />
<br><br />
===Meeting the young minds===<br />
<br><br />
Children are the future, and will follow our footsteps, if we guide them. We cannot start early enough to awake their interest in research by letting them feel like real scientists in the lab. Children are open minded want to discover everything. It’s easy to inspire them with our work. The earlier we start to introduce people to science the better are the chances to create endorsement and interest instead of fear and refusal of synthetic biology.<br />
<br><br />
Therefore, we took the initiative and invited kids to our lab. We gave them a short and easy description of our work and aims at an adequate level. They assisted us at the lab and enjoyed tasks like pipetting. It was such a pleasure to see the fascination in their bright eyes. We think that such positive memories will last a lifetime. <br />
<br><br />
<br />
[[File:UP_kids-1.JPG|left|400px|thumb|]] <br />
[[File:UP_kids-2.JPG|right|400px|thumb|]]</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_ModelingTeam:Potsdam Bioware/Project/Details Modeling2011-10-28T22:38:48Z<p>UP Stefan: /* Results */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
<br />
<br />
<br />
== Modeling ==<br />
<br />
There is no synthetic biology without modeling, of course. In principle there is structure modeling and system modeling. In structure modeling the conformation and structure of proteins is examined and steric consequences for reactions or the whole system can be estimated. We focused on the second sort of modeling: The system modeling in which the reaction kinetics of the whole system is analyzed, outcomes are predicted and parameters correlated to measurements. Thus, a synthetic biology approach can be chosen because a better understanding of the system is achieved and further changes can be planed - just like in engineering.<br />
<br />
=== Model ===<br />
<br />
We built a model of our <i>in vivo</i> selection system to be able to engineer our system effectively. The following schema shows the major reactions taking place in our cell system. The Romanic numbers indicate the system relevant (partially triggered) reactions that were written down as equations and then numerically propagated through time.<br />
<br> <br />
[[File:UP_schema_modeling2v.png|center|650px|thumb|'''Figure 1:'''Simplified schema of our cell system including labels and markers for the chemical reactions in it. Marked reactions are considered in our system modeling.]]<br />
<br />
The schema shows the triggered expression of mircoviridin (our inhibitor), the protease (that needs to be inhibited for medical reasons) and the β-lactamase (that protects the cell from an antibiotic). The protease cleaves at a specific recognition site in the linker peptide. This will abolish the β-lactamase export and in consequnece the cell will die. <br />
<br><br />
There are three important trigger activation times:<br> <br />
* (t0) - start (microviridin added already)<br> <br />
* t1 - start expression of protease<br> <br />
* t2 - start expression of β-lactamase<br> <br />
* t3 - Ampicillin added into medium<br> <br />
* (t4) - end of the experiment: cell cultures survive or die.<br><br />
Keeping these times in mind, the reactions can be written down in seven chemical reaction equations of different sort and order.<br />
<br><br />
[[File:UP_reaction_equations.png|center|700px|thumb|'''Figure 2:''' Reaction equations between relevant molecules in the microviridin-inhibitor-concept including indications about the triggered time period (t1,t2,t3). The Romanic numbers correspond to the reactions marked in the above schema.]]<br />
<br />
=== Concentration equations ===<br />
<br />
From the above reaction equations differential equations can be derived that describe the change of substance concentrations. Three concentrations are fixed, however:<br />
<br><br />
[[File: UP_given_concentrations.png|center|550px|thumb|'''Figure 3:''' Given substance concentrations for differential equations. Those concentrations are added to the system and will not increase over time.]]<br />
<br />
<br />
All other concentrations can be represented in form of differential equations. Between time t1 and t2 four factors are introduced: <br><br />
* k+1 in (1/s*molecules)- factor for association of MdnA (microviridin) and Prot (protease)<br><br />
* k-1 in (1/s) - factor for dissociation of the inhibited protease<br><br />
* kexpr.prot in (molecules/s) - factor for the expression of protease<br><br />
* kdeg1 in (1/s) -factor for degradation of protease<br />
<br><br />
[[File: UP_ode_t1onwards.png|center|550px|thumb|'''Figure 4:''' Differential equations for substance concentrations from t1 until t2. At t1 the expression of protease begins.]]<br />
<br />
<br />
Between time t2 and t3 six additional factors are introduced: <br><br />
* k+2 in (1/s*molecules)- factor for association of Prot (protease) and TorABla (β-lactamase)<br><br />
* k-2 in (1/s) - factor for dissociation of protease and substrate<br><br />
* kcat in (1/s) – factor for the catalytic enzyme reaction that cleaves the single TorA sequence from the β-lactamase inactivating the export<br><br />
* kexpr.Tor in (molecules/s) - factor for the expression of β-lactamase<br><br />
* kdeg2 in (1/s) – factor for degradation of β-lactamase<br><br />
* ktransTor (1/s) – factor by which β-lactamase in the cytoplasm is able to pass the membrane and get into the periplasm<br />
<br><br />
[[File: UP_ode_t2onwards.png|center|550px|thumb|'''Figure 5:''' Differential equations for substance concentrations from t1 until t2. At t2 the expression of lactamase begins.]]<br />
<br />
<br />
After t3 there are only two more factors that we should introduce: <br><br />
* ktransAmp (1/s) – factor by which the added Ampicillin in the medium is able to pass the outer membrane and get into the periplasm<br><br />
* kcat2 in (1/s*molecules) – factor for the catalytic enzyme reaction that cleaves the single TorA sequence from the β-lactamase <br />
<br><br />
[[File: UP_ode_t3onwards.png|center|700px|thumb|'''Figure 6:''' Differential equations for substance concentrations from t1 until t2. At t1 ampicillin is added.]]<br />
<br />
=== Results ===<br />
<br />
The equations above were solved using MATLAB. WARNING: These equations are moderately very stiff! A solution can only be obtained using the functions ''ode23t'' or ''ode23s''!<br><br />
Using the Avogadro constant and the volume of a ''E.coli'' cell (10^-15L) and a periplasm-volume of about 5%, following constants were calculated and compared with similar literature values:<br><br />
k+1 = 1.e-6 (1/molecules*s)<br><br />
k-1 = 2.e-4 (1/s)<br><br />
kexpr.Prot = 1 (molecules/s)<br><br />
kdegr1 = 3.e-3 (1/s)<br><br />
k+2 = 5.e-7 (1/molecules*s)<br><br />
k-2 = 4.e-4 (1/s)<br><br />
kcat = 8 (1/s)<br><br />
kexpr.Tor = 1.4 (1/s)<br><br />
kdegr2 = 3.e-3 (1/s)<br><br />
ktransTor = 2.e-3 (1/s)<br><br />
ktransAmp = 3.e-3 (1/s)<br><br />
Kcat2 = 0.3 (1/molecules*s)<br><br />
MIC(0) = 10000 (molecules)<br><br />
AMP(0) = 2.e+6 (molecules)<br />
<br><br />
[[File: UP_odet_solution_all_times.png|center|700px|thumb|'''Figure 7a:''' MATLAB numerical solution to our system of differential equations for substance concentrations (above) over all time segments.]]<br />
<br />
[[File: UP_odet_solution_all_times_without_mdnA.png|center|700px|thumb|'''Figure 7b:''' MATLAB numerical solution to our system of differential equations for substance concentrations under absense of Microviridin.]]<br />
<br />
[[File: UP_odet_solution_all_times_without_protease.png|center|700px|thumb|'''Figure 7c:''' MATLAB numerical solution to our system of differential equations for substance concentrations under absense of Protease.]]<br />
<br />
<br />
It can be seen that there is only a negligible amount of Ampicillin in the periplasm if the expression works as indicated. This amount of Ampicillin has only a smal effect on the growth rate of the cells. There is also a wide tolerance left for suboptimal conditions. The trigger time t1, t2 and t3 shall be about one hour after one another.<br />
<br><br />
In the first section the effect of the inhibition can clearly be seen: Even though the continuous expression of protease, only a very small number remains active in the cytoplasm. In the second section β-lactamase is expressed. Most of the β-lactamase in the cytoplasm is destroyed by the protease, however the number is by far large enough to steadily release molecules into the periplasm where it cannot be affected by the protease any more. Because the volume of the periplasm is very small, the concentration there is even higher than the absolute amount of molecules in the graph suggests compared to the amount of molecules in the cytoplasm. The third section shows that the concentrations remain very steady and the cells are Ampicillin resistant.<br />
<br><br />
Cells passing this procedure are very Ampicillin resistant and grow fast: They double their cell volume about every 20 minutes. If an error would appear and the Ampicillin concentration inside the periplasma would increase over 2µg/ml, the growth rate would slow down drastically and the cells might die. This way defect cell cultures can easily be separated from the good ones.<br />
<br><br />
The ratio of Ampicillin concentration in ''n µg/ml'' to the growth rate (cells double during this timespan) is approximately this: ''T(growth) = 20min+10*2^(n-1)''.<br />
<br><br />
[[File: UP_celldivision.png|center|600px|thumb|'''Figure 8:''' Starting at t3: Growth (double) rate of cells dependent on the ampicillin concentration in the periplasm. Because of the small amount of ampicillin the growth rate in our assumed system is noraml (25min/double cells) and stays constant over time.]]<br />
<br><br />
One example of how change of a simulation constant impacts the growth of the cells:<br />
<br><br />
[[File: UP_celldivision_combi1.png|center|700px|thumb|'''Figure 9:''' Starting at t3: Growth (double) rate of cells dependent on the ampicillin concentration in the periplasm. Big change of one simulation factor can result in less fitness of the cell and a reduction of its growth rate. In this case the factor of ampicillin transmission through the membrane into the periplasm was changed. If it is much too high, the cells do not survive anymore.]]<br />
<br><br />
[[File: UP_3Dplot_Lact_06.png|center|600px|thumb|'''Figure 11:''' Starting at t3: Lactamase concentration inside the periplasm. A significant change of the simulation factor K_D (Dissociation of microviridin and protease divided by the association) can result in a lower lactamase concentration and thus less fitness of the cell (because the lactamase protects the cell from the antibiotic ampicillin).]]<br />
<br><br />
[[File: UP_3Dplot_cells_06.png|center|600px|thumb|'''Figure 10:''' Starting at t3: Growth (double) rate of cells dependent on the ampicillin concentration in the periplasm. A significant change of the simulation factor K_D (Dissociation of microviridin and protease divided by the association) can result in less fitness of the cell and a reduction of its growth rate.]]<br />
<br />
=== Parameter estimation ===<br />
<br />
To be filled...<br />
<br />
=== MATLAB code ===<br />
<br />
(update uploaded soon)<br><br />
[[Media:conzt1.m]]<br><br />
[[Media:conzt2.m]]<br><br />
[[Media:conzt3.m]]<br><br />
[[Media:UP_plottfuntionendreiaufeinmal2.m]]<br><br />
[[Media:UP_celldivision2.m]]</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_ModelingTeam:Potsdam Bioware/Project/Details Modeling2011-10-28T22:37:50Z<p>UP Stefan: /* Model */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
<br />
<br />
<br />
== Modeling ==<br />
<br />
There is no synthetic biology without modeling, of course. In principle there is structure modeling and system modeling. In structure modeling the conformation and structure of proteins is examined and steric consequences for reactions or the whole system can be estimated. We focused on the second sort of modeling: The system modeling in which the reaction kinetics of the whole system is analyzed, outcomes are predicted and parameters correlated to measurements. Thus, a synthetic biology approach can be chosen because a better understanding of the system is achieved and further changes can be planed - just like in engineering.<br />
<br />
=== Model ===<br />
<br />
We built a model of our <i>in vivo</i> selection system to be able to engineer our system effectively. The following schema shows the major reactions taking place in our cell system. The Romanic numbers indicate the system relevant (partially triggered) reactions that were written down as equations and then numerically propagated through time.<br />
<br> <br />
[[File:UP_schema_modeling2v.png|center|650px|thumb|'''Figure 1:'''Simplified schema of our cell system including labels and markers for the chemical reactions in it. Marked reactions are considered in our system modeling.]]<br />
<br />
The schema shows the triggered expression of mircoviridin (our inhibitor), the protease (that needs to be inhibited for medical reasons) and the β-lactamase (that protects the cell from an antibiotic). The protease cleaves at a specific recognition site in the linker peptide. This will abolish the β-lactamase export and in consequnece the cell will die. <br />
<br><br />
There are three important trigger activation times:<br> <br />
* (t0) - start (microviridin added already)<br> <br />
* t1 - start expression of protease<br> <br />
* t2 - start expression of β-lactamase<br> <br />
* t3 - Ampicillin added into medium<br> <br />
* (t4) - end of the experiment: cell cultures survive or die.<br><br />
Keeping these times in mind, the reactions can be written down in seven chemical reaction equations of different sort and order.<br />
<br><br />
[[File:UP_reaction_equations.png|center|700px|thumb|'''Figure 2:''' Reaction equations between relevant molecules in the microviridin-inhibitor-concept including indications about the triggered time period (t1,t2,t3). The Romanic numbers correspond to the reactions marked in the above schema.]]<br />
<br />
=== Concentration equations ===<br />
<br />
From the above reaction equations differential equations can be derived that describe the change of substance concentrations. Three concentrations are fixed, however:<br />
<br><br />
[[File: UP_given_concentrations.png|center|550px|thumb|'''Figure 3:''' Given substance concentrations for differential equations. Those concentrations are added to the system and will not increase over time.]]<br />
<br />
<br />
All other concentrations can be represented in form of differential equations. Between time t1 and t2 four factors are introduced: <br><br />
* k+1 in (1/s*molecules)- factor for association of MdnA (microviridin) and Prot (protease)<br><br />
* k-1 in (1/s) - factor for dissociation of the inhibited protease<br><br />
* kexpr.prot in (molecules/s) - factor for the expression of protease<br><br />
* kdeg1 in (1/s) -factor for degradation of protease<br />
<br><br />
[[File: UP_ode_t1onwards.png|center|550px|thumb|'''Figure 4:''' Differential equations for substance concentrations from t1 until t2. At t1 the expression of protease begins.]]<br />
<br />
<br />
Between time t2 and t3 six additional factors are introduced: <br><br />
* k+2 in (1/s*molecules)- factor for association of Prot (protease) and TorABla (β-lactamase)<br><br />
* k-2 in (1/s) - factor for dissociation of protease and substrate<br><br />
* kcat in (1/s) – factor for the catalytic enzyme reaction that cleaves the single TorA sequence from the β-lactamase inactivating the export<br><br />
* kexpr.Tor in (molecules/s) - factor for the expression of β-lactamase<br><br />
* kdeg2 in (1/s) – factor for degradation of β-lactamase<br><br />
* ktransTor (1/s) – factor by which β-lactamase in the cytoplasm is able to pass the membrane and get into the periplasm<br />
<br><br />
[[File: UP_ode_t2onwards.png|center|550px|thumb|'''Figure 5:''' Differential equations for substance concentrations from t1 until t2. At t2 the expression of lactamase begins.]]<br />
<br />
<br />
After t3 there are only two more factors that we should introduce: <br><br />
* ktransAmp (1/s) – factor by which the added Ampicillin in the medium is able to pass the outer membrane and get into the periplasm<br><br />
* kcat2 in (1/s*molecules) – factor for the catalytic enzyme reaction that cleaves the single TorA sequence from the β-lactamase <br />
<br><br />
[[File: UP_ode_t3onwards.png|center|700px|thumb|'''Figure 6:''' Differential equations for substance concentrations from t1 until t2. At t1 ampicillin is added.]]<br />
<br />
=== Results ===<br />
<br />
The equations above were solved using MATLAB. WARNING: These equations are moderately to very stiff! A solution an only be obtained using the functions ''ode23t'' or ''ode23s''!<br><br />
Using the Avogadro constant and the volume of a ''E.coli'' cell (10^-15L) and a periplasm-volume of about 5%, following constants were calculated and compared with similar literature values:<br><br />
k+1 = 1.e-6 (1/molecules*s)<br><br />
k-1 = 2.e-4 (1/s)<br><br />
kexpr.Prot = 1 (molecules/s)<br><br />
kdegr1 = 3.e-3 (1/s)<br><br />
k+2 = 5.e-7 (1/molecules*s)<br><br />
k-2 = 4.e-4 (1/s)<br><br />
kcat = 8 (1/s)<br><br />
kexpr.Tor = 1.4 (1/s)<br><br />
kdegr2 = 3.e-3 (1/s)<br><br />
ktransTor = 2.e-3 (1/s)<br><br />
ktransAmp = 3.e-3 (1/s)<br><br />
Kcat2 = 0.3 (1/molecules*s)<br><br />
MIC(0) = 10000 (molecules)<br><br />
AMP(0) = 2.e+6 (molecules)<br />
<br><br />
[[File: UP_odet_solution_all_times.png|center|700px|thumb|'''Figure 7a:''' MATLAB numerical solution to our system of differential equations for substance concentrations (above) over all time segments.]]<br />
<br />
[[File: UP_odet_solution_all_times_without_mdnA.png|center|700px|thumb|'''Figure 7b:''' MATLAB numerical solution to our system of differential equations for substance concentrations under absense of Microviridin.]]<br />
<br />
[[File: UP_odet_solution_all_times_without_protease.png|center|700px|thumb|'''Figure 7c:''' MATLAB numerical solution to our system of differential equations for substance concentrations under absense of Protease.]]<br />
<br />
<br />
It can be seen that there is only a negligible amount of Ampicillin in the periplasm if the expression works as indicated. This amount of Ampicillin has only a smal effect on the growth rate of the cells. There is also a wide tolerance left for suboptimal conditions. The trigger time t1, t2 and t3 shall be about one hour after one another.<br />
<br><br />
In the first section the effect of the inhibition can clearly be seen: Even though the continuous expression of protease, only a very small number remains active in the cytoplasm. In the second section β-lactamase is expressed. Most of the β-lactamase in the cytoplasm is destroyed by the protease, however the number is by far large enough to steadily release molecules into the periplasm where it cannot be affected by the protease any more. Because the volume of the periplasm is very small, the concentration there is even higher than the absolute amount of molecules in the graph suggests compared to the amount of molecules in the cytoplasm. The third section shows that the concentrations remain very steady and the cells are Ampicillin resistant.<br />
<br><br />
Cells passing this procedure are very Ampicillin resistant and grow fast: They double their cell volume about every 20 minutes. If an error would appear and the Ampicillin concentration inside the periplasma would increase over 2µg/ml, the growth rate would slow down drastically and the cells might die. This way defect cell cultures can easily be separated from the good ones.<br />
<br><br />
The ratio of Ampicillin concentration in ''n µg/ml'' to the growth rate (cells double during this timespan) is approximately this: ''T(growth) = 20min+10*2^(n-1)''.<br />
<br><br />
[[File: UP_celldivision.png|center|600px|thumb|'''Figure 8:''' Starting at t3: Growth (double) rate of cells dependent on the ampicillin concentration in the periplasm. Because of the small amount of ampicillin the growth rate in our assumed system is noraml (25min/double cells) and stays constant over time.]]<br />
<br><br />
One example of how change of a simulation constant impacts the growth of the cells:<br />
<br><br />
[[File: UP_celldivision_combi1.png|center|700px|thumb|'''Figure 9:''' Starting at t3: Growth (double) rate of cells dependent on the ampicillin concentration in the periplasm. Big change of one simulation factor can result in less fitness of the cell and a reduction of its growth rate. In this case the factor of ampicillin transmission through the membrane into the periplasm was changed. If it is much too high, the cells do not survive anymore.]]<br />
<br><br />
[[File: UP_3Dplot_Lact_06.png|center|600px|thumb|'''Figure 11:''' Starting at t3: Lactamase concentration inside the periplasm. A significant change of the simulation factor K_D (Dissociation of microviridin and protease divided by the association) can result in a lower lactamase concentration and thus less fitness of the cell (because the lactamase protects the cell from the antibiotic ampicillin).]]<br />
<br><br />
[[File: UP_3Dplot_cells_06.png|center|600px|thumb|'''Figure 10:''' Starting at t3: Growth (double) rate of cells dependent on the ampicillin concentration in the periplasm. A significant change of the simulation factor K_D (Dissociation of microviridin and protease divided by the association) can result in less fitness of the cell and a reduction of its growth rate.]]<br />
<br />
=== Parameter estimation ===<br />
<br />
To be filled...<br />
<br />
=== MATLAB code ===<br />
<br />
(update uploaded soon)<br><br />
[[Media:conzt1.m]]<br><br />
[[Media:conzt2.m]]<br><br />
[[Media:conzt3.m]]<br><br />
[[Media:UP_plottfuntionendreiaufeinmal2.m]]<br><br />
[[Media:UP_celldivision2.m]]</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_SelectionTeam:Potsdam Bioware/Project/Details Selection2011-10-28T22:33:01Z<p>UP Stefan: /* Testing the "Kill Switch" by activating the protease */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==<i>In Vivo</i> Selection==<br />
===Introduction===<br />
In addition to the phage display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an <i>in vitro</i> screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for any protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator. For the protease activity detector we modified the TAT-hitchhiker system developed by DeLisa <i>et al.</i> <br />
We used the BioBrick <partinfo>I757010</partinfo> (β-lactamase) as well as <partinfo>K208005</partinfo> (ssTorA) and fused them together via a linker peptide. For the protease generator we cloned the arabinose induction system in front of a protease in iGEM standard.<br> <br />
<br />
[[File:UP_images_TorA-construct-design2_28.10.2011.png.jpg|center|500px|thumb|'''Figure 1:''' Schema of our constructed selection vector containing protease generator and protease activity detector]]<br> <br />
The system works in a combined manner of the two devices. In order to confer β-lactam antibiotic resistance to cells the β-lactamase has to be exported in the periplasm. This transport is mediated by the TorA export sequence via the Twin-Arginine Translocation (TAT) system (DeLisa, 2009). The linker peptide between the TorA export sequence and the β-lactamase displays the corresponding protease cleavage site. In addition the linker peptide is chosen as short as possible to imitate folding because the TAT pathway only allows transport of correctly folded substrates. The protease as well as the linker peptide are designed as exchangeable parts.<br><br />
<br />
If the protease device is functional, it will cleave the linker peptide between TorA and the β-lactamase construct which leaves the cell without any antibiotic resistance. The construct was tested with increasing ampicillin concentrations. The number of surviving colonies was depended on the export rate of the β-lactamase into the periplasm. A high cleavage rate of the linker peptide leads to a reduced ampicillin resistance. With expressed protease a dramatically drop of the number of surviving colonies could be observed.<br><br />
The survival assay was carried out with and without expressed protease. By increasing of the ampicillin concentration we could detect a cutoff ampicillin concentration. The colony forming units (CFU) were counted and compared. To detected whether our constructed library is able to block the protease we co-transformed the library into <i>E. coli</i> carrying our pUP_SG1 plasmid.<br><br />
<br />
===Site-directed mutagenesis of Tobacco Etch Virus and 14_3C proteases in iGEM standard===<br />
<br />
We chose two model proteases, the Tobacco Etch Virus protease, further along called TEV, and the 14_3C protease from the human rhinovirus, also known as PreScission. Both proteases contained iGEM restriction sites, one in case of the TEV and three for 14 3C, respectively. <br><br />
[[File:UP_Pic_SG_Pre-Complete.png|center|800px|thumb|'''Figure 2:''' Complete sequence alignment of the 14_3C protease]]<br />
[[File:UP_Pic_SG_TEV-Complete.png|center|800px|thumb|'''Figure 3:''' Complete sequence alignment of the 14_3C protease]]<br />
<br><br><br />
Thus, site-directed mutagenesis was applied to mutate each restriction site in E. coli. For every mutation a forward and a reverse primer had to be designed. The following pictures show the forward primers for the side directed mutagenesis, the reverse primers are the reverse complement sequence of the forward primers.<br><br />
<br />
For the 14_3C protease:<br />
[[File:UP_Pic_SG_Pre-End.png|left|400px|thumb|'''Figure 4:''' Reverse primer to add the iGEM suffix on the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaSpeI.png|right|400px|thumb|'''Figure 5:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-1.png|left|400px|thumb|'''Figure 6:''' Forward primer to mutate the first XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-2.png|right|400px|thumb|'''Figure 7:''' Forward primer to mutate the second XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_Pre-Start.png|left|400px|thumb|'''Figure 8:''' Forward primer to add the iGEM prefix to the HRV 14_3C protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
For the TEV protease:<br />
<br />
[[File:UP_Pic_SG_TEV-End.png|left|400px|thumb|'''Figure 9:''' Reverse primer to add the iGEM suffix to the TEV protease]]<br />
[[File:UP_Pic_SG_TEVMutaSpeI.png|right|400px|thumb|'''Figure 10:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the TEV protease]]<br />
[[File:UP_Pic_SG_TEV-Start.png|left|400px|thumb|'''Figure 11:''' Forward primer to add the iGEM prefix to the TEV protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
We used assembly PCR to gain the final product. In case of 14_3C two assembly PCRs had to be done. The expected size of the fragments are shown in the gel picture. In the following picture all fragments contain the correct sizes.<br />
<br><br />
[[File:UP_Fragments_14_3C_TEV.png|center|200px|thumb|'''Figure 12:''' Picture of the, via PCR amplified and mutated Fragment of both protease]]<br />
<br><br />
<br />
===Construction of two component for the ''in vivo'' selection assay===<br />
<br />
The ''in vivo'' assay is based on two devices. On the one hand the protease activity detector device, on the other a protease generator device. The detector device is based on the enzyme β-lactamase, which is only active inside the periplasm and provides the resistance against lactam antibiotics like ampicillin and penicillin. For the protease generator device we needed a second induction system on our plasmid, which is quite uncommon for any plasmid. So several problems had to solved to set up an effective ''in vivo'' selection assay:<br />
*a second, very tight induction system has to be found<br />
*a way to control the export rate of β-lactamase into the periplasm, which can be reduced by the protease<br />
<br />
We solved these problems by putting the protease detector device under the control of the lac-operon. The protease device is induced by the arabinose system of the pBAD_iGEMexpress vector. The idea to control the survival of the cells via the amount of exported protein is inspired by the Hitchhiker assay of DeLisa ''et al''. Using the TAT pathway for the export of β-lactamase into the periplasm allows us to introduce a protease specific cleavage site in front of the signal sequence regulating the export.<br />
<br />
====Introducing the specific protease cleavage site====<br />
To adapt the protease activity detector device to any protease we want to screen, we had to modularize it. Our detector device was set up on a vector for the Hitchhiker selection. The plasmid offers two unique restriction sites on the plasmid, after the signal sequence of TorA one restriction site for <i>Xho</i>I and a <i>Nhe</i>I restriction site in front of the β-lactamase. Both were used to introduce the specific protease cleavage site in between the TorA signal sequence and the β-lactamase. By designing two complementary oligonucleotides containing the specific protease cleavage site flanked by two short linker regions. After the digest of the vector with <i>Xho</i>I and <i>Nhe</i>I the hybridized oligonucleotides, containing the overhangs for <i>Xho</i>I and <i>Nhe</i>I can be ligated into the vector.<br />
<br />
====Adding the arabinose inducible induction system to control the proteases, TEV and 14_3C====<br />
We needed a time independent induction of the protease device. Therefore we cloned the arabionse induction system (<i>araC</i>) in front of the protease device out of the pBAD_iGEMexpress vector. The fusion of gene construct and induction system was always performed via an <i>NgoM</i>IV restriction site. This restriction site was introduced with our amplification primers.<br />
[[File:UP_Pic_SG_AraC-End.png|center|800px|thumb|'''Figure 13:''' Reverse primer for the amplification of the arabinose inducible induction system]]<br />
[[File:UP_Pic_SG_AraC-Start.png|center|1400px|thumb|'''Figure 14:''' Forward primer for the amplification of the arabinose inducible induction system]]<br />
We used the RFC 23 cloning strategy to get a fusion protein of our desired protease and the induction system, which allows us to induce the protease. Both genes were amplified with PCR using two special primers generating a <i>NgoM</i>IV restrictioin site at the end of the induction system and in front of the protease. The amplified protease were digested with <i>BamH</i>I and <i>NgoM</i>IV, the arabinose inducible induction system with <i>NgoM</i>IV and <i>Hind</i>III. The vector, which contains the ssTorA_CS-Protease_β-lactamase device was digested with <i>Hind</i>III and <i>BamH</i>I. A triple ligation yielded the final construct with both devices: the protease detector and the protease generator. The two pictures below show the final vector constructs , which where used for the survival screening.<br />
[[File:UP_Pic_SG_TEV_SG1-Plasmid.png|center|800px|thumb|'''Figure 15:''' Final vector construct containing arabinose inducible TEV protease fusion and the protease detector device]]<br />
[[File:UP_Pic_SG_PreSciccion_SG2-Plasmid.png|center|800px|thumb|'''Figure 16:''' Final vector construct containing arabinose inducible HRV 14_3C protease fusion and the protease detector device]]<br />
<br />
===Investigation of the ''in vivo'' selection assay===<br />
<p><br />
<br />
Two major points have to be demonstrated: First, the export of β-lactamase over the TAT pathway has to be efficient enough to provide a certain resistance against ampicillin and second, the activated protease is able to intimately reduce the resistance against ampicillin by cleavage of the TorA signal sequence from the β-lactamase.</p><br />
====Testing the export efficiency of the protease activity detector====<br />
To test the export rate of the β-lactamse of the protease activity detector device we grew an <i>E. coli</i> culture up to an OD<sub>600</sub> of 0.6. The cells were then diluted to an OD of 0.002 and 100 µL were plated in triplicates with increasing ampicillin concentrations. The graph below shows the amount of surviving cells in percentage. <br><br />
<br />
[[Image:UP_Survial-screen_bla-construct.png|center|600px|thumb|'''Figure 17:''' Amount of survived cells. The construct was induced with 1 mM IPTG. The rate of surviving cells without IPTG and without ampicilin was set to 100%. ]]<br />
<br />
[[Image:UP_TEV-Survival.png|center|600px|thumb|'''Figure 18:''' Survival test at different ampicillin concentrations: A) 1000 cfu plated on agar plates containing chloramphenicol; B) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 400µg/mL ampicillin; C) no culture growth of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 600µg/mL ampicillin]]<br />
[[Image:UP_14_3C_SurvTest.png|center|600px|thumb|'''Figure 19:''' Survival test at different ampicillin concentrations: <br />
A) 1000 cfu plated on agar plates containing chloramphenicol; <br />
B) Influence of resistance induction with 1 mM IPTG, 1000 cfu where plated on agar plates containing chloramphenicol and 1mM IPTG; C) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 50 µg/mL ampicillin; D) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 100 µg/mL ampicillin; E) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 200 µg/mL ampicillin; F) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 400 µg/mL ampicillin; G) No culture growth of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 800 µg/mL ampicillin]]<br />
<br />
This assay was performed at two temperatures. At 37°C, the optimal growth temperature and at 30°C for best protein expression conditions. All assays show the same results. The cells were able to grow at ampicilin concentrations up to 400 µg/ml. Cells transformed with the final construct containing the detection device and the protease generator device were able to survive up to 800 µg/ml ampicilin (incubated at 30°C over 2 days).<br />
<br />
This leads to the conclusion, that the export of β-lactamase into the periplasm is very effective. It is also important to mention, that there is no cell survival when ampicilin is added to the media without the induction of the protease activity detection device by IPTG.<br />
<br />
====Testing the "Kill Switch" by activating the protease====<br />
<br />
The second major point which needs to be demonstrated is that we are able to kill the ''E.coli'' cells by activating the protease. This is referred as the "Kill Switch". In the final assay we want to have the ability to induce the protease activity detector device and the protease generate independently from each other. But first of all we have to check if our system is working and if we are able to kill the cells by activating the protease.<br><br />
To proof the principle we used the natural TEV protease and make a co-transformation with the protease activity detector device containing the cleavage site for the TEV protease. Both were induced by IPTG and so we can't control the expression rate neither the time of the induction of the protease generator device.<br><br />
After the incubation at 37°C we were able to show the desired proof of principle: when we activate the protease the resistance against ampicillin; based on the export rate of β-lactamase into the periplasm via the TAT pathway, was intimately reduced. The cells with activated TEV protease were able to survive up to concentration of 50 µg/mL ampicillin.<br><br />
[[Image:UP_bla-TEV_double-trans.png|center|500px|thumb|'''Figure 20:''' Percentage of surviving cells on different ampicillin concentration. Blue - cells transformed with the single protease activity detector device for TEV protease; Red - cell transformed with both, the protease activity detector device for TEV protease and the TEV protease itself]]<br><br />
Based on the ampicillin resistance of the plasmid of HRV14_3C protease, we were unable to show the proof of principle for this protease.<br><br />
The next step was to use the complete test vector which contains the protease activity detector device and the protease generator device. We introduced the arabinose inducible induction system fused to the protease flanked by the restriction sites of <i>BamH</i>I and <i>Hind</i>III into the vector of the modified Hitchhiker selection system.<br><br />
With these final vectors we had to repeat the survival screening to show that the system is also working when there is a dual induction based on only one plasmid. The cells were grown to an OD<sub>600</sub> of 0.6 and diluted to an OD<sub>600</sub> of 0.002. 100 µL of this dilution were plated on agar plates with different ampicillin concentrations and induced and induced protease. The results are shown in figure 20.<br><br />
[[Image:UP_SurvTEV_onepla.jpg|center|500px|thumb|'''Figure 21:''' Cell growth on different ampicillin concentration. Blue - cells without induction of the TEV protease; Red - cells with induced TEV protease]]<br><br />
<br><br />
The survival screening assay for TEV protease was performed 30 °C and all plates were incubated for 2 days. The ''E.coli'' XL1 blue cells were able to survive up to ampicilin concentration of 800 µg/mL, which is an increase of 100% of the original resistance capacity. When 2% arabinose is added to the media, the cells showed an intimately reduce surviving rate. The cells were able to survive at ampicilin concentrations up 25 µg/mL.<br><br />
This shows, that we got a high dynamic range from 50 up to 800 µg/mL ampicilin to screen for inhibition of the protease.<br />
<br />
===Transferring and testing the library===<br />
After the successful test of the protease activity we transferred the generated <i>mdnA</i> library into our cells. The following pictures show the amazing results. Cell growth could be detected up to 400 µg Ampicillin/mL.<br />
[[Image:UP_2011-06-01_CM-Kan-Amp_labeled02.jpg|left|420px|thumb|'''Figure 22:''' Cell growth on Ampicillin without induced protease]]<br />
[[Image:UP_2011-06-01_CM-Kan-Ara-Amp_labeled01.jpg|right|420px|thumb|'''Figure 23:''' Cell growth on Ampicillin with induced protease]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
===Background===<br />
====Twin Arginine Translocon (TAT)====<br />
The TAT pathway is responsible for the transport of folded proteins across energy-transducing membranes and it is able of discriminating of unfolded proteins. This transport pathway is common for bacteria, archea and plants and translocates circa 6% of <i>E.coli</i> produced secreted proteins. The translocated proteins must have a signal peptide for targeting of the TAT-transporter, such proteins are e.g. hydrogenases, dehydrogenases and reductases. The signal sequence itself is composed of an N-terminal positive charged domain, a hydrophobic domain and a C-terminal domain.<br><br />
The translocon is composed of three parts: TatA, TatB and TatC. The transport-pore is proposed to be formed during substrate binding by these three parts. The TatA, TatB and TatC proteins may form complexes of different sizes, which on the other hand form pores matching the size of the folded substrate. The transport of proteins through the TAT pathway depends on the proton motive force. Calculations by Adler & Theg showed that the transport of one folded substrate molecule requires the release of approximately 7.9x10<sup>4</sup> protons, which equals 10.000 ATP molecules. The signal sequence is cleaved off after translocation.<br />
<br><br />
[[File:UP_TorA_TATSeq2.jpg|center|450px|thumb|'''Figure 24:''' Schematic representation of TorA-signal sequence. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br><br />
The used signal sequence originates from the TorA protein (Trimethylamin-N-Oxid-Reductase). It is the main respiratory enzyme which reduces TMAO under anaerobic conditions in the periplasm, where it is transported by the TAT pathway.<br />
<br> <br />
[[File:UP_TAT_Pathway2.jpg|center|450px|thumb|'''Figure 25:''' Schematic representation of TAT-dependent pathway. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br />
<br />
<br />
<br><br />
<br />
====Tobacco Etch Virus (TEV) protease====<br />
TEV protease is the common name for the 27 kDa catalytic domain of the Nuclear Inclusion a endopeptidase (NIa) encoded by the tobacco etch virus. TEV protease is a useful reagent for cleaving fusion proteins. It recognizes a linear epitope of the general form E-Xaa-Xaa-Y -Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. In TEV protease the serine nucleophile of the conventional Ser-Asp-His triad is a cysteine instead. This probably explains why TEV protease is resistant to many commonly used protease inhibitors.<br><br />
<br />
====14_3C-Protease====<br />
<br />
The 14_3C protease originates from the human rhinovirus. Rhinoviruses are the most frequent reason for infections of the upper and lower respiratory tract, also known as the cold. Because the 3C protease of human rhinovirus is necessary for the cleavage of the polyprotein translated from viral RNA it may serve as a potential target for development of antiviral targets. The recombinant type 14_3C protease from human rhinovirus (HRV 3C) recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln↓GlyPro. The small, 22-kDa size of the protease got its optimal activity at 4°C but is still very active at 37 °C. It is commonly used for an easy tag removal after the purification of recombinant proteins carrying his-tag. The 14_3C works with a catalytic triade, containing the amino acid residues Ser-Asp-His at its active site.<br><br />
<br />
===References===<br />
*Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. and Bottomley, S.P. (2007) Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-2367<br><br />
*Genest O., Ilbert M., Méjean V., Iobbi-Nivol C.,(2005) TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature. J. Biol. Chem. 280: 15644-15648.<br />
*Kapust R. B., Tözsér J., Fox J. D., Anderson D. E., Cherry S., Copeland T. D., Waugh D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 12:993-1000.<br><br />
*Lee P. A., Tullman-Ercekand D., Georgiou G., (2006) The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60:373–95<br />
*Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30:544-550.<br><br />
*Shih S. R., Chen S. J., Hakimelahi G. H., Liu H. J., Tseng C. T., Shia K. S., (2004) Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting molecules. Med Res Rev 24(4):449-74.<br><br />
*Wanga QM, Chen SH., (2007) Human rhinovirus 3C protease as a potential target for the development of antiviral agents. Curr Protein Pept Sci. 8(1):19-27. <br><br />
*Waraho D., DeLisa M. P., (2009). Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism. PNAS 106(10):3692-7</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_SelectionTeam:Potsdam Bioware/Project/Details Selection2011-10-28T22:31:35Z<p>UP Stefan: /* Testing the export efficiency of the protease activity detector */</p>
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<br />
==<i>In Vivo</i> Selection==<br />
===Introduction===<br />
In addition to the phage display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an <i>in vitro</i> screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for any protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator. For the protease activity detector we modified the TAT-hitchhiker system developed by DeLisa <i>et al.</i> <br />
We used the BioBrick <partinfo>I757010</partinfo> (β-lactamase) as well as <partinfo>K208005</partinfo> (ssTorA) and fused them together via a linker peptide. For the protease generator we cloned the arabinose induction system in front of a protease in iGEM standard.<br> <br />
<br />
[[File:UP_images_TorA-construct-design2_28.10.2011.png.jpg|center|500px|thumb|'''Figure 1:''' Schema of our constructed selection vector containing protease generator and protease activity detector]]<br> <br />
The system works in a combined manner of the two devices. In order to confer β-lactam antibiotic resistance to cells the β-lactamase has to be exported in the periplasm. This transport is mediated by the TorA export sequence via the Twin-Arginine Translocation (TAT) system (DeLisa, 2009). The linker peptide between the TorA export sequence and the β-lactamase displays the corresponding protease cleavage site. In addition the linker peptide is chosen as short as possible to imitate folding because the TAT pathway only allows transport of correctly folded substrates. The protease as well as the linker peptide are designed as exchangeable parts.<br><br />
<br />
If the protease device is functional, it will cleave the linker peptide between TorA and the β-lactamase construct which leaves the cell without any antibiotic resistance. The construct was tested with increasing ampicillin concentrations. The number of surviving colonies was depended on the export rate of the β-lactamase into the periplasm. A high cleavage rate of the linker peptide leads to a reduced ampicillin resistance. With expressed protease a dramatically drop of the number of surviving colonies could be observed.<br><br />
The survival assay was carried out with and without expressed protease. By increasing of the ampicillin concentration we could detect a cutoff ampicillin concentration. The colony forming units (CFU) were counted and compared. To detected whether our constructed library is able to block the protease we co-transformed the library into <i>E. coli</i> carrying our pUP_SG1 plasmid.<br><br />
<br />
===Site-directed mutagenesis of Tobacco Etch Virus and 14_3C proteases in iGEM standard===<br />
<br />
We chose two model proteases, the Tobacco Etch Virus protease, further along called TEV, and the 14_3C protease from the human rhinovirus, also known as PreScission. Both proteases contained iGEM restriction sites, one in case of the TEV and three for 14 3C, respectively. <br><br />
[[File:UP_Pic_SG_Pre-Complete.png|center|800px|thumb|'''Figure 2:''' Complete sequence alignment of the 14_3C protease]]<br />
[[File:UP_Pic_SG_TEV-Complete.png|center|800px|thumb|'''Figure 3:''' Complete sequence alignment of the 14_3C protease]]<br />
<br><br><br />
Thus, site-directed mutagenesis was applied to mutate each restriction site in E. coli. For every mutation a forward and a reverse primer had to be designed. The following pictures show the forward primers for the side directed mutagenesis, the reverse primers are the reverse complement sequence of the forward primers.<br><br />
<br />
For the 14_3C protease:<br />
[[File:UP_Pic_SG_Pre-End.png|left|400px|thumb|'''Figure 4:''' Reverse primer to add the iGEM suffix on the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaSpeI.png|right|400px|thumb|'''Figure 5:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-1.png|left|400px|thumb|'''Figure 6:''' Forward primer to mutate the first XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-2.png|right|400px|thumb|'''Figure 7:''' Forward primer to mutate the second XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_Pre-Start.png|left|400px|thumb|'''Figure 8:''' Forward primer to add the iGEM prefix to the HRV 14_3C protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
For the TEV protease:<br />
<br />
[[File:UP_Pic_SG_TEV-End.png|left|400px|thumb|'''Figure 9:''' Reverse primer to add the iGEM suffix to the TEV protease]]<br />
[[File:UP_Pic_SG_TEVMutaSpeI.png|right|400px|thumb|'''Figure 10:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the TEV protease]]<br />
[[File:UP_Pic_SG_TEV-Start.png|left|400px|thumb|'''Figure 11:''' Forward primer to add the iGEM prefix to the TEV protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
We used assembly PCR to gain the final product. In case of 14_3C two assembly PCRs had to be done. The expected size of the fragments are shown in the gel picture. In the following picture all fragments contain the correct sizes.<br />
<br><br />
[[File:UP_Fragments_14_3C_TEV.png|center|200px|thumb|'''Figure 12:''' Picture of the, via PCR amplified and mutated Fragment of both protease]]<br />
<br><br />
<br />
===Construction of two component for the ''in vivo'' selection assay===<br />
<br />
The ''in vivo'' assay is based on two devices. On the one hand the protease activity detector device, on the other a protease generator device. The detector device is based on the enzyme β-lactamase, which is only active inside the periplasm and provides the resistance against lactam antibiotics like ampicillin and penicillin. For the protease generator device we needed a second induction system on our plasmid, which is quite uncommon for any plasmid. So several problems had to solved to set up an effective ''in vivo'' selection assay:<br />
*a second, very tight induction system has to be found<br />
*a way to control the export rate of β-lactamase into the periplasm, which can be reduced by the protease<br />
<br />
We solved these problems by putting the protease detector device under the control of the lac-operon. The protease device is induced by the arabinose system of the pBAD_iGEMexpress vector. The idea to control the survival of the cells via the amount of exported protein is inspired by the Hitchhiker assay of DeLisa ''et al''. Using the TAT pathway for the export of β-lactamase into the periplasm allows us to introduce a protease specific cleavage site in front of the signal sequence regulating the export.<br />
<br />
====Introducing the specific protease cleavage site====<br />
To adapt the protease activity detector device to any protease we want to screen, we had to modularize it. Our detector device was set up on a vector for the Hitchhiker selection. The plasmid offers two unique restriction sites on the plasmid, after the signal sequence of TorA one restriction site for <i>Xho</i>I and a <i>Nhe</i>I restriction site in front of the β-lactamase. Both were used to introduce the specific protease cleavage site in between the TorA signal sequence and the β-lactamase. By designing two complementary oligonucleotides containing the specific protease cleavage site flanked by two short linker regions. After the digest of the vector with <i>Xho</i>I and <i>Nhe</i>I the hybridized oligonucleotides, containing the overhangs for <i>Xho</i>I and <i>Nhe</i>I can be ligated into the vector.<br />
<br />
====Adding the arabinose inducible induction system to control the proteases, TEV and 14_3C====<br />
We needed a time independent induction of the protease device. Therefore we cloned the arabionse induction system (<i>araC</i>) in front of the protease device out of the pBAD_iGEMexpress vector. The fusion of gene construct and induction system was always performed via an <i>NgoM</i>IV restriction site. This restriction site was introduced with our amplification primers.<br />
[[File:UP_Pic_SG_AraC-End.png|center|800px|thumb|'''Figure 13:''' Reverse primer for the amplification of the arabinose inducible induction system]]<br />
[[File:UP_Pic_SG_AraC-Start.png|center|1400px|thumb|'''Figure 14:''' Forward primer for the amplification of the arabinose inducible induction system]]<br />
We used the RFC 23 cloning strategy to get a fusion protein of our desired protease and the induction system, which allows us to induce the protease. Both genes were amplified with PCR using two special primers generating a <i>NgoM</i>IV restrictioin site at the end of the induction system and in front of the protease. The amplified protease were digested with <i>BamH</i>I and <i>NgoM</i>IV, the arabinose inducible induction system with <i>NgoM</i>IV and <i>Hind</i>III. The vector, which contains the ssTorA_CS-Protease_β-lactamase device was digested with <i>Hind</i>III and <i>BamH</i>I. A triple ligation yielded the final construct with both devices: the protease detector and the protease generator. The two pictures below show the final vector constructs , which where used for the survival screening.<br />
[[File:UP_Pic_SG_TEV_SG1-Plasmid.png|center|800px|thumb|'''Figure 15:''' Final vector construct containing arabinose inducible TEV protease fusion and the protease detector device]]<br />
[[File:UP_Pic_SG_PreSciccion_SG2-Plasmid.png|center|800px|thumb|'''Figure 16:''' Final vector construct containing arabinose inducible HRV 14_3C protease fusion and the protease detector device]]<br />
<br />
===Investigation of the ''in vivo'' selection assay===<br />
<p><br />
<br />
Two major points have to be demonstrated: First, the export of β-lactamase over the TAT pathway has to be efficient enough to provide a certain resistance against ampicillin and second, the activated protease is able to intimately reduce the resistance against ampicillin by cleavage of the TorA signal sequence from the β-lactamase.</p><br />
====Testing the export efficiency of the protease activity detector====<br />
To test the export rate of the β-lactamse of the protease activity detector device we grew an <i>E. coli</i> culture up to an OD<sub>600</sub> of 0.6. The cells were then diluted to an OD of 0.002 and 100 µL were plated in triplicates with increasing ampicillin concentrations. The graph below shows the amount of surviving cells in percentage. <br><br />
<br />
[[Image:UP_Survial-screen_bla-construct.png|center|600px|thumb|'''Figure 17:''' Amount of survived cells. The construct was induced with 1 mM IPTG. The rate of surviving cells without IPTG and without ampicilin was set to 100%. ]]<br />
<br />
[[Image:UP_TEV-Survival.png|center|600px|thumb|'''Figure 18:''' Survival test at different ampicillin concentrations: A) 1000 cfu plated on agar plates containing chloramphenicol; B) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 400µg/mL ampicillin; C) no culture growth of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 600µg/mL ampicillin]]<br />
[[Image:UP_14_3C_SurvTest.png|center|600px|thumb|'''Figure 19:''' Survival test at different ampicillin concentrations: <br />
A) 1000 cfu plated on agar plates containing chloramphenicol; <br />
B) Influence of resistance induction with 1 mM IPTG, 1000 cfu where plated on agar plates containing chloramphenicol and 1mM IPTG; C) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 50 µg/mL ampicillin; D) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 100 µg/mL ampicillin; E) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 200 µg/mL ampicillin; F) Reduced amount of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 400 µg/mL ampicillin; G) No culture growth of <i>E.coli</i> colonies, 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 800 µg/mL ampicillin]]<br />
<br />
This assay was performed at two temperatures. At 37°C, the optimal growth temperature and at 30°C for best protein expression conditions. All assays show the same results. The cells were able to grow at ampicilin concentrations up to 400 µg/ml. Cells transformed with the final construct containing the detection device and the protease generator device were able to survive up to 800 µg/ml ampicilin (incubated at 30°C over 2 days).<br />
<br />
This leads to the conclusion, that the export of β-lactamase into the periplasm is very effective. It is also important to mention, that there is no cell survival when ampicilin is added to the media without the induction of the protease activity detection device by IPTG.<br />
<br />
====Testing the "Kill Switch" by activating the protease====<br />
<br />
The second major point which needs to be demonstrated is that we are able to kill the ''E.coli'' cells by activating the protease. This is referred as the "Kill Switch". In the final assay we want to have the ability to induce the protease activity detector device and the protease generate independently from each other. But first of all we have to check if our system is working and if we are able to kill the cells by activating the protease.<br><br />
To proof the principle we used the natural TEV protease and make a co-transformation with the protease activity detector device containing the cleavage site for the TEV protease. Both were induced by IPTG and so we can't control the expression rate neither the time of the induction of the protease generator device.<br><br />
After the incubation at 37°C we were able to show the desired proof of principle: when we activate the protease the resistance against ampicillin; based on the export rate of β-lactamase into the periplasm via the TAT pathway, was intimately reduced. The cells with activated TEV protease were able to survive up to concentration of 50 µg/ml ampicillin.<br><br />
[[Image:UP_bla-TEV_double-trans.png|center|500px|thumb|'''Figure 20:''' Percentage of surviving cells on different ampicillin concentration. Blue - cells transformed with the single protease activity detector device for TEV protease; Red - cell transformed with both, the protease activity detector device for TEV protease and the TEV protease itself]]<br><br />
Based on the ampicillin resistance of the plasmid of HRV14_3C protease, we were unable to show the proof of principle for this protease.<br><br />
The next step was to use the complete test vector which contains the protease activity detector device and the protease generator device. We introduced the arabinose inducible induction system fused to the protease flanked by the restriction sites of <i>BamH</i>I and <i>Hind</i>III into the vector of the modified Hitchhiker selection system.<br><br />
With these final vectors we had to repeat the survival screening to show that the system is also working when there is a dual induction based on only one plasmid. The cells were grown to an OD<sub>600</sub> of 0.6 and diluted to an OD<sub>600</sub> of 0.002. 100 µL of this dilution were plated on agar plates with different ampicillin concentrations and induced and induced protease. The results are shown in figure 20.<br><br />
[[Image:UP_SurvTEV_onepla.jpg|center|500px|thumb|'''Figure 21:''' Cell growth on different ampicillin concentration. Blue - cells without induction of the TEV protease; Red - cells with induced TEV protease]]<br><br />
<br><br />
The survival screening assay for TEV protease was performed 30 °C and all plates were incubated for 2 days. The ''E.coli'' XL1 blue cells were able to survive up to ampicilin concentration of 800 µg/mL, which is an increase of 100% of the original resistance capacity. When 2% arabinose is added to the media, the cells showed an intimately reduce surviving rate. The cells were able to survive at ampicilin concentrations up 25 µg/mL.<br><br />
This shows, that we got a high dynamic range from 50 up to 800 µg/mL ampicilin to screen for inhibition of the protease.<br />
<br />
===Transferring and testing the library===<br />
After the successful test of the protease activity we transferred the generated <i>mdnA</i> library into our cells. The following pictures show the amazing results. Cell growth could be detected up to 400 µg Ampicillin/mL.<br />
[[Image:UP_2011-06-01_CM-Kan-Amp_labeled02.jpg|left|420px|thumb|'''Figure 22:''' Cell growth on Ampicillin without induced protease]]<br />
[[Image:UP_2011-06-01_CM-Kan-Ara-Amp_labeled01.jpg|right|420px|thumb|'''Figure 23:''' Cell growth on Ampicillin with induced protease]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
===Background===<br />
====Twin Arginine Translocon (TAT)====<br />
The TAT pathway is responsible for the transport of folded proteins across energy-transducing membranes and it is able of discriminating of unfolded proteins. This transport pathway is common for bacteria, archea and plants and translocates circa 6% of <i>E.coli</i> produced secreted proteins. The translocated proteins must have a signal peptide for targeting of the TAT-transporter, such proteins are e.g. hydrogenases, dehydrogenases and reductases. The signal sequence itself is composed of an N-terminal positive charged domain, a hydrophobic domain and a C-terminal domain.<br><br />
The translocon is composed of three parts: TatA, TatB and TatC. The transport-pore is proposed to be formed during substrate binding by these three parts. The TatA, TatB and TatC proteins may form complexes of different sizes, which on the other hand form pores matching the size of the folded substrate. The transport of proteins through the TAT pathway depends on the proton motive force. Calculations by Adler & Theg showed that the transport of one folded substrate molecule requires the release of approximately 7.9x10<sup>4</sup> protons, which equals 10.000 ATP molecules. The signal sequence is cleaved off after translocation.<br />
<br><br />
[[File:UP_TorA_TATSeq2.jpg|center|450px|thumb|'''Figure 24:''' Schematic representation of TorA-signal sequence. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br><br />
The used signal sequence originates from the TorA protein (Trimethylamin-N-Oxid-Reductase). It is the main respiratory enzyme which reduces TMAO under anaerobic conditions in the periplasm, where it is transported by the TAT pathway.<br />
<br> <br />
[[File:UP_TAT_Pathway2.jpg|center|450px|thumb|'''Figure 25:''' Schematic representation of TAT-dependent pathway. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br />
<br />
<br />
<br><br />
<br />
====Tobacco Etch Virus (TEV) protease====<br />
TEV protease is the common name for the 27 kDa catalytic domain of the Nuclear Inclusion a endopeptidase (NIa) encoded by the tobacco etch virus. TEV protease is a useful reagent for cleaving fusion proteins. It recognizes a linear epitope of the general form E-Xaa-Xaa-Y -Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. In TEV protease the serine nucleophile of the conventional Ser-Asp-His triad is a cysteine instead. This probably explains why TEV protease is resistant to many commonly used protease inhibitors.<br><br />
<br />
====14_3C-Protease====<br />
<br />
The 14_3C protease originates from the human rhinovirus. Rhinoviruses are the most frequent reason for infections of the upper and lower respiratory tract, also known as the cold. Because the 3C protease of human rhinovirus is necessary for the cleavage of the polyprotein translated from viral RNA it may serve as a potential target for development of antiviral targets. The recombinant type 14_3C protease from human rhinovirus (HRV 3C) recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln↓GlyPro. The small, 22-kDa size of the protease got its optimal activity at 4°C but is still very active at 37 °C. It is commonly used for an easy tag removal after the purification of recombinant proteins carrying his-tag. The 14_3C works with a catalytic triade, containing the amino acid residues Ser-Asp-His at its active site.<br><br />
<br />
===References===<br />
*Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. and Bottomley, S.P. (2007) Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-2367<br><br />
*Genest O., Ilbert M., Méjean V., Iobbi-Nivol C.,(2005) TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature. J. Biol. Chem. 280: 15644-15648.<br />
*Kapust R. B., Tözsér J., Fox J. D., Anderson D. E., Cherry S., Copeland T. D., Waugh D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 12:993-1000.<br><br />
*Lee P. A., Tullman-Ercekand D., Georgiou G., (2006) The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60:373–95<br />
*Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30:544-550.<br><br />
*Shih S. R., Chen S. J., Hakimelahi G. H., Liu H. J., Tseng C. T., Shia K. S., (2004) Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting molecules. Med Res Rev 24(4):449-74.<br><br />
*Wanga QM, Chen SH., (2007) Human rhinovirus 3C protease as a potential target for the development of antiviral agents. Curr Protein Pept Sci. 8(1):19-27. <br><br />
*Waraho D., DeLisa M. P., (2009). Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism. PNAS 106(10):3692-7</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_SelectionTeam:Potsdam Bioware/Project/Details Selection2011-10-28T22:25:54Z<p>UP Stefan: /* Construction of two component for the in vivo selection assay */</p>
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<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==<i>In Vivo</i> Selection==<br />
===Introduction===<br />
In addition to the phage display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an <i>in vitro</i> screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for any protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator. For the protease activity detector we modified the TAT-hitchhiker system developed by DeLisa <i>et al.</i> <br />
We used the BioBrick <partinfo>I757010</partinfo> (β-lactamase) as well as <partinfo>K208005</partinfo> (ssTorA) and fused them together via a linker peptide. For the protease generator we cloned the arabinose induction system in front of a protease in iGEM standard.<br> <br />
<br />
[[File:UP_images_TorA-construct-design2_28.10.2011.png.jpg|center|500px|thumb|'''Figure 1:''' Schema of our constructed selection vector containing protease generator and protease activity detector]]<br> <br />
The system works in a combined manner of the two devices. In order to confer β-lactam antibiotic resistance to cells the β-lactamase has to be exported in the periplasm. This transport is mediated by the TorA export sequence via the Twin-Arginine Translocation (TAT) system (DeLisa, 2009). The linker peptide between the TorA export sequence and the β-lactamase displays the corresponding protease cleavage site. In addition the linker peptide is chosen as short as possible to imitate folding because the TAT pathway only allows transport of correctly folded substrates. The protease as well as the linker peptide are designed as exchangeable parts.<br><br />
<br />
If the protease device is functional, it will cleave the linker peptide between TorA and the β-lactamase construct which leaves the cell without any antibiotic resistance. The construct was tested with increasing ampicillin concentrations. The number of surviving colonies was depended on the export rate of the β-lactamase into the periplasm. A high cleavage rate of the linker peptide leads to a reduced ampicillin resistance. With expressed protease a dramatically drop of the number of surviving colonies could be observed.<br><br />
The survival assay was carried out with and without expressed protease. By increasing of the ampicillin concentration we could detect a cutoff ampicillin concentration. The colony forming units (CFU) were counted and compared. To detected whether our constructed library is able to block the protease we co-transformed the library into <i>E. coli</i> carrying our pUP_SG1 plasmid.<br><br />
<br />
===Site-directed mutagenesis of Tobacco Etch Virus and 14_3C proteases in iGEM standard===<br />
<br />
We chose two model proteases, the Tobacco Etch Virus protease, further along called TEV, and the 14_3C protease from the human rhinovirus, also known as PreScission. Both proteases contained iGEM restriction sites, one in case of the TEV and three for 14 3C, respectively. <br><br />
[[File:UP_Pic_SG_Pre-Complete.png|center|800px|thumb|'''Figure 2:''' Complete sequence alignment of the 14_3C protease]]<br />
[[File:UP_Pic_SG_TEV-Complete.png|center|800px|thumb|'''Figure 3:''' Complete sequence alignment of the 14_3C protease]]<br />
<br><br><br />
Thus, site-directed mutagenesis was applied to mutate each restriction site in E. coli. For every mutation a forward and a reverse primer had to be designed. The following pictures show the forward primers for the side directed mutagenesis, the reverse primers are the reverse complement sequence of the forward primers.<br><br />
<br />
For the 14_3C protease:<br />
[[File:UP_Pic_SG_Pre-End.png|left|400px|thumb|'''Figure 4:''' Reverse primer to add the iGEM suffix on the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaSpeI.png|right|400px|thumb|'''Figure 5:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-1.png|left|400px|thumb|'''Figure 6:''' Forward primer to mutate the first XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-2.png|right|400px|thumb|'''Figure 7:''' Forward primer to mutate the second XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_Pre-Start.png|left|400px|thumb|'''Figure 8:''' Forward primer to add the iGEM prefix to the HRV 14_3C protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
For the TEV protease:<br />
<br />
[[File:UP_Pic_SG_TEV-End.png|left|400px|thumb|'''Figure 9:''' Reverse primer to add the iGEM suffix to the TEV protease]]<br />
[[File:UP_Pic_SG_TEVMutaSpeI.png|right|400px|thumb|'''Figure 10:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the TEV protease]]<br />
[[File:UP_Pic_SG_TEV-Start.png|left|400px|thumb|'''Figure 11:''' Forward primer to add the iGEM prefix to the TEV protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
We used assembly PCR to gain the final product. In case of 14_3C two assembly PCRs had to be done. The expected size of the fragments are shown in the gel picture. In the following picture all fragments contain the correct sizes.<br />
<br><br />
[[File:UP_Fragments_14_3C_TEV.png|center|200px|thumb|'''Figure 12:''' Picture of the, via PCR amplified and mutated Fragment of both protease]]<br />
<br><br />
<br />
===Construction of two component for the ''in vivo'' selection assay===<br />
<br />
The ''in vivo'' assay is based on two devices. On the one hand the protease activity detector device, on the other a protease generator device. The detector device is based on the enzyme β-lactamase, which is only active inside the periplasm and provides the resistance against lactam antibiotics like ampicillin and penicillin. For the protease generator device we needed a second induction system on our plasmid, which is quite uncommon for any plasmid. So several problems had to solved to set up an effective ''in vivo'' selection assay:<br />
*a second, very tight induction system has to be found<br />
*a way to control the export rate of β-lactamase into the periplasm, which can be reduced by the protease<br />
<br />
We solved these problems by putting the protease detector device under the control of the lac-operon. The protease device is induced by the arabinose system of the pBAD_iGEMexpress vector. The idea to control the survival of the cells via the amount of exported protein is inspired by the Hitchhiker assay of DeLisa ''et al''. Using the TAT pathway for the export of β-lactamase into the periplasm allows us to introduce a protease specific cleavage site in front of the signal sequence regulating the export.<br />
<br />
====Introducing the specific protease cleavage site====<br />
To adapt the protease activity detector device to any protease we want to screen, we had to modularize it. Our detector device was set up on a vector for the Hitchhiker selection. The plasmid offers two unique restriction sites on the plasmid, after the signal sequence of TorA one restriction site for <i>Xho</i>I and a <i>Nhe</i>I restriction site in front of the β-lactamase. Both were used to introduce the specific protease cleavage site in between the TorA signal sequence and the β-lactamase. By designing two complementary oligonucleotides containing the specific protease cleavage site flanked by two short linker regions. After the digest of the vector with <i>Xho</i>I and <i>Nhe</i>I the hybridized oligonucleotides, containing the overhangs for <i>Xho</i>I and <i>Nhe</i>I can be ligated into the vector.<br />
<br />
====Adding the arabinose inducible induction system to control the proteases, TEV and 14_3C====<br />
We needed a time independent induction of the protease device. Therefore we cloned the arabionse induction system (<i>araC</i>) in front of the protease device out of the pBAD_iGEMexpress vector. The fusion of gene construct and induction system was always performed via an <i>NgoM</i>IV restriction site. This restriction site was introduced with our amplification primers.<br />
[[File:UP_Pic_SG_AraC-End.png|center|800px|thumb|'''Figure 13:''' Reverse primer for the amplification of the arabinose inducible induction system]]<br />
[[File:UP_Pic_SG_AraC-Start.png|center|1400px|thumb|'''Figure 14:''' Forward primer for the amplification of the arabinose inducible induction system]]<br />
We used the RFC 23 cloning strategy to get a fusion protein of our desired protease and the induction system, which allows us to induce the protease. Both genes were amplified with PCR using two special primers generating a <i>NgoM</i>IV restrictioin site at the end of the induction system and in front of the protease. The amplified protease were digested with <i>BamH</i>I and <i>NgoM</i>IV, the arabinose inducible induction system with <i>NgoM</i>IV and <i>Hind</i>III. The vector, which contains the ssTorA_CS-Protease_β-lactamase device was digested with <i>Hind</i>III and <i>BamH</i>I. A triple ligation yielded the final construct with both devices: the protease detector and the protease generator. The two pictures below show the final vector constructs , which where used for the survival screening.<br />
[[File:UP_Pic_SG_TEV_SG1-Plasmid.png|center|800px|thumb|'''Figure 15:''' Final vector construct containing arabinose inducible TEV protease fusion and the protease detector device]]<br />
[[File:UP_Pic_SG_PreSciccion_SG2-Plasmid.png|center|800px|thumb|'''Figure 16:''' Final vector construct containing arabinose inducible HRV 14_3C protease fusion and the protease detector device]]<br />
<br />
===Investigation of the ''in vivo'' selection assay===<br />
<p><br />
<br />
Two major points have to be demonstrated: First, the export of β-lactamase over the TAT pathway has to be efficient enough to provide a certain resistance against ampicillin and second, the activated protease is able to intimately reduce the resistance against ampicillin by cleavage of the TorA signal sequence from the β-lactamase.</p><br />
====Testing the export efficiency of the protease activity detector====<br />
To test the export rate of the β-lactamse of the protease activity detector device we grew an <i>E. coli</i> culture up to an OD<sub>600</sub> of 0.6. The cells were then diluted to an OD of 0.002 and 100 µL were plated in triplicates with increasing ampicillin concentrations. The graph below shows the amount of surviving cells in percentage. <br><br />
<br />
[[Image:UP_Survial-screen_bla-construct.png|center|600px|thumb|'''Figure 17:''' Amount of survived cells. The construct was induced with 1 mM IPTG. The rate of surviving cells without IPTG and without ampicilin was set to 100%. ]]<br />
<br />
[[Image:UP_TEV-Survival.png|center|600px|thumb|'''Figure 18:''' Survival test at different ampicillin concentrations: A) approx. 1000 cfu plated on agar plates containing chloramphenicol; B) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 400µg/ml ampicillin; C) no culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 600µg/ml ampicillin]]<br />
[[Image:UP_14_3C_SurvTest.png|center|600px|thumb|'''Figure 19:''' Survival test at different ampicillin concentrations: <br />
A) approx. 1000 cfu plated on agar plates containing chloramphenicol; <br />
B) Influence of resistance induction with 1 mM IPTG, approx. 1000 cfu where plated on agar plates containing chloramphenicol and 1mM IPTG; C) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 50 µg/ml ampicillin; D) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 100 µg/ml ampicillin; E) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 200 µg/ml ampicillin; F) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 400 µg/ml ampicillin; G) No culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 800 µg/ml ampicillin]]<br />
<br />
This assay was performed at two temperatures. At 37°C, the optimal growth temperature and at 30°C for best protein expression conditions. All assays show the same results. The cells were able to grow at ampicilin concentrations up to 400 µg/ml. Cells transformed with the final construct containing the detection device and the protease generator device were able to survive up to 800 µg/ml ampicilin (incubated at 30°C over 2 days).<br />
<br />
This leads to the conclusion, that the export of β-lactamase into the periplasm is very effective. It is also important to mention, that there is no cell survival when ampicilin is added to the media without the induction of the protease activity detection device by IPTG.<br />
<br />
====Testing the "Kill Switch" by activating the protease====<br />
<br />
The second major point which needs to be demonstrated is that we are able to kill the ''E.coli'' cells by activating the protease. This is referred as the "Kill Switch". In the final assay we want to have the ability to induce the protease activity detector device and the protease generate independently from each other. But first of all we have to check if our system is working and if we are able to kill the cells by activating the protease.<br><br />
To proof the principle we used the natural TEV protease and make a co-transformation with the protease activity detector device containing the cleavage site for the TEV protease. Both were induced by IPTG and so we can't control the expression rate neither the time of the induction of the protease generator device.<br><br />
After the incubation at 37°C we were able to show the desired proof of principle: when we activate the protease the resistance against ampicillin; based on the export rate of β-lactamase into the periplasm via the TAT pathway, was intimately reduced. The cells with activated TEV protease were able to survive up to concentration of 50 µg/ml ampicillin.<br><br />
[[Image:UP_bla-TEV_double-trans.png|center|500px|thumb|'''Figure 20:''' Percentage of surviving cells on different ampicillin concentration. Blue - cells transformed with the single protease activity detector device for TEV protease; Red - cell transformed with both, the protease activity detector device for TEV protease and the TEV protease itself]]<br><br />
Based on the ampicillin resistance of the plasmid of HRV14_3C protease, we were unable to show the proof of principle for this protease.<br><br />
The next step was to use the complete test vector which contains the protease activity detector device and the protease generator device. We introduced the arabinose inducible induction system fused to the protease flanked by the restriction sites of <i>BamH</i>I and <i>Hind</i>III into the vector of the modified Hitchhiker selection system.<br><br />
With these final vectors we had to repeat the survival screening to show that the system is also working when there is a dual induction based on only one plasmid. The cells were grown to an OD<sub>600</sub> of 0.6 and diluted to an OD<sub>600</sub> of 0.002. 100 µL of this dilution were plated on agar plates with different ampicillin concentrations and induced and induced protease. The results are shown in figure 20.<br><br />
[[Image:UP_SurvTEV_onepla.jpg|center|500px|thumb|'''Figure 21:''' Cell growth on different ampicillin concentration. Blue - cells without induction of the TEV protease; Red - cells with induced TEV protease]]<br><br />
<br><br />
The survival screening assay for TEV protease was performed 30 °C and all plates were incubated for 2 days. The ''E.coli'' XL1 blue cells were able to survive up to ampicilin concentration of 800 µg/mL, which is an increase of 100% of the original resistance capacity. When 2% arabinose is added to the media, the cells showed an intimately reduce surviving rate. The cells were able to survive at ampicilin concentrations up 25 µg/mL.<br><br />
This shows, that we got a high dynamic range from 50 up to 800 µg/mL ampicilin to screen for inhibition of the protease.<br />
<br />
===Transferring and testing the library===<br />
After the successful test of the protease activity we transferred the generated <i>mdnA</i> library into our cells. The following pictures show the amazing results. Cell growth could be detected up to 400 µg Ampicillin/mL.<br />
[[Image:UP_2011-06-01_CM-Kan-Amp_labeled02.jpg|left|420px|thumb|'''Figure 22:''' Cell growth on Ampicillin without induced protease]]<br />
[[Image:UP_2011-06-01_CM-Kan-Ara-Amp_labeled01.jpg|right|420px|thumb|'''Figure 23:''' Cell growth on Ampicillin with induced protease]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
===Background===<br />
====Twin Arginine Translocon (TAT)====<br />
The TAT pathway is responsible for the transport of folded proteins across energy-transducing membranes and it is able of discriminating of unfolded proteins. This transport pathway is common for bacteria, archea and plants and translocates circa 6% of <i>E.coli</i> produced secreted proteins. The translocated proteins must have a signal peptide for targeting of the TAT-transporter, such proteins are e.g. hydrogenases, dehydrogenases and reductases. The signal sequence itself is composed of an N-terminal positive charged domain, a hydrophobic domain and a C-terminal domain.<br><br />
The translocon is composed of three parts: TatA, TatB and TatC. The transport-pore is proposed to be formed during substrate binding by these three parts. The TatA, TatB and TatC proteins may form complexes of different sizes, which on the other hand form pores matching the size of the folded substrate. The transport of proteins through the TAT pathway depends on the proton motive force. Calculations by Adler & Theg showed that the transport of one folded substrate molecule requires the release of approximately 7.9x10<sup>4</sup> protons, which equals 10.000 ATP molecules. The signal sequence is cleaved off after translocation.<br />
<br><br />
[[File:UP_TorA_TATSeq2.jpg|center|450px|thumb|'''Figure 24:''' Schematic representation of TorA-signal sequence. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br><br />
The used signal sequence originates from the TorA protein (Trimethylamin-N-Oxid-Reductase). It is the main respiratory enzyme which reduces TMAO under anaerobic conditions in the periplasm, where it is transported by the TAT pathway.<br />
<br> <br />
[[File:UP_TAT_Pathway2.jpg|center|450px|thumb|'''Figure 25:''' Schematic representation of TAT-dependent pathway. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br />
<br />
<br />
<br><br />
<br />
====Tobacco Etch Virus (TEV) protease====<br />
TEV protease is the common name for the 27 kDa catalytic domain of the Nuclear Inclusion a endopeptidase (NIa) encoded by the tobacco etch virus. TEV protease is a useful reagent for cleaving fusion proteins. It recognizes a linear epitope of the general form E-Xaa-Xaa-Y -Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. In TEV protease the serine nucleophile of the conventional Ser-Asp-His triad is a cysteine instead. This probably explains why TEV protease is resistant to many commonly used protease inhibitors.<br><br />
<br />
====14_3C-Protease====<br />
<br />
The 14_3C protease originates from the human rhinovirus. Rhinoviruses are the most frequent reason for infections of the upper and lower respiratory tract, also known as the cold. Because the 3C protease of human rhinovirus is necessary for the cleavage of the polyprotein translated from viral RNA it may serve as a potential target for development of antiviral targets. The recombinant type 14_3C protease from human rhinovirus (HRV 3C) recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln↓GlyPro. The small, 22-kDa size of the protease got its optimal activity at 4°C but is still very active at 37 °C. It is commonly used for an easy tag removal after the purification of recombinant proteins carrying his-tag. The 14_3C works with a catalytic triade, containing the amino acid residues Ser-Asp-His at its active site.<br><br />
<br />
===References===<br />
*Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. and Bottomley, S.P. (2007) Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-2367<br><br />
*Genest O., Ilbert M., Méjean V., Iobbi-Nivol C.,(2005) TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature. J. Biol. Chem. 280: 15644-15648.<br />
*Kapust R. B., Tözsér J., Fox J. D., Anderson D. E., Cherry S., Copeland T. D., Waugh D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 12:993-1000.<br><br />
*Lee P. A., Tullman-Ercekand D., Georgiou G., (2006) The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60:373–95<br />
*Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30:544-550.<br><br />
*Shih S. R., Chen S. J., Hakimelahi G. H., Liu H. J., Tseng C. T., Shia K. S., (2004) Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting molecules. Med Res Rev 24(4):449-74.<br><br />
*Wanga QM, Chen SH., (2007) Human rhinovirus 3C protease as a potential target for the development of antiviral agents. Curr Protein Pept Sci. 8(1):19-27. <br><br />
*Waraho D., DeLisa M. P., (2009). Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism. PNAS 106(10):3692-7</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_SelectionTeam:Potsdam Bioware/Project/Details Selection2011-10-28T22:19:26Z<p>UP Stefan: /* Construction of two component for the in vivo selection assay */</p>
<hr />
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<br />
==<i>In Vivo</i> Selection==<br />
===Introduction===<br />
In addition to the phage display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an <i>in vitro</i> screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for any protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator. For the protease activity detector we modified the TAT-hitchhiker system developed by DeLisa <i>et al.</i> <br />
We used the BioBrick <partinfo>I757010</partinfo> (β-lactamase) as well as <partinfo>K208005</partinfo> (ssTorA) and fused them together via a linker peptide. For the protease generator we cloned the arabinose induction system in front of a protease in iGEM standard.<br> <br />
<br />
[[File:UP_images_TorA-construct-design2_28.10.2011.png.jpg|center|500px|thumb|'''Figure 1:''' Schema of our constructed selection vector containing protease generator and protease activity detector]]<br> <br />
The system works in a combined manner of the two devices. In order to confer β-lactam antibiotic resistance to cells the β-lactamase has to be exported in the periplasm. This transport is mediated by the TorA export sequence via the Twin-Arginine Translocation (TAT) system (DeLisa, 2009). The linker peptide between the TorA export sequence and the β-lactamase displays the corresponding protease cleavage site. In addition the linker peptide is chosen as short as possible to imitate folding because the TAT pathway only allows transport of correctly folded substrates. The protease as well as the linker peptide are designed as exchangeable parts.<br><br />
<br />
If the protease device is functional, it will cleave the linker peptide between TorA and the β-lactamase construct which leaves the cell without any antibiotic resistance. The construct was tested with increasing ampicillin concentrations. The number of surviving colonies was depended on the export rate of the β-lactamase into the periplasm. A high cleavage rate of the linker peptide leads to a reduced ampicillin resistance. With expressed protease a dramatically drop of the number of surviving colonies could be observed.<br><br />
The survival assay was carried out with and without expressed protease. By increasing of the ampicillin concentration we could detect a cutoff ampicillin concentration. The colony forming units (CFU) were counted and compared. To detected whether our constructed library is able to block the protease we co-transformed the library into <i>E. coli</i> carrying our pUP_SG1 plasmid.<br><br />
<br />
===Site-directed mutagenesis of Tobacco Etch Virus and 14_3C proteases in iGEM standard===<br />
<br />
We chose two model proteases, the Tobacco Etch Virus protease, further along called TEV, and the 14_3C protease from the human rhinovirus, also known as PreScission. Both proteases contained iGEM restriction sites, one in case of the TEV and three for 14 3C, respectively. <br><br />
[[File:UP_Pic_SG_Pre-Complete.png|center|800px|thumb|'''Figure 2:''' Complete sequence alignment of the 14_3C protease]]<br />
[[File:UP_Pic_SG_TEV-Complete.png|center|800px|thumb|'''Figure 3:''' Complete sequence alignment of the 14_3C protease]]<br />
<br><br><br />
Thus, site-directed mutagenesis was applied to mutate each restriction site in E. coli. For every mutation a forward and a reverse primer had to be designed. The following pictures show the forward primers for the side directed mutagenesis, the reverse primers are the reverse complement sequence of the forward primers.<br><br />
<br />
For the 14_3C protease:<br />
[[File:UP_Pic_SG_Pre-End.png|left|400px|thumb|'''Figure 4:''' Reverse primer to add the iGEM suffix on the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaSpeI.png|right|400px|thumb|'''Figure 5:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-1.png|left|400px|thumb|'''Figure 6:''' Forward primer to mutate the first XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-2.png|right|400px|thumb|'''Figure 7:''' Forward primer to mutate the second XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_Pre-Start.png|left|400px|thumb|'''Figure 8:''' Forward primer to add the iGEM prefix to the HRV 14_3C protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
For the TEV protease:<br />
<br />
[[File:UP_Pic_SG_TEV-End.png|left|400px|thumb|'''Figure 9:''' Reverse primer to add the iGEM suffix to the TEV protease]]<br />
[[File:UP_Pic_SG_TEVMutaSpeI.png|right|400px|thumb|'''Figure 10:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the TEV protease]]<br />
[[File:UP_Pic_SG_TEV-Start.png|left|400px|thumb|'''Figure 11:''' Forward primer to add the iGEM prefix to the TEV protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
We used assembly PCR to gain the final product. In case of 14_3C two assembly PCRs had to be done. The expected size of the fragments are shown in the gel picture. In the following picture all fragments contain the correct sizes.<br />
<br><br />
[[File:UP_Fragments_14_3C_TEV.png|center|200px|thumb|'''Figure 12:''' Picture of the, via PCR amplified and mutated Fragment of both protease]]<br />
<br><br />
<br />
===Construction of two component for the ''in vivo'' selection assay===<br />
<br />
The ''in vivo'' assay is based on two devices. On the one hand the protease activity detector device, on the other a protease generator device. The detector device is based on the enzyme β-lactamase, which is only active inside the periplasm and provides the resistance against lactam antibiotics like ampicillin and penicillin. For the protease generator device we needed a second induction system on our plasmid, which is quite uncommon for any plasmid. So several problems had to solved to set up an effective ''in vivo'' selection assay:<br />
*a second, very tight induction system has to be found<br />
*a way to control the export rate of β-lactamase into the periplasm, which can be reduced by the protease<br />
<br />
We solved these problems by putting the protease detector device under the control of the lac-operon. The protease device is induced by the arabinose system of the pBAD_iGEMexpress vector. The solution for the export problem of β-lactamase is inspired by Hitchhiker assay of DeLisa ''et al''. Using the TAT pathway for the export of β-lactamase into the periplasm gives us the ability to introduce a protease specific cleavage site in front of the signal sequence to regulate the export.<br />
<br />
====Introducing the specific protease cleavage site====<br />
To adapt the protease activity detector device to any protease we want to screen, we had to modularize it. Our detector device was set up on a vector for the Hitchhiker selection. The plasmid offers two unique restriction sites on the plasmid, after the signal sequence of TorA one restriction site for <i>Xho</i>I and a <i>Nhe</i>I restriction site in front of the β-lactamase. Both were used to introduce the cleavage site in between the TorA signal sequence and the β-lactamase. By designing two complementary oligonucleotides containing the cleavage site flanked by two short linker regions. After the digest of the vector with XhoI and NheI the hybridized oligonucleotides, containing the overhangs for <i>Xho</i>I and <i>Nhe</i>I can be ligated into the vector.<br />
<br />
====Adding the arabinose inducible induction system to control the proteases, TEV and 14_3C====<br />
We needed a time independent induction of the protease device. Therefore we cloned the arabionse induction system (<i>araC</i>) in front of the protease device out of the pBAD_iGEMexpress vector. The fusion of gene construct and induction system was always performed via an NgoMIV restriction site. This restriction site was introduced with our amplification primers.<br />
[[File:UP_Pic_SG_AraC-End.png|center|800px|thumb|'''Figure 13:''' Reverse primer for the amplification of the arabinose inducible induction system]]<br />
[[File:UP_Pic_SG_AraC-Start.png|center|1400px|thumb|'''Figure 14:''' Forward primer for the amplification of the arabinose inducible induction system]]<br />
We used the RFC 23 cloning strategy to get a fusion protein of our desired protease and the induction system, which allows us to induce the protease. Both genes were amplified with PCR using two special primers generating a NgoMIV restrictioin site at the end of the induction system and in front of the protease. The amplified protease were digested with BamHI and <i>NgoM</i>IV, the arabinose inducible induction system with <i>NgoM</i>IV and <i>Hind</i>III. The vector, which contains the ssTorA_CS-Protease_β-lactamase device was digested with <i>Hind</i>III and <i>BamH</i>I. A triple ligation yielded the final construct with both devices: the protease detector and the protease generator. The two pictures below show the final vector constructs , which where used for the survival screening.<br />
[[File:UP_Pic_SG_TEV_SG1-Plasmid.png|center|800px|thumb|'''Figure 15:''' Final vector construct containing arabinose inducible TEV protease fusion and the protease detector device]]<br />
[[File:UP_Pic_SG_PreSciccion_SG2-Plasmid.png|center|800px|thumb|'''Figure 16:''' Final vector construct containing arabinose inducible HRV 14_3C protease fusion and the protease detector device]]<br />
<br />
===Investigation of the ''in vivo'' selection assay===<br />
<p><br />
<br />
Two major points have to be demonstrated: First, the export of β-lactamase over the TAT pathway has to be efficient enough to provide a certain resistance against ampicillin and second, the activated protease is able to intimately reduce the resistance against ampicillin by cleavage of the TorA signal sequence from the β-lactamase.</p><br />
====Testing the export efficiency of the protease activity detector====<br />
To test the export rate of the β-lactamse of the protease activity detector device we grew an <i>E. coli</i> culture up to an OD<sub>600</sub> of 0.6. The cells were then diluted to an OD of 0.002 and 100 µL were plated in triplicates with increasing ampicillin concentrations. The graph below shows the amount of surviving cells in percentage. <br><br />
<br />
[[Image:UP_Survial-screen_bla-construct.png|center|600px|thumb|'''Figure 17:''' Amount of survived cells. The construct was induced with 1 mM IPTG. The rate of surviving cells without IPTG and without ampicilin was set to 100%. ]]<br />
<br />
[[Image:UP_TEV-Survival.png|center|600px|thumb|'''Figure 18:''' Survival test at different ampicillin concentrations: A) approx. 1000 cfu plated on agar plates containing chloramphenicol; B) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 400µg/ml ampicillin; C) no culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 600µg/ml ampicillin]]<br />
[[Image:UP_14_3C_SurvTest.png|center|600px|thumb|'''Figure 19:''' Survival test at different ampicillin concentrations: <br />
A) approx. 1000 cfu plated on agar plates containing chloramphenicol; <br />
B) Influence of resistance induction with 1 mM IPTG, approx. 1000 cfu where plated on agar plates containing chloramphenicol and 1mM IPTG; C) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 50 µg/ml ampicillin; D) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 100 µg/ml ampicillin; E) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 200 µg/ml ampicillin; F) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 400 µg/ml ampicillin; G) No culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 800 µg/ml ampicillin]]<br />
<br />
This assay was performed at two temperatures. At 37°C, the optimal growth temperature and at 30°C for best protein expression conditions. All assays show the same results. The cells were able to grow at ampicilin concentrations up to 400 µg/ml. Cells transformed with the final construct containing the detection device and the protease generator device were able to survive up to 800 µg/ml ampicilin (incubated at 30°C over 2 days).<br />
<br />
This leads to the conclusion, that the export of β-lactamase into the periplasm is very effective. It is also important to mention, that there is no cell survival when ampicilin is added to the media without the induction of the protease activity detection device by IPTG.<br />
<br />
====Testing the "Kill Switch" by activating the protease====<br />
<br />
The second major point which needs to be demonstrated is that we are able to kill the ''E.coli'' cells by activating the protease. This is referred as the "Kill Switch". In the final assay we want to have the ability to induce the protease activity detector device and the protease generate independently from each other. But first of all we have to check if our system is working and if we are able to kill the cells by activating the protease.<br><br />
To proof the principle we used the natural TEV protease and make a co-transformation with the protease activity detector device containing the cleavage site for the TEV protease. Both were induced by IPTG and so we can't control the expression rate neither the time of the induction of the protease generator device.<br><br />
After the incubation at 37°C we were able to show the desired proof of principle: when we activate the protease the resistance against ampicillin; based on the export rate of β-lactamase into the periplasm via the TAT pathway, was intimately reduced. The cells with activated TEV protease were able to survive up to concentration of 50 µg/ml ampicillin.<br><br />
[[Image:UP_bla-TEV_double-trans.png|center|500px|thumb|'''Figure 20:''' Percentage of surviving cells on different ampicillin concentration. Blue - cells transformed with the single protease activity detector device for TEV protease; Red - cell transformed with both, the protease activity detector device for TEV protease and the TEV protease itself]]<br><br />
Based on the ampicillin resistance of the plasmid of HRV14_3C protease, we were unable to show the proof of principle for this protease.<br><br />
The next step was to use the complete test vector which contains the protease activity detector device and the protease generator device. We introduced the arabinose inducible induction system fused to the protease flanked by the restriction sites of <i>BamH</i>I and <i>Hind</i>III into the vector of the modified Hitchhiker selection system.<br><br />
With these final vectors we had to repeat the survival screening to show that the system is also working when there is a dual induction based on only one plasmid. The cells were grown to an OD<sub>600</sub> of 0.6 and diluted to an OD<sub>600</sub> of 0.002. 100 µL of this dilution were plated on agar plates with different ampicillin concentrations and induced and induced protease. The results are shown in figure 20.<br><br />
[[Image:UP_SurvTEV_onepla.jpg|center|500px|thumb|'''Figure 21:''' Cell growth on different ampicillin concentration. Blue - cells without induction of the TEV protease; Red - cells with induced TEV protease]]<br><br />
<br><br />
The survival screening assay for TEV protease was performed 30 °C and all plates were incubated for 2 days. The ''E.coli'' XL1 blue cells were able to survive up to ampicilin concentration of 800 µg/mL, which is an increase of 100% of the original resistance capacity. When 2% arabinose is added to the media, the cells showed an intimately reduce surviving rate. The cells were able to survive at ampicilin concentrations up 25 µg/mL.<br><br />
This shows, that we got a high dynamic range from 50 up to 800 µg/mL ampicilin to screen for inhibition of the protease.<br />
<br />
===Transferring and testing the library===<br />
After the successful test of the protease activity we transferred the generated <i>mdnA</i> library into our cells. The following pictures show the amazing results. Cell growth could be detected up to 400 µg Ampicillin/mL.<br />
[[Image:UP_2011-06-01_CM-Kan-Amp_labeled02.jpg|left|420px|thumb|'''Figure 22:''' Cell growth on Ampicillin without induced protease]]<br />
[[Image:UP_2011-06-01_CM-Kan-Ara-Amp_labeled01.jpg|right|420px|thumb|'''Figure 23:''' Cell growth on Ampicillin with induced protease]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
===Background===<br />
====Twin Arginine Translocon (TAT)====<br />
The TAT pathway is responsible for the transport of folded proteins across energy-transducing membranes and it is able of discriminating of unfolded proteins. This transport pathway is common for bacteria, archea and plants and translocates circa 6% of <i>E.coli</i> produced secreted proteins. The translocated proteins must have a signal peptide for targeting of the TAT-transporter, such proteins are e.g. hydrogenases, dehydrogenases and reductases. The signal sequence itself is composed of an N-terminal positive charged domain, a hydrophobic domain and a C-terminal domain.<br><br />
The translocon is composed of three parts: TatA, TatB and TatC. The transport-pore is proposed to be formed during substrate binding by these three parts. The TatA, TatB and TatC proteins may form complexes of different sizes, which on the other hand form pores matching the size of the folded substrate. The transport of proteins through the TAT pathway depends on the proton motive force. Calculations by Adler & Theg showed that the transport of one folded substrate molecule requires the release of approximately 7.9x10<sup>4</sup> protons, which equals 10.000 ATP molecules. The signal sequence is cleaved off after translocation.<br />
<br><br />
[[File:UP_TorA_TATSeq2.jpg|center|450px|thumb|'''Figure 24:''' Schematic representation of TorA-signal sequence. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br><br />
The used signal sequence originates from the TorA protein (Trimethylamin-N-Oxid-Reductase). It is the main respiratory enzyme which reduces TMAO under anaerobic conditions in the periplasm, where it is transported by the TAT pathway.<br />
<br> <br />
[[File:UP_TAT_Pathway2.jpg|center|450px|thumb|'''Figure 25:''' Schematic representation of TAT-dependent pathway. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br />
<br />
<br />
<br><br />
<br />
====Tobacco Etch Virus (TEV) protease====<br />
TEV protease is the common name for the 27 kDa catalytic domain of the Nuclear Inclusion a endopeptidase (NIa) encoded by the tobacco etch virus. TEV protease is a useful reagent for cleaving fusion proteins. It recognizes a linear epitope of the general form E-Xaa-Xaa-Y -Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. In TEV protease the serine nucleophile of the conventional Ser-Asp-His triad is a cysteine instead. This probably explains why TEV protease is resistant to many commonly used protease inhibitors.<br><br />
<br />
====14_3C-Protease====<br />
<br />
The 14_3C protease originates from the human rhinovirus. Rhinoviruses are the most frequent reason for infections of the upper and lower respiratory tract, also known as the cold. Because the 3C protease of human rhinovirus is necessary for the cleavage of the polyprotein translated from viral RNA it may serve as a potential target for development of antiviral targets. The recombinant type 14_3C protease from human rhinovirus (HRV 3C) recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln↓GlyPro. The small, 22-kDa size of the protease got its optimal activity at 4°C but is still very active at 37 °C. It is commonly used for an easy tag removal after the purification of recombinant proteins carrying his-tag. The 14_3C works with a catalytic triade, containing the amino acid residues Ser-Asp-His at its active site.<br><br />
<br />
===References===<br />
*Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. and Bottomley, S.P. (2007) Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-2367<br><br />
*Genest O., Ilbert M., Méjean V., Iobbi-Nivol C.,(2005) TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature. J. Biol. Chem. 280: 15644-15648.<br />
*Kapust R. B., Tözsér J., Fox J. D., Anderson D. E., Cherry S., Copeland T. D., Waugh D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 12:993-1000.<br><br />
*Lee P. A., Tullman-Ercekand D., Georgiou G., (2006) The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60:373–95<br />
*Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30:544-550.<br><br />
*Shih S. R., Chen S. J., Hakimelahi G. H., Liu H. J., Tseng C. T., Shia K. S., (2004) Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting molecules. Med Res Rev 24(4):449-74.<br><br />
*Wanga QM, Chen SH., (2007) Human rhinovirus 3C protease as a potential target for the development of antiviral agents. Curr Protein Pept Sci. 8(1):19-27. <br><br />
*Waraho D., DeLisa M. P., (2009). Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism. PNAS 106(10):3692-7</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_SelectionTeam:Potsdam Bioware/Project/Details Selection2011-10-28T22:19:10Z<p>UP Stefan: /* Construction of two component for the in vivo selection assay */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==<i>In Vivo</i> Selection==<br />
===Introduction===<br />
In addition to the phage display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an <i>in vitro</i> screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for any protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator. For the protease activity detector we modified the TAT-hitchhiker system developed by DeLisa <i>et al.</i> <br />
We used the BioBrick <partinfo>I757010</partinfo> (β-lactamase) as well as <partinfo>K208005</partinfo> (ssTorA) and fused them together via a linker peptide. For the protease generator we cloned the arabinose induction system in front of a protease in iGEM standard.<br> <br />
<br />
[[File:UP_images_TorA-construct-design2_28.10.2011.png.jpg|center|500px|thumb|'''Figure 1:''' Schema of our constructed selection vector containing protease generator and protease activity detector]]<br> <br />
The system works in a combined manner of the two devices. In order to confer β-lactam antibiotic resistance to cells the β-lactamase has to be exported in the periplasm. This transport is mediated by the TorA export sequence via the Twin-Arginine Translocation (TAT) system (DeLisa, 2009). The linker peptide between the TorA export sequence and the β-lactamase displays the corresponding protease cleavage site. In addition the linker peptide is chosen as short as possible to imitate folding because the TAT pathway only allows transport of correctly folded substrates. The protease as well as the linker peptide are designed as exchangeable parts.<br><br />
<br />
If the protease device is functional, it will cleave the linker peptide between TorA and the β-lactamase construct which leaves the cell without any antibiotic resistance. The construct was tested with increasing ampicillin concentrations. The number of surviving colonies was depended on the export rate of the β-lactamase into the periplasm. A high cleavage rate of the linker peptide leads to a reduced ampicillin resistance. With expressed protease a dramatically drop of the number of surviving colonies could be observed.<br><br />
The survival assay was carried out with and without expressed protease. By increasing of the ampicillin concentration we could detect a cutoff ampicillin concentration. The colony forming units (CFU) were counted and compared. To detected whether our constructed library is able to block the protease we co-transformed the library into <i>E. coli</i> carrying our pUP_SG1 plasmid.<br><br />
<br />
===Site-directed mutagenesis of Tobacco Etch Virus and 14_3C proteases in iGEM standard===<br />
<br />
We chose two model proteases, the Tobacco Etch Virus protease, further along called TEV, and the 14_3C protease from the human rhinovirus, also known as PreScission. Both proteases contained iGEM restriction sites, one in case of the TEV and three for 14 3C, respectively. <br><br />
[[File:UP_Pic_SG_Pre-Complete.png|center|800px|thumb|'''Figure 2:''' Complete sequence alignment of the 14_3C protease]]<br />
[[File:UP_Pic_SG_TEV-Complete.png|center|800px|thumb|'''Figure 3:''' Complete sequence alignment of the 14_3C protease]]<br />
<br><br><br />
Thus, site-directed mutagenesis was applied to mutate each restriction site in E. coli. For every mutation a forward and a reverse primer had to be designed. The following pictures show the forward primers for the side directed mutagenesis, the reverse primers are the reverse complement sequence of the forward primers.<br><br />
<br />
For the 14_3C protease:<br />
[[File:UP_Pic_SG_Pre-End.png|left|400px|thumb|'''Figure 4:''' Reverse primer to add the iGEM suffix on the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaSpeI.png|right|400px|thumb|'''Figure 5:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-1.png|left|400px|thumb|'''Figure 6:''' Forward primer to mutate the first XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-2.png|right|400px|thumb|'''Figure 7:''' Forward primer to mutate the second XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_Pre-Start.png|left|400px|thumb|'''Figure 8:''' Forward primer to add the iGEM prefix to the HRV 14_3C protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
For the TEV protease:<br />
<br />
[[File:UP_Pic_SG_TEV-End.png|left|400px|thumb|'''Figure 9:''' Reverse primer to add the iGEM suffix to the TEV protease]]<br />
[[File:UP_Pic_SG_TEVMutaSpeI.png|right|400px|thumb|'''Figure 10:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the TEV protease]]<br />
[[File:UP_Pic_SG_TEV-Start.png|left|400px|thumb|'''Figure 11:''' Forward primer to add the iGEM prefix to the TEV protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
We used assembly PCR to gain the final product. In case of 14_3C two assembly PCRs had to be done. The expected size of the fragments are shown in the gel picture. In the following picture all fragments contain the correct sizes.<br />
<br><br />
[[File:UP_Fragments_14_3C_TEV.png|center|200px|thumb|'''Figure 12:''' Picture of the, via PCR amplified and mutated Fragment of both protease]]<br />
<br><br />
<br />
===Construction of two component for the ''in vivo'' selection assay===<br />
<br />
The ''in vivo'' assay is based on two devices. On the one hand the protease activity detector device, on the other a protease generator device. The detector device is based on the enzyme β-lactamase, which is only active inside the periplasm and provides the resistance against lactam antibiotics like ampicillin and penicillin. For the protease generator device we needed a second induction system on our plasmid, which is quite uncommon for any plasmid. So several problems had to solved to set up an effective ''in vivo'' selection assay:<br />
*a second, very tight induction system has to be found<br />
*a way to control the export rate of β-lactamase into the periplasm, which can be reduced by the protease<br />
<br />
We solved these problems by putting the protease detector device under the control of the lac-operon. The protease device is induced by the arabinose system of the pBAD_iGEMexpress vector. The solution for the export problem of β-lactamase is inspired by Hitchhiker assay of De'Lisa ''et al''. Using the TAT pathway for the export of β-lactamase into the periplasm gives us the ability to introduce a protease specific cleavage site in front of the signal sequence to regulate the export.<br />
<br />
====Introducing the specific protease cleavage site====<br />
To adapt the protease activity detector device to any protease we want to screen, we had to modularize it. Our detector device was set up on a vector for the Hitchhiker selection. The plasmid offers two unique restriction sites on the plasmid, after the signal sequence of TorA one restriction site for <i>Xho</i>I and a <i>Nhe</i>I restriction site in front of the β-lactamase. Both were used to introduce the cleavage site in between the TorA signal sequence and the β-lactamase. By designing two complementary oligonucleotides containing the cleavage site flanked by two short linker regions. After the digest of the vector with XhoI and NheI the hybridized oligonucleotides, containing the overhangs for <i>Xho</i>I and <i>Nhe</i>I can be ligated into the vector.<br />
<br />
====Adding the arabinose inducible induction system to control the proteases, TEV and 14_3C====<br />
We needed a time independent induction of the protease device. Therefore we cloned the arabionse induction system (<i>araC</i>) in front of the protease device out of the pBAD_iGEMexpress vector. The fusion of gene construct and induction system was always performed via an NgoMIV restriction site. This restriction site was introduced with our amplification primers.<br />
[[File:UP_Pic_SG_AraC-End.png|center|800px|thumb|'''Figure 13:''' Reverse primer for the amplification of the arabinose inducible induction system]]<br />
[[File:UP_Pic_SG_AraC-Start.png|center|1400px|thumb|'''Figure 14:''' Forward primer for the amplification of the arabinose inducible induction system]]<br />
We used the RFC 23 cloning strategy to get a fusion protein of our desired protease and the induction system, which allows us to induce the protease. Both genes were amplified with PCR using two special primers generating a NgoMIV restrictioin site at the end of the induction system and in front of the protease. The amplified protease were digested with BamHI and <i>NgoM</i>IV, the arabinose inducible induction system with <i>NgoM</i>IV and <i>Hind</i>III. The vector, which contains the ssTorA_CS-Protease_β-lactamase device was digested with <i>Hind</i>III and <i>BamH</i>I. A triple ligation yielded the final construct with both devices: the protease detector and the protease generator. The two pictures below show the final vector constructs , which where used for the survival screening.<br />
[[File:UP_Pic_SG_TEV_SG1-Plasmid.png|center|800px|thumb|'''Figure 15:''' Final vector construct containing arabinose inducible TEV protease fusion and the protease detector device]]<br />
[[File:UP_Pic_SG_PreSciccion_SG2-Plasmid.png|center|800px|thumb|'''Figure 16:''' Final vector construct containing arabinose inducible HRV 14_3C protease fusion and the protease detector device]]<br />
<br />
===Investigation of the ''in vivo'' selection assay===<br />
<p><br />
<br />
Two major points have to be demonstrated: First, the export of β-lactamase over the TAT pathway has to be efficient enough to provide a certain resistance against ampicillin and second, the activated protease is able to intimately reduce the resistance against ampicillin by cleavage of the TorA signal sequence from the β-lactamase.</p><br />
====Testing the export efficiency of the protease activity detector====<br />
To test the export rate of the β-lactamse of the protease activity detector device we grew an <i>E. coli</i> culture up to an OD<sub>600</sub> of 0.6. The cells were then diluted to an OD of 0.002 and 100 µL were plated in triplicates with increasing ampicillin concentrations. The graph below shows the amount of surviving cells in percentage. <br><br />
<br />
[[Image:UP_Survial-screen_bla-construct.png|center|600px|thumb|'''Figure 17:''' Amount of survived cells. The construct was induced with 1 mM IPTG. The rate of surviving cells without IPTG and without ampicilin was set to 100%. ]]<br />
<br />
[[Image:UP_TEV-Survival.png|center|600px|thumb|'''Figure 18:''' Survival test at different ampicillin concentrations: A) approx. 1000 cfu plated on agar plates containing chloramphenicol; B) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 400µg/ml ampicillin; C) no culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 600µg/ml ampicillin]]<br />
[[Image:UP_14_3C_SurvTest.png|center|600px|thumb|'''Figure 19:''' Survival test at different ampicillin concentrations: <br />
A) approx. 1000 cfu plated on agar plates containing chloramphenicol; <br />
B) Influence of resistance induction with 1 mM IPTG, approx. 1000 cfu where plated on agar plates containing chloramphenicol and 1mM IPTG; C) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 50 µg/ml ampicillin; D) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 100 µg/ml ampicillin; E) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 200 µg/ml ampicillin; F) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 400 µg/ml ampicillin; G) No culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 800 µg/ml ampicillin]]<br />
<br />
This assay was performed at two temperatures. At 37°C, the optimal growth temperature and at 30°C for best protein expression conditions. All assays show the same results. The cells were able to grow at ampicilin concentrations up to 400 µg/ml. Cells transformed with the final construct containing the detection device and the protease generator device were able to survive up to 800 µg/ml ampicilin (incubated at 30°C over 2 days).<br />
<br />
This leads to the conclusion, that the export of β-lactamase into the periplasm is very effective. It is also important to mention, that there is no cell survival when ampicilin is added to the media without the induction of the protease activity detection device by IPTG.<br />
<br />
====Testing the "Kill Switch" by activating the protease====<br />
<br />
The second major point which needs to be demonstrated is that we are able to kill the ''E.coli'' cells by activating the protease. This is referred as the "Kill Switch". In the final assay we want to have the ability to induce the protease activity detector device and the protease generate independently from each other. But first of all we have to check if our system is working and if we are able to kill the cells by activating the protease.<br><br />
To proof the principle we used the natural TEV protease and make a co-transformation with the protease activity detector device containing the cleavage site for the TEV protease. Both were induced by IPTG and so we can't control the expression rate neither the time of the induction of the protease generator device.<br><br />
After the incubation at 37°C we were able to show the desired proof of principle: when we activate the protease the resistance against ampicillin; based on the export rate of β-lactamase into the periplasm via the TAT pathway, was intimately reduced. The cells with activated TEV protease were able to survive up to concentration of 50 µg/ml ampicillin.<br><br />
[[Image:UP_bla-TEV_double-trans.png|center|500px|thumb|'''Figure 20:''' Percentage of surviving cells on different ampicillin concentration. Blue - cells transformed with the single protease activity detector device for TEV protease; Red - cell transformed with both, the protease activity detector device for TEV protease and the TEV protease itself]]<br><br />
Based on the ampicillin resistance of the plasmid of HRV14_3C protease, we were unable to show the proof of principle for this protease.<br><br />
The next step was to use the complete test vector which contains the protease activity detector device and the protease generator device. We introduced the arabinose inducible induction system fused to the protease flanked by the restriction sites of <i>BamH</i>I and <i>Hind</i>III into the vector of the modified Hitchhiker selection system.<br><br />
With these final vectors we had to repeat the survival screening to show that the system is also working when there is a dual induction based on only one plasmid. The cells were grown to an OD<sub>600</sub> of 0.6 and diluted to an OD<sub>600</sub> of 0.002. 100 µL of this dilution were plated on agar plates with different ampicillin concentrations and induced and induced protease. The results are shown in figure 20.<br><br />
[[Image:UP_SurvTEV_onepla.jpg|center|500px|thumb|'''Figure 21:''' Cell growth on different ampicillin concentration. Blue - cells without induction of the TEV protease; Red - cells with induced TEV protease]]<br><br />
<br><br />
The survival screening assay for TEV protease was performed 30 °C and all plates were incubated for 2 days. The ''E.coli'' XL1 blue cells were able to survive up to ampicilin concentration of 800 µg/mL, which is an increase of 100% of the original resistance capacity. When 2% arabinose is added to the media, the cells showed an intimately reduce surviving rate. The cells were able to survive at ampicilin concentrations up 25 µg/mL.<br><br />
This shows, that we got a high dynamic range from 50 up to 800 µg/mL ampicilin to screen for inhibition of the protease.<br />
<br />
===Transferring and testing the library===<br />
After the successful test of the protease activity we transferred the generated <i>mdnA</i> library into our cells. The following pictures show the amazing results. Cell growth could be detected up to 400 µg Ampicillin/mL.<br />
[[Image:UP_2011-06-01_CM-Kan-Amp_labeled02.jpg|left|420px|thumb|'''Figure 22:''' Cell growth on Ampicillin without induced protease]]<br />
[[Image:UP_2011-06-01_CM-Kan-Ara-Amp_labeled01.jpg|right|420px|thumb|'''Figure 23:''' Cell growth on Ampicillin with induced protease]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
===Background===<br />
====Twin Arginine Translocon (TAT)====<br />
The TAT pathway is responsible for the transport of folded proteins across energy-transducing membranes and it is able of discriminating of unfolded proteins. This transport pathway is common for bacteria, archea and plants and translocates circa 6% of <i>E.coli</i> produced secreted proteins. The translocated proteins must have a signal peptide for targeting of the TAT-transporter, such proteins are e.g. hydrogenases, dehydrogenases and reductases. The signal sequence itself is composed of an N-terminal positive charged domain, a hydrophobic domain and a C-terminal domain.<br><br />
The translocon is composed of three parts: TatA, TatB and TatC. The transport-pore is proposed to be formed during substrate binding by these three parts. The TatA, TatB and TatC proteins may form complexes of different sizes, which on the other hand form pores matching the size of the folded substrate. The transport of proteins through the TAT pathway depends on the proton motive force. Calculations by Adler & Theg showed that the transport of one folded substrate molecule requires the release of approximately 7.9x10<sup>4</sup> protons, which equals 10.000 ATP molecules. The signal sequence is cleaved off after translocation.<br />
<br><br />
[[File:UP_TorA_TATSeq2.jpg|center|450px|thumb|'''Figure 24:''' Schematic representation of TorA-signal sequence. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br><br />
The used signal sequence originates from the TorA protein (Trimethylamin-N-Oxid-Reductase). It is the main respiratory enzyme which reduces TMAO under anaerobic conditions in the periplasm, where it is transported by the TAT pathway.<br />
<br> <br />
[[File:UP_TAT_Pathway2.jpg|center|450px|thumb|'''Figure 25:''' Schematic representation of TAT-dependent pathway. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br />
<br />
<br />
<br><br />
<br />
====Tobacco Etch Virus (TEV) protease====<br />
TEV protease is the common name for the 27 kDa catalytic domain of the Nuclear Inclusion a endopeptidase (NIa) encoded by the tobacco etch virus. TEV protease is a useful reagent for cleaving fusion proteins. It recognizes a linear epitope of the general form E-Xaa-Xaa-Y -Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. In TEV protease the serine nucleophile of the conventional Ser-Asp-His triad is a cysteine instead. This probably explains why TEV protease is resistant to many commonly used protease inhibitors.<br><br />
<br />
====14_3C-Protease====<br />
<br />
The 14_3C protease originates from the human rhinovirus. Rhinoviruses are the most frequent reason for infections of the upper and lower respiratory tract, also known as the cold. Because the 3C protease of human rhinovirus is necessary for the cleavage of the polyprotein translated from viral RNA it may serve as a potential target for development of antiviral targets. The recombinant type 14_3C protease from human rhinovirus (HRV 3C) recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln↓GlyPro. The small, 22-kDa size of the protease got its optimal activity at 4°C but is still very active at 37 °C. It is commonly used for an easy tag removal after the purification of recombinant proteins carrying his-tag. The 14_3C works with a catalytic triade, containing the amino acid residues Ser-Asp-His at its active site.<br><br />
<br />
===References===<br />
*Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. and Bottomley, S.P. (2007) Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-2367<br><br />
*Genest O., Ilbert M., Méjean V., Iobbi-Nivol C.,(2005) TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature. J. Biol. Chem. 280: 15644-15648.<br />
*Kapust R. B., Tözsér J., Fox J. D., Anderson D. E., Cherry S., Copeland T. D., Waugh D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 12:993-1000.<br><br />
*Lee P. A., Tullman-Ercekand D., Georgiou G., (2006) The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60:373–95<br />
*Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30:544-550.<br><br />
*Shih S. R., Chen S. J., Hakimelahi G. H., Liu H. J., Tseng C. T., Shia K. S., (2004) Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting molecules. Med Res Rev 24(4):449-74.<br><br />
*Wanga QM, Chen SH., (2007) Human rhinovirus 3C protease as a potential target for the development of antiviral agents. Curr Protein Pept Sci. 8(1):19-27. <br><br />
*Waraho D., DeLisa M. P., (2009). Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism. PNAS 106(10):3692-7</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_SelectionTeam:Potsdam Bioware/Project/Details Selection2011-10-28T22:18:23Z<p>UP Stefan: /* Site-directed mutagenesis of Tobacco Etch Virus and 14_3C proteases in iGEM standard */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==<i>In Vivo</i> Selection==<br />
===Introduction===<br />
In addition to the phage display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an <i>in vitro</i> screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for any protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator. For the protease activity detector we modified the TAT-hitchhiker system developed by DeLisa <i>et al.</i> <br />
We used the BioBrick <partinfo>I757010</partinfo> (β-lactamase) as well as <partinfo>K208005</partinfo> (ssTorA) and fused them together via a linker peptide. For the protease generator we cloned the arabinose induction system in front of a protease in iGEM standard.<br> <br />
<br />
[[File:UP_images_TorA-construct-design2_28.10.2011.png.jpg|center|500px|thumb|'''Figure 1:''' Schema of our constructed selection vector containing protease generator and protease activity detector]]<br> <br />
The system works in a combined manner of the two devices. In order to confer β-lactam antibiotic resistance to cells the β-lactamase has to be exported in the periplasm. This transport is mediated by the TorA export sequence via the Twin-Arginine Translocation (TAT) system (DeLisa, 2009). The linker peptide between the TorA export sequence and the β-lactamase displays the corresponding protease cleavage site. In addition the linker peptide is chosen as short as possible to imitate folding because the TAT pathway only allows transport of correctly folded substrates. The protease as well as the linker peptide are designed as exchangeable parts.<br><br />
<br />
If the protease device is functional, it will cleave the linker peptide between TorA and the β-lactamase construct which leaves the cell without any antibiotic resistance. The construct was tested with increasing ampicillin concentrations. The number of surviving colonies was depended on the export rate of the β-lactamase into the periplasm. A high cleavage rate of the linker peptide leads to a reduced ampicillin resistance. With expressed protease a dramatically drop of the number of surviving colonies could be observed.<br><br />
The survival assay was carried out with and without expressed protease. By increasing of the ampicillin concentration we could detect a cutoff ampicillin concentration. The colony forming units (CFU) were counted and compared. To detected whether our constructed library is able to block the protease we co-transformed the library into <i>E. coli</i> carrying our pUP_SG1 plasmid.<br><br />
<br />
===Site-directed mutagenesis of Tobacco Etch Virus and 14_3C proteases in iGEM standard===<br />
<br />
We chose two model proteases, the Tobacco Etch Virus protease, further along called TEV, and the 14_3C protease from the human rhinovirus, also known as PreScission. Both proteases contained iGEM restriction sites, one in case of the TEV and three for 14 3C, respectively. <br><br />
[[File:UP_Pic_SG_Pre-Complete.png|center|800px|thumb|'''Figure 2:''' Complete sequence alignment of the 14_3C protease]]<br />
[[File:UP_Pic_SG_TEV-Complete.png|center|800px|thumb|'''Figure 3:''' Complete sequence alignment of the 14_3C protease]]<br />
<br><br><br />
Thus, site-directed mutagenesis was applied to mutate each restriction site in E. coli. For every mutation a forward and a reverse primer had to be designed. The following pictures show the forward primers for the side directed mutagenesis, the reverse primers are the reverse complement sequence of the forward primers.<br><br />
<br />
For the 14_3C protease:<br />
[[File:UP_Pic_SG_Pre-End.png|left|400px|thumb|'''Figure 4:''' Reverse primer to add the iGEM suffix on the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaSpeI.png|right|400px|thumb|'''Figure 5:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-1.png|left|400px|thumb|'''Figure 6:''' Forward primer to mutate the first XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-2.png|right|400px|thumb|'''Figure 7:''' Forward primer to mutate the second XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_Pre-Start.png|left|400px|thumb|'''Figure 8:''' Forward primer to add the iGEM prefix to the HRV 14_3C protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
For the TEV protease:<br />
<br />
[[File:UP_Pic_SG_TEV-End.png|left|400px|thumb|'''Figure 9:''' Reverse primer to add the iGEM suffix to the TEV protease]]<br />
[[File:UP_Pic_SG_TEVMutaSpeI.png|right|400px|thumb|'''Figure 10:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the TEV protease]]<br />
[[File:UP_Pic_SG_TEV-Start.png|left|400px|thumb|'''Figure 11:''' Forward primer to add the iGEM prefix to the TEV protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
We used assembly PCR to gain the final product. In case of 14_3C two assembly PCRs had to be done. The expected size of the fragments are shown in the gel picture. In the following picture all fragments contain the correct sizes.<br />
<br><br />
[[File:UP_Fragments_14_3C_TEV.png|center|200px|thumb|'''Figure 12:''' Picture of the, via PCR amplified and mutated Fragment of both protease]]<br />
<br><br />
<br />
===Construction of two component for the ''in vivo'' selection assay===<br />
<br />
The ''in vivo'' assay is based on two devices. On the one hand the protease activity detector device, on the other a protease generator device. The detector device is based on the enzyme β-lactamase, which is only active inside the periplasm and provides the resistance against lactam antibiotics like ampicillin and penicillin. For the protease generator device we needed a second induction system on our plasmid, which is quite uncommon for any plasmid. So several problems had to solved to set up an effective ''in vivo'' selection assay:<br />
*a second, very tight induction system has to be found<br />
*a way to control the export rate of β-lactamase into the periplasm, which can be reduce by the protease<br />
<br />
We solved these problems by putting the protease detector device under the control of the lac-operon. The protease device is induced by the arabinose system of the pBAD_iGEMexpress vector. The solution for the export problem of β-lactamase is inspired by Hitchhiker assay of De'Lisa ''et al''. Using the TAT pathway for the export of β-lactamase into the periplasm gives us the ability to introduce a protease specific cleavage site in front of the signal sequence to regulate the export.<br />
<br />
====Introducing the specific protease cleavage site====<br />
To adapt the protease activity detector device to any protease we want to screen, we had to modularize it. Our detector device was set up on a vector for the Hitchhiker selection. The plasmid offers two unique restriction sites on the plasmid, after the signal sequence of TorA one restriction site for <i>Xho</i>I and a <i>Nhe</i>I restriction site in front of the β-lactamase. Both were used to introduce the cleavage site in between the TorA signal sequence and the β-lactamase. By designing two complementary oligonucleotides containing the cleavage site flanked by two short linker regions. After the digest of the vector with XhoI and NheI the hybridized oligonucleotides, containing the overhangs for <i>Xho</i>I and <i>Nhe</i>I can be ligated into the vector.<br />
<br />
====Adding the arabinose inducible induction system to control the proteases, TEV and 14_3C====<br />
We needed a time independent induction of the protease device. Therefore we cloned the arabionse induction system (<i>araC</i>) in front of the protease device out of the pBAD_iGEMexpress vector. The fusion of gene construct and induction system was always performed via an NgoMIV restriction site. This restriction site was introduced with our amplification primers.<br />
[[File:UP_Pic_SG_AraC-End.png|center|800px|thumb|'''Figure 13:''' Reverse primer for the amplification of the arabinose inducible induction system]]<br />
[[File:UP_Pic_SG_AraC-Start.png|center|1400px|thumb|'''Figure 14:''' Forward primer for the amplification of the arabinose inducible induction system]]<br />
We used the RFC 23 cloning strategy to get a fusion protein of our desired protease and the induction system, which allows us to induce the protease. Both genes were amplified with PCR using two special primers generating a NgoMIV restrictioin site at the end of the induction system and in front of the protease. The amplified protease were digested with BamHI and <i>NgoM</i>IV, the arabinose inducible induction system with <i>NgoM</i>IV and <i>Hind</i>III. The vector, which contains the ssTorA_CS-Protease_β-lactamase device was digested with <i>Hind</i>III and <i>BamH</i>I. A triple ligation yielded the final construct with both devices: the protease detector and the protease generator. The two pictures below show the final vector constructs , which where used for the survival screening.<br />
[[File:UP_Pic_SG_TEV_SG1-Plasmid.png|center|800px|thumb|'''Figure 15:''' Final vector construct containing arabinose inducible TEV protease fusion and the protease detector device]]<br />
[[File:UP_Pic_SG_PreSciccion_SG2-Plasmid.png|center|800px|thumb|'''Figure 16:''' Final vector construct containing arabinose inducible HRV 14_3C protease fusion and the protease detector device]]<br />
<br />
===Investigation of the ''in vivo'' selection assay===<br />
<p><br />
<br />
Two major points have to be demonstrated: First, the export of β-lactamase over the TAT pathway has to be efficient enough to provide a certain resistance against ampicillin and second, the activated protease is able to intimately reduce the resistance against ampicillin by cleavage of the TorA signal sequence from the β-lactamase.</p><br />
====Testing the export efficiency of the protease activity detector====<br />
To test the export rate of the β-lactamse of the protease activity detector device we grew an <i>E. coli</i> culture up to an OD<sub>600</sub> of 0.6. The cells were then diluted to an OD of 0.002 and 100 µL were plated in triplicates with increasing ampicillin concentrations. The graph below shows the amount of surviving cells in percentage. <br><br />
<br />
[[Image:UP_Survial-screen_bla-construct.png|center|600px|thumb|'''Figure 17:''' Amount of survived cells. The construct was induced with 1 mM IPTG. The rate of surviving cells without IPTG and without ampicilin was set to 100%. ]]<br />
<br />
[[Image:UP_TEV-Survival.png|center|600px|thumb|'''Figure 18:''' Survival test at different ampicillin concentrations: A) approx. 1000 cfu plated on agar plates containing chloramphenicol; B) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 400µg/ml ampicillin; C) no culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 600µg/ml ampicillin]]<br />
[[Image:UP_14_3C_SurvTest.png|center|600px|thumb|'''Figure 19:''' Survival test at different ampicillin concentrations: <br />
A) approx. 1000 cfu plated on agar plates containing chloramphenicol; <br />
B) Influence of resistance induction with 1 mM IPTG, approx. 1000 cfu where plated on agar plates containing chloramphenicol and 1mM IPTG; C) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 50 µg/ml ampicillin; D) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 100 µg/ml ampicillin; E) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 200 µg/ml ampicillin; F) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 400 µg/ml ampicillin; G) No culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 800 µg/ml ampicillin]]<br />
<br />
This assay was performed at two temperatures. At 37°C, the optimal growth temperature and at 30°C for best protein expression conditions. All assays show the same results. The cells were able to grow at ampicilin concentrations up to 400 µg/ml. Cells transformed with the final construct containing the detection device and the protease generator device were able to survive up to 800 µg/ml ampicilin (incubated at 30°C over 2 days).<br />
<br />
This leads to the conclusion, that the export of β-lactamase into the periplasm is very effective. It is also important to mention, that there is no cell survival when ampicilin is added to the media without the induction of the protease activity detection device by IPTG.<br />
<br />
====Testing the "Kill Switch" by activating the protease====<br />
<br />
The second major point which needs to be demonstrated is that we are able to kill the ''E.coli'' cells by activating the protease. This is referred as the "Kill Switch". In the final assay we want to have the ability to induce the protease activity detector device and the protease generate independently from each other. But first of all we have to check if our system is working and if we are able to kill the cells by activating the protease.<br><br />
To proof the principle we used the natural TEV protease and make a co-transformation with the protease activity detector device containing the cleavage site for the TEV protease. Both were induced by IPTG and so we can't control the expression rate neither the time of the induction of the protease generator device.<br><br />
After the incubation at 37°C we were able to show the desired proof of principle: when we activate the protease the resistance against ampicillin; based on the export rate of β-lactamase into the periplasm via the TAT pathway, was intimately reduced. The cells with activated TEV protease were able to survive up to concentration of 50 µg/ml ampicillin.<br><br />
[[Image:UP_bla-TEV_double-trans.png|center|500px|thumb|'''Figure 20:''' Percentage of surviving cells on different ampicillin concentration. Blue - cells transformed with the single protease activity detector device for TEV protease; Red - cell transformed with both, the protease activity detector device for TEV protease and the TEV protease itself]]<br><br />
Based on the ampicillin resistance of the plasmid of HRV14_3C protease, we were unable to show the proof of principle for this protease.<br><br />
The next step was to use the complete test vector which contains the protease activity detector device and the protease generator device. We introduced the arabinose inducible induction system fused to the protease flanked by the restriction sites of <i>BamH</i>I and <i>Hind</i>III into the vector of the modified Hitchhiker selection system.<br><br />
With these final vectors we had to repeat the survival screening to show that the system is also working when there is a dual induction based on only one plasmid. The cells were grown to an OD<sub>600</sub> of 0.6 and diluted to an OD<sub>600</sub> of 0.002. 100 µL of this dilution were plated on agar plates with different ampicillin concentrations and induced and induced protease. The results are shown in figure 20.<br><br />
[[Image:UP_SurvTEV_onepla.jpg|center|500px|thumb|'''Figure 21:''' Cell growth on different ampicillin concentration. Blue - cells without induction of the TEV protease; Red - cells with induced TEV protease]]<br><br />
<br><br />
The survival screening assay for TEV protease was performed 30 °C and all plates were incubated for 2 days. The ''E.coli'' XL1 blue cells were able to survive up to ampicilin concentration of 800 µg/mL, which is an increase of 100% of the original resistance capacity. When 2% arabinose is added to the media, the cells showed an intimately reduce surviving rate. The cells were able to survive at ampicilin concentrations up 25 µg/mL.<br><br />
This shows, that we got a high dynamic range from 50 up to 800 µg/mL ampicilin to screen for inhibition of the protease.<br />
<br />
===Transferring and testing the library===<br />
After the successful test of the protease activity we transferred the generated <i>mdnA</i> library into our cells. The following pictures show the amazing results. Cell growth could be detected up to 400 µg Ampicillin/mL.<br />
[[Image:UP_2011-06-01_CM-Kan-Amp_labeled02.jpg|left|420px|thumb|'''Figure 22:''' Cell growth on Ampicillin without induced protease]]<br />
[[Image:UP_2011-06-01_CM-Kan-Ara-Amp_labeled01.jpg|right|420px|thumb|'''Figure 23:''' Cell growth on Ampicillin with induced protease]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
===Background===<br />
====Twin Arginine Translocon (TAT)====<br />
The TAT pathway is responsible for the transport of folded proteins across energy-transducing membranes and it is able of discriminating of unfolded proteins. This transport pathway is common for bacteria, archea and plants and translocates circa 6% of <i>E.coli</i> produced secreted proteins. The translocated proteins must have a signal peptide for targeting of the TAT-transporter, such proteins are e.g. hydrogenases, dehydrogenases and reductases. The signal sequence itself is composed of an N-terminal positive charged domain, a hydrophobic domain and a C-terminal domain.<br><br />
The translocon is composed of three parts: TatA, TatB and TatC. The transport-pore is proposed to be formed during substrate binding by these three parts. The TatA, TatB and TatC proteins may form complexes of different sizes, which on the other hand form pores matching the size of the folded substrate. The transport of proteins through the TAT pathway depends on the proton motive force. Calculations by Adler & Theg showed that the transport of one folded substrate molecule requires the release of approximately 7.9x10<sup>4</sup> protons, which equals 10.000 ATP molecules. The signal sequence is cleaved off after translocation.<br />
<br><br />
[[File:UP_TorA_TATSeq2.jpg|center|450px|thumb|'''Figure 24:''' Schematic representation of TorA-signal sequence. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br><br />
The used signal sequence originates from the TorA protein (Trimethylamin-N-Oxid-Reductase). It is the main respiratory enzyme which reduces TMAO under anaerobic conditions in the periplasm, where it is transported by the TAT pathway.<br />
<br> <br />
[[File:UP_TAT_Pathway2.jpg|center|450px|thumb|'''Figure 25:''' Schematic representation of TAT-dependent pathway. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br />
<br />
<br />
<br><br />
<br />
====Tobacco Etch Virus (TEV) protease====<br />
TEV protease is the common name for the 27 kDa catalytic domain of the Nuclear Inclusion a endopeptidase (NIa) encoded by the tobacco etch virus. TEV protease is a useful reagent for cleaving fusion proteins. It recognizes a linear epitope of the general form E-Xaa-Xaa-Y -Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. In TEV protease the serine nucleophile of the conventional Ser-Asp-His triad is a cysteine instead. This probably explains why TEV protease is resistant to many commonly used protease inhibitors.<br><br />
<br />
====14_3C-Protease====<br />
<br />
The 14_3C protease originates from the human rhinovirus. Rhinoviruses are the most frequent reason for infections of the upper and lower respiratory tract, also known as the cold. Because the 3C protease of human rhinovirus is necessary for the cleavage of the polyprotein translated from viral RNA it may serve as a potential target for development of antiviral targets. The recombinant type 14_3C protease from human rhinovirus (HRV 3C) recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln↓GlyPro. The small, 22-kDa size of the protease got its optimal activity at 4°C but is still very active at 37 °C. It is commonly used for an easy tag removal after the purification of recombinant proteins carrying his-tag. The 14_3C works with a catalytic triade, containing the amino acid residues Ser-Asp-His at its active site.<br><br />
<br />
===References===<br />
*Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. and Bottomley, S.P. (2007) Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-2367<br><br />
*Genest O., Ilbert M., Méjean V., Iobbi-Nivol C.,(2005) TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature. J. Biol. Chem. 280: 15644-15648.<br />
*Kapust R. B., Tözsér J., Fox J. D., Anderson D. E., Cherry S., Copeland T. D., Waugh D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 12:993-1000.<br><br />
*Lee P. A., Tullman-Ercekand D., Georgiou G., (2006) The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60:373–95<br />
*Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30:544-550.<br><br />
*Shih S. R., Chen S. J., Hakimelahi G. H., Liu H. J., Tseng C. T., Shia K. S., (2004) Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting molecules. Med Res Rev 24(4):449-74.<br><br />
*Wanga QM, Chen SH., (2007) Human rhinovirus 3C protease as a potential target for the development of antiviral agents. Curr Protein Pept Sci. 8(1):19-27. <br><br />
*Waraho D., DeLisa M. P., (2009). Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism. PNAS 106(10):3692-7</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_SelectionTeam:Potsdam Bioware/Project/Details Selection2011-10-28T22:16:53Z<p>UP Stefan: /* Introduction */</p>
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<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
<br />
==<i>In Vivo</i> Selection==<br />
===Introduction===<br />
In addition to the phage display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an <i>in vitro</i> screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for any protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator. For the protease activity detector we modified the TAT-hitchhiker system developed by DeLisa <i>et al.</i> <br />
We used the BioBrick <partinfo>I757010</partinfo> (β-lactamase) as well as <partinfo>K208005</partinfo> (ssTorA) and fused them together via a linker peptide. For the protease generator we cloned the arabinose induction system in front of a protease in iGEM standard.<br> <br />
<br />
[[File:UP_images_TorA-construct-design2_28.10.2011.png.jpg|center|500px|thumb|'''Figure 1:''' Schema of our constructed selection vector containing protease generator and protease activity detector]]<br> <br />
The system works in a combined manner of the two devices. In order to confer β-lactam antibiotic resistance to cells the β-lactamase has to be exported in the periplasm. This transport is mediated by the TorA export sequence via the Twin-Arginine Translocation (TAT) system (DeLisa, 2009). The linker peptide between the TorA export sequence and the β-lactamase displays the corresponding protease cleavage site. In addition the linker peptide is chosen as short as possible to imitate folding because the TAT pathway only allows transport of correctly folded substrates. The protease as well as the linker peptide are designed as exchangeable parts.<br><br />
<br />
If the protease device is functional, it will cleave the linker peptide between TorA and the β-lactamase construct which leaves the cell without any antibiotic resistance. The construct was tested with increasing ampicillin concentrations. The number of surviving colonies was depended on the export rate of the β-lactamase into the periplasm. A high cleavage rate of the linker peptide leads to a reduced ampicillin resistance. With expressed protease a dramatically drop of the number of surviving colonies could be observed.<br><br />
The survival assay was carried out with and without expressed protease. By increasing of the ampicillin concentration we could detect a cutoff ampicillin concentration. The colony forming units (CFU) were counted and compared. To detected whether our constructed library is able to block the protease we co-transformed the library into <i>E. coli</i> carrying our pUP_SG1 plasmid.<br><br />
<br />
===Site-directed mutagenesis of Tobacco Etch Virus and 14_3C proteases in iGEM standard===<br />
<br />
We chose two model proteases, the Tobacco Etch Virus protease, further along called TEV, and the 14_3C protease from the human rhinovirus, also known as PreScission. Both proteases contained iGEM restriction sites, one in case of the TEV and three for 14 3C, respectively. <br><br />
[[File:UP_Pic_SG_Pre-Complete.png|center|800px|thumb|'''Figure 2:''' Complete sequence alignment of the 14_3C protease]]<br />
[[File:UP_Pic_SG_TEV-Complete.png|center|800px|thumb|'''Figure 3:''' Complete sequence alignment of the 14_3C protease]]<br />
<br><br><br />
Thus, site-directed mutagenesis was applied to mutate each restriction site in E. coli. For every mutation a forward and a reverse primer had to be designed. The following pictures show the forward primers for the side directed mutagenesis, the reverse primers are the reverse complement sequence of the forward primers.<br><br />
<br />
For the 14_3C protease:<br />
[[File:UP_Pic_SG_Pre-End.png|left|400px|thumb|'''Figure 4:''' Reverse primer to add the iGEM suffix on the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaSpeI.png|right|400px|thumb|'''Figure 5:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-1.png|left|400px|thumb|'''Figure 6:''' Forward primer to mutate the first XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_PreMutaXbaI-2.png|right|400px|thumb|'''Figure 7:''' Forward primer to mutate the second XbaI restriction site in the gene sequence of the HRV 14_3C protease]]<br />
[[File:UP_Pic_SG_Pre-Start.png|left|400px|thumb|'''Figure 8:''' Forward primer to add the iGEM prefix to the HRV 14_3C protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
For the TEV protease:<br />
<br />
[[File:UP_Pic_SG_TEV-End.png|left|400px|thumb|'''Figure 9:''' Reverse primer to add the iGEM suffix to the TEV protease]]<br />
[[File:UP_Pic_SG_TEVMutaSpeI.png|right|400px|thumb|'''Figure 10:''' Forward primer to mutate the SpeI restriction site in the gene sequence of the TEV protease]]<br />
[[File:UP_Pic_SG_TEV-Start.png|left|400px|thumb|'''Figure 11:''' Forward primer to add the iGEM prefix to the TEV protease]]<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
We used assembly PCR to gain the final product. In case of PreScission two assembly PCRs had to be done. The expected size of the fragments are shown in the gel picture. In the following picture all fragments contain the correct sizes.<br />
<br><br />
[[File:UP_Fragments_14_3C_TEV.png|center|200px|thumb|'''Figure 12:''' Picture of the, via PCR amplified and mutated Fragment of both protease]]<br />
<br><br />
<br />
===Construction of two component for the ''in vivo'' selection assay===<br />
<br />
The ''in vivo'' assay is based on two devices. On the one hand the protease activity detector device, on the other a protease generator device. The detector device is based on the enzyme β-lactamase, which is only active inside the periplasm and provides the resistance against lactam antibiotics like ampicillin and penicillin. For the protease generator device we needed a second induction system on our plasmid, which is quite uncommon for any plasmid. So several problems had to solved to set up an effective ''in vivo'' selection assay:<br />
*a second, very tight induction system has to be found<br />
*a way to control the export rate of β-lactamase into the periplasm, which can be reduce by the protease<br />
<br />
We solved these problems by putting the protease detector device under the control of the lac-operon. The protease device is induced by the arabinose system of the pBAD_iGEMexpress vector. The solution for the export problem of β-lactamase is inspired by Hitchhiker assay of De'Lisa ''et al''. Using the TAT pathway for the export of β-lactamase into the periplasm gives us the ability to introduce a protease specific cleavage site in front of the signal sequence to regulate the export.<br />
<br />
====Introducing the specific protease cleavage site====<br />
To adapt the protease activity detector device to any protease we want to screen, we had to modularize it. Our detector device was set up on a vector for the Hitchhiker selection. The plasmid offers two unique restriction sites on the plasmid, after the signal sequence of TorA one restriction site for <i>Xho</i>I and a <i>Nhe</i>I restriction site in front of the β-lactamase. Both were used to introduce the cleavage site in between the TorA signal sequence and the β-lactamase. By designing two complementary oligonucleotides containing the cleavage site flanked by two short linker regions. After the digest of the vector with XhoI and NheI the hybridized oligonucleotides, containing the overhangs for <i>Xho</i>I and <i>Nhe</i>I can be ligated into the vector.<br />
<br />
====Adding the arabinose inducible induction system to control the proteases, TEV and 14_3C====<br />
We needed a time independent induction of the protease device. Therefore we cloned the arabionse induction system (<i>araC</i>) in front of the protease device out of the pBAD_iGEMexpress vector. The fusion of gene construct and induction system was always performed via an NgoMIV restriction site. This restriction site was introduced with our amplification primers.<br />
[[File:UP_Pic_SG_AraC-End.png|center|800px|thumb|'''Figure 13:''' Reverse primer for the amplification of the arabinose inducible induction system]]<br />
[[File:UP_Pic_SG_AraC-Start.png|center|1400px|thumb|'''Figure 14:''' Forward primer for the amplification of the arabinose inducible induction system]]<br />
We used the RFC 23 cloning strategy to get a fusion protein of our desired protease and the induction system, which allows us to induce the protease. Both genes were amplified with PCR using two special primers generating a NgoMIV restrictioin site at the end of the induction system and in front of the protease. The amplified protease were digested with BamHI and <i>NgoM</i>IV, the arabinose inducible induction system with <i>NgoM</i>IV and <i>Hind</i>III. The vector, which contains the ssTorA_CS-Protease_β-lactamase device was digested with <i>Hind</i>III and <i>BamH</i>I. A triple ligation yielded the final construct with both devices: the protease detector and the protease generator. The two pictures below show the final vector constructs , which where used for the survival screening.<br />
[[File:UP_Pic_SG_TEV_SG1-Plasmid.png|center|800px|thumb|'''Figure 15:''' Final vector construct containing arabinose inducible TEV protease fusion and the protease detector device]]<br />
[[File:UP_Pic_SG_PreSciccion_SG2-Plasmid.png|center|800px|thumb|'''Figure 16:''' Final vector construct containing arabinose inducible HRV 14_3C protease fusion and the protease detector device]]<br />
<br />
===Investigation of the ''in vivo'' selection assay===<br />
<p><br />
<br />
Two major points have to be demonstrated: First, the export of β-lactamase over the TAT pathway has to be efficient enough to provide a certain resistance against ampicillin and second, the activated protease is able to intimately reduce the resistance against ampicillin by cleavage of the TorA signal sequence from the β-lactamase.</p><br />
====Testing the export efficiency of the protease activity detector====<br />
To test the export rate of the β-lactamse of the protease activity detector device we grew an <i>E. coli</i> culture up to an OD<sub>600</sub> of 0.6. The cells were then diluted to an OD of 0.002 and 100 µL were plated in triplicates with increasing ampicillin concentrations. The graph below shows the amount of surviving cells in percentage. <br><br />
<br />
[[Image:UP_Survial-screen_bla-construct.png|center|600px|thumb|'''Figure 17:''' Amount of survived cells. The construct was induced with 1 mM IPTG. The rate of surviving cells without IPTG and without ampicilin was set to 100%. ]]<br />
<br />
[[Image:UP_TEV-Survival.png|center|600px|thumb|'''Figure 18:''' Survival test at different ampicillin concentrations: A) approx. 1000 cfu plated on agar plates containing chloramphenicol; B) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 400µg/ml ampicillin; C) no culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1mM IPTG to induce ampicillin resistance via BBa_K627012 and 600µg/ml ampicillin]]<br />
[[Image:UP_14_3C_SurvTest.png|center|600px|thumb|'''Figure 19:''' Survival test at different ampicillin concentrations: <br />
A) approx. 1000 cfu plated on agar plates containing chloramphenicol; <br />
B) Influence of resistance induction with 1 mM IPTG, approx. 1000 cfu where plated on agar plates containing chloramphenicol and 1mM IPTG; C) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 50 µg/ml ampicillin; D) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 100 µg/ml ampicillin; E) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 200 µg/ml ampicillin; F) Reduced amount of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 400 µg/ml ampicillin; G) No culture growth of <i>E.coli</i> colonies, approx. 1000 cfu were plated on agar plates containing chloramphenicol, 1 mM IPTG to induce ampicillin resistance via BBa_K627013 and 800 µg/ml ampicillin]]<br />
<br />
This assay was performed at two temperatures. At 37°C, the optimal growth temperature and at 30°C for best protein expression conditions. All assays show the same results. The cells were able to grow at ampicilin concentrations up to 400 µg/ml. Cells transformed with the final construct containing the detection device and the protease generator device were able to survive up to 800 µg/ml ampicilin (incubated at 30°C over 2 days).<br />
<br />
This leads to the conclusion, that the export of β-lactamase into the periplasm is very effective. It is also important to mention, that there is no cell survival when ampicilin is added to the media without the induction of the protease activity detection device by IPTG.<br />
<br />
====Testing the "Kill Switch" by activating the protease====<br />
<br />
The second major point which needs to be demonstrated is that we are able to kill the ''E.coli'' cells by activating the protease. This is referred as the "Kill Switch". In the final assay we want to have the ability to induce the protease activity detector device and the protease generate independently from each other. But first of all we have to check if our system is working and if we are able to kill the cells by activating the protease.<br><br />
To proof the principle we used the natural TEV protease and make a co-transformation with the protease activity detector device containing the cleavage site for the TEV protease. Both were induced by IPTG and so we can't control the expression rate neither the time of the induction of the protease generator device.<br><br />
After the incubation at 37°C we were able to show the desired proof of principle: when we activate the protease the resistance against ampicillin; based on the export rate of β-lactamase into the periplasm via the TAT pathway, was intimately reduced. The cells with activated TEV protease were able to survive up to concentration of 50 µg/ml ampicillin.<br><br />
[[Image:UP_bla-TEV_double-trans.png|center|500px|thumb|'''Figure 20:''' Percentage of surviving cells on different ampicillin concentration. Blue - cells transformed with the single protease activity detector device for TEV protease; Red - cell transformed with both, the protease activity detector device for TEV protease and the TEV protease itself]]<br><br />
Based on the ampicillin resistance of the plasmid of HRV14_3C protease, we were unable to show the proof of principle for this protease.<br><br />
The next step was to use the complete test vector which contains the protease activity detector device and the protease generator device. We introduced the arabinose inducible induction system fused to the protease flanked by the restriction sites of <i>BamH</i>I and <i>Hind</i>III into the vector of the modified Hitchhiker selection system.<br><br />
With these final vectors we had to repeat the survival screening to show that the system is also working when there is a dual induction based on only one plasmid. The cells were grown to an OD<sub>600</sub> of 0.6 and diluted to an OD<sub>600</sub> of 0.002. 100 µL of this dilution were plated on agar plates with different ampicillin concentrations and induced and induced protease. The results are shown in figure 20.<br><br />
[[Image:UP_SurvTEV_onepla.jpg|center|500px|thumb|'''Figure 21:''' Cell growth on different ampicillin concentration. Blue - cells without induction of the TEV protease; Red - cells with induced TEV protease]]<br><br />
<br><br />
The survival screening assay for TEV protease was performed 30 °C and all plates were incubated for 2 days. The ''E.coli'' XL1 blue cells were able to survive up to ampicilin concentration of 800 µg/mL, which is an increase of 100% of the original resistance capacity. When 2% arabinose is added to the media, the cells showed an intimately reduce surviving rate. The cells were able to survive at ampicilin concentrations up 25 µg/mL.<br><br />
This shows, that we got a high dynamic range from 50 up to 800 µg/mL ampicilin to screen for inhibition of the protease.<br />
<br />
===Transferring and testing the library===<br />
After the successful test of the protease activity we transferred the generated <i>mdnA</i> library into our cells. The following pictures show the amazing results. Cell growth could be detected up to 400 µg Ampicillin/mL.<br />
[[Image:UP_2011-06-01_CM-Kan-Amp_labeled02.jpg|left|420px|thumb|'''Figure 22:''' Cell growth on Ampicillin without induced protease]]<br />
[[Image:UP_2011-06-01_CM-Kan-Ara-Amp_labeled01.jpg|right|420px|thumb|'''Figure 23:''' Cell growth on Ampicillin with induced protease]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br><br><br><br><br />
<br />
===Background===<br />
====Twin Arginine Translocon (TAT)====<br />
The TAT pathway is responsible for the transport of folded proteins across energy-transducing membranes and it is able of discriminating of unfolded proteins. This transport pathway is common for bacteria, archea and plants and translocates circa 6% of <i>E.coli</i> produced secreted proteins. The translocated proteins must have a signal peptide for targeting of the TAT-transporter, such proteins are e.g. hydrogenases, dehydrogenases and reductases. The signal sequence itself is composed of an N-terminal positive charged domain, a hydrophobic domain and a C-terminal domain.<br><br />
The translocon is composed of three parts: TatA, TatB and TatC. The transport-pore is proposed to be formed during substrate binding by these three parts. The TatA, TatB and TatC proteins may form complexes of different sizes, which on the other hand form pores matching the size of the folded substrate. The transport of proteins through the TAT pathway depends on the proton motive force. Calculations by Adler & Theg showed that the transport of one folded substrate molecule requires the release of approximately 7.9x10<sup>4</sup> protons, which equals 10.000 ATP molecules. The signal sequence is cleaved off after translocation.<br />
<br><br />
[[File:UP_TorA_TATSeq2.jpg|center|450px|thumb|'''Figure 24:''' Schematic representation of TorA-signal sequence. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br><br />
The used signal sequence originates from the TorA protein (Trimethylamin-N-Oxid-Reductase). It is the main respiratory enzyme which reduces TMAO under anaerobic conditions in the periplasm, where it is transported by the TAT pathway.<br />
<br> <br />
[[File:UP_TAT_Pathway2.jpg|center|450px|thumb|'''Figure 25:''' Schematic representation of TAT-dependent pathway. Figure adopted from: Philip A. Lee, Danielle Tullman-Ercekand George Georgiou Annu. Rev. Microbiol. 2006. 60:373–95]]<br />
<br />
<br />
<br />
<br><br />
<br />
====Tobacco Etch Virus (TEV) protease====<br />
TEV protease is the common name for the 27 kDa catalytic domain of the Nuclear Inclusion a endopeptidase (NIa) encoded by the tobacco etch virus. TEV protease is a useful reagent for cleaving fusion proteins. It recognizes a linear epitope of the general form E-Xaa-Xaa-Y -Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. In TEV protease the serine nucleophile of the conventional Ser-Asp-His triad is a cysteine instead. This probably explains why TEV protease is resistant to many commonly used protease inhibitors.<br><br />
<br />
====14_3C-Protease====<br />
<br />
The 14_3C protease originates from the human rhinovirus. Rhinoviruses are the most frequent reason for infections of the upper and lower respiratory tract, also known as the cold. Because the 3C protease of human rhinovirus is necessary for the cleavage of the polyprotein translated from viral RNA it may serve as a potential target for development of antiviral targets. The recombinant type 14_3C protease from human rhinovirus (HRV 3C) recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln↓GlyPro. The small, 22-kDa size of the protease got its optimal activity at 4°C but is still very active at 37 °C. It is commonly used for an easy tag removal after the purification of recombinant proteins carrying his-tag. The 14_3C works with a catalytic triade, containing the amino acid residues Ser-Asp-His at its active site.<br><br />
<br />
===References===<br />
*Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M. and Bottomley, S.P. (2007) Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16: 2360-2367<br><br />
*Genest O., Ilbert M., Méjean V., Iobbi-Nivol C.,(2005) TorD, an Essential Chaperone for TorA Molybdoenzyme Maturation at High Temperature. J. Biol. Chem. 280: 15644-15648.<br />
*Kapust R. B., Tözsér J., Fox J. D., Anderson D. E., Cherry S., Copeland T. D., Waugh D. S. (2001) Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 12:993-1000.<br><br />
*Lee P. A., Tullman-Ercekand D., Georgiou G., (2006) The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol. 60:373–95<br />
*Lucast, L. J., Batey, R. T., and Doudna, J. A. (2001). Large-scale purification of a stable form of recombinant tobacco etch virus protease. Biotechniques 30:544-550.<br><br />
*Shih S. R., Chen S. J., Hakimelahi G. H., Liu H. J., Tseng C. T., Shia K. S., (2004) Selective human enterovirus and rhinovirus inhibitors: An overview of capsid-binding and protease-inhibiting molecules. Med Res Rev 24(4):449-74.<br><br />
*Wanga QM, Chen SH., (2007) Human rhinovirus 3C protease as a potential target for the development of antiviral agents. Curr Protein Pept Sci. 8(1):19-27. <br><br />
*Waraho D., DeLisa M. P., (2009). Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial hitchhiker transport mechanism. PNAS 106(10):3692-7</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/SummaryTeam:Potsdam Bioware/Project/Summary2011-10-28T22:15:15Z<p>UP Stefan: /* In Vivo Selection */</p>
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<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Summary ==<br />
<br />
===Modification, Selection and Production of Cyclic Peptides for Therapy===<br />
One key task of biopharmaceuticals is the binding and blocking of deregulated proteins. Picking the right lead structure for biopharmaceuticals is very important for success. This year, we developed a novel system for the modification, selection and optimization of peptides showing potential for protease inhibition. These promising inhibitors are found in cyanobacteria and are called Microviridins. They belong to the class peptides characterized by unusual ω-ester and ω-amide bonds between the amino-acid side chains, which are also referred to as depsipeptides. These modifications are introduced post translationally by a set of enzymes and result in an extraordinary tricyclic cage structure. <br />
<br><br>Proteases are a large enzyme family and comprise about 647 human gene products. Therefore they are important drug targets. Additional many harmful bacteria, viruses and fungi use proteasess in their reproduction cycle and growth. The ability of blocking these proteases is a highly relevant therapeutic application. Prominent examples of targets are the angiotensin-converting enzyme (ACE) and HIV proteases.<br />
<br><br>We chose a 6.5 kb Microviridin gene cluster named ''mdnABCDE'' from ''Mycrocystis aeruginosa'' NIES843. The mdn gene cluster comprises a gene encoding a precursor peptide named MdnA, which is then modified to form the Microviridin, two genes encoding ATP-grasp-type ligases named ''mdnB'' and ''mdnC'', an ABC- transporter encoding gene named ''mdnE'' as well as one gene encoding an N- acetyltransferase of the GNAT family named ''mdnD'' (Ziemert et al., 2008). Our major aim was to modify the MdnA precursor peptide and optimize its protease inhibiting properties. Towards this goal, we synthesized semi-rational ''mdnA'' gene libraries with partially randomized oligonucleotides. The library was successfully cloned and verified by mass spectrometry.<br />
<br><br>To identify the best candidates inhibiting various given proteases, we established two different selection systems: Phage Display and a new developed ''in-vivo'' selection assay. Phage display is a frequently used technique in laboratories, yet to our knowledge, phage display of cellularly cyclized peptides is new. We constructed a fusion between the surface protein geneIII of the phage and the ''mdnA'' gene within the ''mdnA'' gene cluster. This was verified by western blotting, an ELISA test and a trypsin assay.<br />
<br><br>We also constructed a recombinant ''in-vivo'' selection system linking protease degradation to antibiotic resistance. The designed device is divided into two parts: first a protease activity detector device and second a protease generator device. For the protease activity detector device we fused various protease cleavage sites between a signal sequence and the antibiotic resistance conferring enzyme β-lactamse. β-lactamse confers only resistance when transferred to the periplasm of ''E. coli''. Thus, when the protease cleaves the signal sequence from the lactamase antibiotic resistance is abolished and cells die under selective pressure. However, when our Microviridin variants inhibit the protease, cells survive, allowing an easy and efficient inhibitor selection. We achieved different growth rates with increasing ampicillin concentrations. And were able to show cell death when the protease is activated. After cotransformation with the ''mdnA''-libary we were able to isolate several clones, which will be sequenced.<br />
<br><br />
<br><br />
In addition to the practical work we established a mathematical model of the ''in-vivo'' selection system, which helped us to understand the selection process. We modeled the reaction kinetics by ordinary differential equations and coded them in matlab, to predict, later based on our experimental data, several constants like dissoziation and assoziation constants of the interaction between microviridin and the protease.<br><br />
<br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br><br><br />
<br />
== Highlights ==<br />
<br />
<br />
=== Microviridin ===<br />
[[File:UP_HPLC_mvd_01.jpg|left|300px|thumb|'''Figure 1:''' HPLC chromatogram of mdnA]] <br />
<br />
The major aim of the microviridin group was to modify the mdnA such that the protease inhibiting activity is enhanced. Therefore we used random mutagenesis as well as focused oligonucleotids for creating a library, which is ready for being screened for mdnA with a therapeutically promising set of mutations. For further experiments we also fused the mdnA to a myc-tag. So in the future we will be able to purify and isolate the mdnA.<br><br />
Due to the applicability of the whole mdn-cluster the creation of several BioBricks was possible. The construction was done using a given template vector containing the mdn-genes and sophisticated design of primers. Characterization of the BioBricks was done via HPLC analysis, mass spectrometry and western blot.<br><br />
In a subproject we also built auxiliary expression backbones with inducible promoters for easy cloning via the iGEM restriction enzyme sites.<br />
'''[[:Team:Potsdam_Bioware/Project/Details_Microviridin|[more]]]'''<br />
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<br><br />
<br><br />
<br />
=== Phage Display ===<br />
[[File:UP_Phage_Display_02.jpg|left|290px|thumb|'''Figure 2: Optimization of phage display.''' After optimized conditions (right) a clear concentration of phages carrying mdnA after one panning round was noted.]]<br />
Phage display is a powerful tool for selecting peptides or proteins that bind and regulate the function of target proteins. It is defined as a system in which the protein and its encoding gene are covalently linked. Because of the therapeutic interest of microviridins as protease inhibitors a selection system for screening recombinant <i>mdnA</i>-libraries is of great importance. In our project the fundamental suitability of phage display for this purpose was shown. Therefore an appropriate phagemid, carrying an mdnA-myc-geneIII fusion gene, was constructed. The expression of the mdnA-myc-geneIII protein in the cells was shown by western blotting. Furthermore the production of phage particles carrying MdnA was determined by ELISA and phage display. '''[[:Team:Potsdam_Bioware/Project/Details_Phage|[more]]]'''<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== In Vivo Selection ===<br />
[[Image:UP_SurvTEV_onepla.jpg|left|290px|thumb|'''Figure 3: Ampicillin resistance test.''' Survival Screen, without induced (blue) and with induced (magenta) protease. From left to right increasing Ampicillin concentrations are shown.]]<br />
In addition to the Phage Display we developed a novel selection system. The design aimed for a cheap and time-saving alternative in contrast to an in vitro screen of protease inhibition kinetics. The assay allows us to select effective inhibitors for a random protease, among the billions of randomly generated mutants of the Microviridin. For this purpose we designed a plasmid containing two devices, first a protease activity detector and second a protease generator.'''[[:Team:Potsdam_Bioware/Project/Details_Selection|[more]]]'''<br />
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<br><br><br> <br><br />
<br />
=== Modeling ===<br />
[[File: UP_3Dplot_Lact_06.png|left|300px|thumb|'''Figure 4:''' Plot that shows the lactamase concentration inside the periplasm (and thus the cell fittness) in dependence of the Enzyme inhibition reaction coefficient K_D.]]<br />
There is no synthetic biology without modeling, of course. We focused on systems modeling of our invivo selection system in which the reaction kinetics are analyzed and outcomes are predicted. Thus a synthetic biology approach can be chosen because a better understanding of the system is achieved and further changes can be planed - just like in engineering.<br> The reactions in our system were written down as equations under consideration of their induction at different times and the substance concentrations were numerically propagated through time. Using our concentration calculations we were able to see that our system works very well in theory - it is robust against changes of the most important system parameters. We learned about correct time-scales for our triggering and we were able to identify expected cell-division rates as a reference for the lab work. In a final step we were able to fit our model to wet-lab measurements so that predictions are more reliable. '''[[:Team:Potsdam_Bioware/Project/Details_Modeling|[more]]]'''<br><br><br><br><br><br><br />
<br />
=== Ethics ===<br />
<br />
For information about an ethics seminar and a survey among politicians see '''[[:Team:Potsdam_Bioware/Safety_Ethics|[here]]]'''.<br />
<br />
=== Software ===<br />
<br />
Have a look at the features and screenshots of our BioLog app on this '''[[:Team:Potsdam_Bioware/Software|[page]]]'''.</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T22:13:55Z<p>UP Stefan: /* Children, the scientists of tomorrow/Meeting the young minds? */</p>
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__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Roespel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or biotechnology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|420px|thumb| Our Team and Mr. Roespel at the right]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|420px|thumb|Our team in front of the German "Bundestag".]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html<br />
<br><br />
<br><br />
===Children, the scientists of tomorrow/Meeting the young minds?===<br />
<br><br />
Children are the future, and will follow our footsteps, if we guide them. We cannot start early enough to awake their interest in research by letting them feel like real scientists in the lab. Children are open minded want to discover everything. It’s easy to inspire them with our work. The earlier we start to introduce people to science the better are the chances to create endorsement and interest instead of fear and refusal of synthetic biology.<br />
<br><br />
Therefore, we took the initiative and invited kids to our lab. We gave them a short and easy description of our work and aims at an adequate level. They assisted us at the lab and enjoyed tasks like pipetting. It was such a pleasure to see the fascination in their bright eyes. We think that such positive memories will last a lifetime. <br />
<br><br />
<br />
[[File:UP_kids-1.JPG|left|400px|thumb|]] <br />
[[File:UP_kids-2.JPG|right|400px|thumb|]]</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T22:09:46Z<p>UP Stefan: /* Children, the scientists of tomorrow/Meeting the young minds? */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Roespel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or biotechnology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|420px|thumb| Our Team and Mr. Roespel at the right]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|420px|thumb|Our team in front of the German "Bundestag".]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html<br />
<br><br />
<br><br />
===Children, the scientists of tomorrow/Meeting the young minds?===<br />
<br><br />
Children are the future, and will follow our footsteps, if we guide them. We cannot start early enough to awake their interest in research by letting them feel like real scientists in the lab. Children are open minded want to discover everything. It’s easy to inspire them with our work. The earlier we start to introduce people to science the better are the chances to create endorsement and interest instead of fear and refusal of synthetic biology.<br />
<br><br />
Therefore, we took the initiative and invited kids to our lab. We gave them a short and easy description of our work and aims at an adequate level. They assisted us at the lab and enjoyed tasks like pipetting. It was such a pleasure to see the fascination in their bright eyes. We think that such positive memories will last a lifetime. <br />
<br><br />
<br />
[[File:UP_kids-1.JPG|left|400px|thumb|'''Figure 2:''']] <br />
[[File:UP_kids-2.JPG|right|400px|thumb|'''Figure 2:''']]</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T22:09:30Z<p>UP Stefan: /* Children, the scientists of tomorrow/Meeting the young minds? */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Roespel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or biotechnology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|420px|thumb| Our Team and Mr. Roespel at the right]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|420px|thumb|Our team in front of the German "Bundestag".]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html<br />
<br><br />
<br><br />
===Children, the scientists of tomorrow/Meeting the young minds?===<br />
<br><br />
Children are the future, and will follow our footsteps, if we guide them. We cannot start early enough to awake their interest in research by letting them feel like real scientists in the lab. Children are open minded want to discover everything. It’s easy to inspire them with our work. The earlier we start to introduce people to science the better are the chances to create endorsement and interest instead of fear and refusal of synthetic biology.<br />
<br><br />
Therefore, we took the initiative and invited kids to our lab. We gave them a short and easy description of our work and aims at an adequate level. They assisted us at the lab and enjoyed tasks like pipetting. It was such a pleasure to see the fascination in their bright eyes. We think that such positive memories will last a lifetime. <br />
<br><br />
<br />
[[File:UP_kids-1.JPG|left|300px|thumb|'''Figure 2:''']] <br />
[[File:UP_kids-2.JPG|right|300px|thumb|'''Figure 2:''']]</div>UP Stefanhttp://2011.igem.org/File:UP_kids-2.JPGFile:UP kids-2.JPG2011-10-28T22:09:16Z<p>UP Stefan: </p>
<hr />
<div></div>UP Stefanhttp://2011.igem.org/File:UP_kids-1.JPGFile:UP kids-1.JPG2011-10-28T22:08:54Z<p>UP Stefan: </p>
<hr />
<div></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T22:05:08Z<p>UP Stefan: /* Children, the scientists of tomorrow */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Roespel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or biotechnology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|420px|thumb| Our Team and Mr. Roespel at the right]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|420px|thumb|Our team in front of the German "Bundestag".]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html<br />
<br><br />
<br><br />
===Children, the scientists of tomorrow/Meeting the young minds?===<br />
<br><br />
Children are the future, and will follow our footsteps, if we guide them. We cannot start early enough to awake their interest in research by letting them feel like real scientists in the lab. Children are open minded want to discover everything. It’s easy to inspire them with our work. The earlier we start to introduce people to science the better are the chances to create endorsement and interest instead of fear and refusal of synthetic biology.<br />
<br><br />
Therefore, we took the initiative and invited kids to our lab. We gave them a short and easy description of our work and aims at an adequate level. They assisted us at the lab and enjoyed tasks like pipetting. It was such a pleasure to see the fascination in their bright eyes. We think that such positive memories will last a lifetime. <br />
<br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:47:09Z<p>UP Stefan: /* Survey */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Roespel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or biotechnology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|420px|thumb| Our Team and Mr. Roespel at the right]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|420px|thumb|Our team in front of the German "Bundestag".]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:45:09Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or biotechnology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|420px|thumb| Our Team and Mr. Roespel at the right]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|420px|thumb|Our team in front of the German "Bundestag".]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Project/Details_PhageTeam:Potsdam Bioware/Project/Details Phage2011-10-28T21:44:10Z<p>UP Stefan: /* Detection of phages carrying mdnA on their surface by ELISA */</p>
<hr />
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== Phage Display ==<br />
===Introduction===<br />
<br />
Phage Display is an efficient tool for selecting protein or peptides with specific binding properties from a large recombinant library. This proteins are represented on the surface of bacteriophages. This enables the coupling of phenotype and stable packaged genotype because the proteins which form the phage including the proteins of interest are coded in its genome. To test the suitability of phage display system as an appropriate screening method for recombinant <i>mdnA</i> libraries a vector containing a <i>mdnA-myc-gene III</i>-fusion gene was generated. This vector contains a plasmid origin of replication, so it can be amplified like plasmids. Additionally it contains a f1 ori which enables the packaging of single strand DNA into phages. The vector also contains the whole <i>mdn</i>-cluster which is needed to produce the MdnA peptide. Between <i>mdnA</i> and <i>gene III</i> is a <i>myc</i>-tag located, which is used for an easy detection. The successful expression of the <i>MdnA-myc-geneIII</i>-fusion protein on the surface of the phage was determined by ELISA using anti-myc antibody 9E10 after transforming ''E. coli'' cells and purifying the produced phages. The next step was to perform a phage display on a know target of the <i>MdnA</i>. To test the fundamental suitability of this screening method, phages representing <i>MdnA</i> on their surface and phages not representing <i>MdnA</i> in a ratio of one to one were incubated with immobilized trypsin which is known as a target of <i>MdnA</i>. After the first panning round a marked concentrating of phages carrying <i>MdnA</i> was recognized.<br><br />
<br />
===Cloning strategy===<br />
<br />
The phage display vector pPDV089 was derived from the plasmid pARW089 which carries the whole <i>mdn</i>-cluster. This plasmid contains a plasmid origin of replication and additionally a f1 ori which enables the packaging of single strand DNA into phages. For selective amplification ampcillin and kanamycin resistance genes are included. To create the phagemid pPDV089 standard cloning procedure were performed. <br />
First <i>mdnA</i> was deleted by excising using the restriction enzymes <i>Sfo</i>I and <i>Aat</i>II. The next step comprised the introduction of a <i>mdnA-geneIII</i>-fusion gene. Therefore <i>gene III</i> was amplified from pak100blaKDIR and <i>mdnA</i> from pARW089 by PCR. The primers were designed to enable the introduction of iGEM and other restriction sites required for further cloning steps. The purified PCR product <i>geneIII</i> was digested by <i>NgoM</i>IV and <i>Aat</i>II whereas the PCR product <i>mdnA</i> was digested by <i>Sfo</i>I and <i>Age</i>I. Subsequent the three fragment ligation of <i>mdnA</i> and <i>geneIII</i> into the digested vector has been conducted. Thus a <i>mdnA-geneIII</i>-fusion part according to RFC25 was generated whereby <i>Age</i>I and <i>NgoM</i>IV overhangs are compatible and placed in frame with the protein sequence. The ligation of <i>Age</i>I and <i>NgoM</i>IV overhangs resulted in a scar coding for the threonine and glycine. Because the introduction of restriction sites before <i>mdnA</i> leaded to a great distance between ribosome binding site (RBS) and <i>mdnA</i> a second RBS was inserted among <i>Sfo</i>I and <i>Xba</i>I recognition sites to ensure a sufficiently expression rate of the <i>mdnA-geneIII</i>-fusion gene. The myc sequence located between <i>mdnA</i> and <i>gene III</i> allows the detection of the expression of the <i>mdnA-geneIII</i>-fusion protein on the surface of the phage using western blot or ELISA. In the last step the kanamycin resistance gene was disturbed because for phage display the selection of cells carriyng both helper phages and the phagemid is beneficial and the helper phages have a kanamycin resistance, too. Therefore a 300 bp fragment of the kanamycin resistance gene was deleted using the restriction enzyme <i>Nsi</i>I which had two recognition sites in the kanamycin gene.<br />
<br />
<br />
<br />
{| border="0" cellspacing="0" cellpadding="2" <br />
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| [[File:UP cloning strategy.png|center|450px|thumb|'''Figure 1: Cloning strategy for creating a plasmid which can be used for phage display with <i>mdnA</i>. '''<i>MdnA</i> was cut out of the vector and a <i>mdnA-myc-geneIII</i> fusion gene was created and ligated with the vector pARW089 containing the <i>mdn</i>-cluster without <i>mdnA</i>.]] || [[File:UP pPDV089.png|center|350px|thumb|'''Figure 2: Designed vector pPDV089 carrying the <i>mdnA-myc-gene III</i> fusion gene.''' Therefore the <i>mdnA</i> sequence was cut out of the vector pARW089 and the ligated <i>mdnA-myc-geneIII</i> gene was inserted]] <br />
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<br />
===Control of expression of ''mdnA''-''myc''-''geneIII'' in ''E. coli''===<br />
<br />
To control expression of the ''mdnA''-''myc''-''geneIII'' fusion gene was analyzed by western blotting. E. coli cells transformed with the phagemid pPDV089 were harvested and lysated. The cell proteins were electrophoretically separated and transferred to a membrane. The ''mdnA''-''myc''-''geneIII''-fusion proteins were detected using specific anti-''myc''-antibodies and horseradish peroxidase (HRP)-linked antibodies as second antibodies. Enhanced chemiluminescence (ECL) was used to visualize the protein. ECL is based on the emission of light during the HRP -catalyzed oxidation of luminol, which was captured by a camera. The western blot analysis resulted in a band of a size just below the 30 kDa mark representing the ''mdnA''-''myc''-''geneIII''-fusion protein (24 kDa).<br />
<br />
[[File:UP_coomassie+western blot.png|center|400px|thumb|'''Figure 3: Control of expression of ''mdnA''-''myc''-''geneIII'' in ''E. coli'' by western blotting.''' For detection anti-''myc''-antibodies and secondary HRP-linked antibodies were used. The resulting band represents the ''mdnA''-''myc''-''geneIII''-fusion protein.]]<br />
<br><br />
<br><br />
<br><br />
<br />
===Detection of phages carrying ''mdnA'' on their surface by ELISA===<br />
<br />
The next step was the detection of the expression of the ''mdnA''-''myc''-''geneIII''-fusion gene on the surface of the phage. So ''E. coli'' cells strain XL1-Blue were first transformed with the phagemid pPDV089 before they were infected with helper phages. ''E. coli'' cells containing both plasmids were selected. An ELISA test was performed to determine whether these cells are able to produce phage particles carrying the MdnA peptide on their surface. To perform this test anti-''c-myc''-antibodies were immobilized on ELISA plates and incubated with purified phages. For detection a second antibody coupled with horse radish peroxidase (HRP) was used which binds the gene VIII protein of the phages. The HRP substrate o-phenyldiamine (OPD) was added and in case of binding a color reaction was expected. The color shift from achromatic to yellow in wells incubated with phages produced in XL1-Blue cells showed the successful expression of ''mdnA''-c-''myc''-''geneIII''-fusion protein on the phages.<br><br />
For more precise results the absorption at 492 nm was measured. The data were presented in a bar plot. As a negative control helper phages were added instead of produced phages. Furthermore two wells were prepared were the secondary antibody was not added.<br />
The graphic shows clearly the much higher absorption measured in wells, which were incubated with phage particles of interest produced in XL1-Blue cells. As has already pointed out this shows the succeeded expression of ''mdnA''-c-''myc'''-''geneIII''-fusion protein on the surface of the phages.<br />
<br />
<br />
<div align="center"><br />
[[File:UP_ELISA3_mit_fehlerindikator.png|center|400px|thumb| '''Figure 4:''' Detection of phages carrying ''mdnA'' on their surface by ELISA. The bar plot shows the absorption at 492 nm. Anti-''myc''-antibodies were immobilized. For detection a second antibody coupled with horse radish peroxidase (HRP) was used which binds the gene VIII coat protein of the phages. The left bar shows the absorption of the wells containing helper phages (negative control), the right bar shows the absorption of wells containing ''mdnA'' carrying phages]]</div><br><br />
<br><br />
<br />
===Testing phage display with unmodified mdnA to examine its suitability as screening method===<br />
<br />
To test the fundamental suitability of this screening method, phages representing unmodified mdnA on their surface and phages not representing mdnA (helper phages) in a ratio of one to one were incubated with immobilized trypsin which is known as a target of mdnA. The display was conducted in ELISA plates. The bound phages were eluted using a buffer with low pH value and neutralized afterwards. To check how many phages interacted with trypsin, ''E. coli'' cells XL1-Blue were re-infected with eluted phages and plated on agar with different antibiotics. Cells infected with phages carrying mdnA are able to grow on agar with ampicillin whereas cells infected with helper phages are able to grow on agar with kanamycin. To control the success of the panning round additionally ''E. coli'' cells were infected with phage mix before panning and plated on agar. Subsequent the number of clones grew on ampicillin and kanamycin before and after panning was compared. During the running of this step it was noticed that much more cells were infected with helper phages than with phages carrying mdnA despite of the engaged 1:1 ratio. This was surprising and indicated that mdnA on the surface of the phages may inhibit their infectivity. After controlling the plates an infection ratio of phages carrying mdnA to helper phages of 1:400 was calculated. This fact should be analyzed in further experiments.<br><br />
The results of the first phage display are plotted in the figure below. After one panning round an enrichment of phages carrying mdnA was expected. This is attributable to the fact that phage particles carrying mdnA-c-myc-gene III-fusion protein on their surface are expected to bind specifically to the immobilized trypsin. Unfortunately this was not observed in this experiment. The ratio of cells growing on kanamycin agar before (4000) to cells growing on kanamycin agar (cells containing helper phages) after panning (750) was determined as 5:1. The ratio of cells growing on ampicillin agar before (12) to cells growing on ampicillin agar (cells containing mdnA carrying phages) after panning (2) was nearly equal. Thus no enrichment of mdnA carrying phages occurred in the first experiment. So it was decided to repeat this experiment under improved conditions. Therefor the number of washing steps during the described experimental procedure was increased. Here the ratio of cells growing on kanamycin agar before (3000) to cells growing on kanamycin agar (cells containing helper phages) after panning (29) was determined as 103:1. The ratio of cells growing on ampicillin agar before (26) to cells growing on ampicillin agar (cells containing mdnA carrying phages) after panning (2) was determined as 13:1. Thus an enrichment factor of eight was reached for the phages displaying mdnA on their surface. <br><br />
These results indicate that the unmodified mdnA expressed on the phages binds specifically to the immobilized trypsin. Therefore it can be deduced that mdnA is presented in a functional 3D structure. These findings suggest that phage display in general is an appropriate method for screening a recombinant mdnA library. Further experiments are required to optimize this system.<br />
<br />
<br />
[[File:UP panning 3.png|center|400px|thumb|'''Figure 5: Optimization of phage display.''' After optimized conditions (right) a clear concentration of phages carrying mdnA after one panning round was noted. ''E. coli'' cells were infected with phage mix (helper phages and phages carrying mdnA) before and after panning and plated on agar containing kanamycin or ampicillin. The ratio of cells growing on ampicillin or kanamycin agar before panning to cells growing on ampicillin or kanamycin agar after was calculated. Helper phages which acted as negative control have a kanamycine resistance whereby phages carrying mdnA have an ampicillin resistance.]]<br />
<br />
===Testing phage display with unmodified mdnA against further proteases===<br />
<br />
Furthermore the interaction of unmodified mdnA with other proteases was determined. From the literature (Ziemert, 2010) the high inhibitory activity of microviridin L, besides trypsin, against chymotrypsin and elastase is also known. So a phage display with these enzymes was performed. Additionally papain, proteinase K, mycolysin and pepsin were tested for which no interaction was shown yet. For all enzymes an equal amount of ''E. coli'' cells and phages were used. In agreement with data from the literature interaction of microviridin with chymotrypsin and elastase was confirmed. Additionally an interaction with papain was noted. All other proteases were not bound by microviridin. <br />
<br />
[[File:UP_test of different enzymes.png|center|400px|thumb|'''Figure 6: Phage display against different proteases.''' After panning the number of clones was counted. In agreement with data from the literature interaction of microviridin with chymotrypsin and elastase was confirmed. Additionally an interaction with papain was noted.]]<br />
<br><br><br />
<br />
===References===<br />
*Fuh G., Sidhu S.S. (2000). Efficient phage display of polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein. FEBS Lett. 480(2-3):231-4<br />
<br />
*Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R., Plückthun, A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Meth. 201(1):35-55 <br />
<br />
*Rakonjac J., Feng J., Model P. (1999). Filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of pIII. J Mol Biol. 289(5):1253-65<br />
*Smith, G.P. (1985). Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virus surface. Science 228: 1315-17<br />
<br />
*Ziemert, N., Ishida, K., Liaimer, A., Hertweck, C. & Dittmann, E. (2008). Ribosomal synthesis of tricyclic depsipeptides in bloom-forming cyanobacteria. Angewandte Chemie (International ed. in English) 47, 7756-9<br />
<br />
*Ziemert, N., Ishida, K., Weiz, A., Hertweck, C. & Dittmann, E. (2010). Exploiting the natural diversity of microviridin gene clusters for discovery of novel tricyclic depsipeptides. Applied and environmental microbiology 76, 3568-74 <br />
<br><br></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:41:56Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
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__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|420px|thumb| Our Team and Mr. Roespel at the right]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|420px|thumb|Our team in front of the German "Bundestag".]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:41:39Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|450px|thumb| Our Team and Mr. Roespel at the right]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|450px|thumb|Our team in front of the German "Bundestag".]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:39:03Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|400px]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|400px]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:38:49Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|400px]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|400px]]<br />
<br><br><br><br><br><br><br><br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:38:20Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|400px]]<br />
[[Image:UP_vor-bundestag_640.JPG|right|400px]]<br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/File:UP_vor-bundestag_640.JPGFile:UP vor-bundestag 640.JPG2011-10-28T21:37:53Z<p>UP Stefan: </p>
<hr />
<div></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:36:32Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG|left|400px]]<br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:35:40Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.JPG]]<br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:35:08Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
[[Image:UP_bundestag_640.jpg]]<br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/File:UP_bundestag_640.JPGFile:UP bundestag 640.JPG2011-10-28T21:35:01Z<p>UP Stefan: </p>
<hr />
<div></div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:30:54Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
<br><br><br />
<br />
== Survey ==<br />
<br />
In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
<br />
In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
<br />
This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
<br />
Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
<br />
<br />
<br />
[[File:UP_survey1.png|center|400px]]<br />
<br><br><br />
[[File:UP_survey2.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey3.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey4.png|center|600px]]<br />
<br><br><br />
[[File:UP_survey5.png|center|500px]]<br />
<br><br><br />
<br />
The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
<br />
This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
<br><br><br />
=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. We were able to present them our project and our perspective about Synthetic Biology.<br><br />
<br />
Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
<br />
=== Statement of the German government ===<br />
<br />
In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
<br />
In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
<br />
On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
<br />
In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
<br />
(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
<br />
The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
<br />
The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
<br />
The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
<br />
In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
<br><br><br />
<br />
== Outreach ==<br />
<br />
<br />
<br />
===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
<br />
[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
<br />
The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
<br> <br />
The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
<br> <br />
Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
<br> <br />
The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
<br />
=== biotechnologie.tv ===<br />
<html><br />
<div align="center"><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
<br />
<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
<br />
<br><br><br />
<br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
</div><br />
</html><br />
<br />
=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
<br><br />
For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
<br />
University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefanhttp://2011.igem.org/Team:Potsdam_Bioware/Safety_EthicsTeam:Potsdam Bioware/Safety Ethics2011-10-28T21:30:37Z<p>UP Stefan: /* Potsdam_Bioware at the German parliament */</p>
<hr />
<div>{{:Team:Potsdam_Bioware/Head}}{{:Team:Potsdam_Bioware/jquery}}{{:Team:Potsdam_Bioware/menu_home}}<br />
__NOTOC__<br />
== Safety & Ethics ==<br />
<br><br />
<br />
=== Safety Assessment ===<br />
<br />
<p style="text-align:justify;">Our iGEM project requires only the handling of the non-pathogenic, non-adherent ''Escherichia coli'' K12 and B strains and the well-established filamentous phage. Both, the bacteria and the phage are commonly used chassis in laboratories and pose no risk when handled according to the mandatory rules. As all of us are well briefed about laboratory safety and biohazard regulations we follow these at all times. In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Following these rules, there should not be a significant danger neither to the environment nor to team members.<br><br />
<br />
The most important issues we discussed are the consequences of the error-prone PCR we use to modify our parts. We tried to estimate the chances of generating highly toxic proteins. Surveying the literature, we found several reports about natural variants of microviridins and one rational mutational study, but no reports on toxic effects. As cyanobacteria can also produce toxic compounds (non-ribosomal peptides named microcystins) toxicity testing is well established in the cyanobacteria research community, and obviously, testing did not identify toxic effects. Therefore, we assume that our mutations will not have any hazardous effects. Additionally, the obtained, constructed, and planned plasmids contain only previously described parts without any known risk potential. Therefore, as far as we can foresee, our constructed BioBricks will not have or trigger any toxic effects or be critical in any way for the environment. This means that only a negligible risk arises from our used methods and constructs to the environment, the public and the team members. Last but not least, we do not see any particular danger of abuse or other security threat of our work, since it is specifically addresses scientific questions. It is our goal that the health of mankind and the environment benefit from our research.</p><br />
<br />
=== Safety Questions ===<br />
<br />
<br />
# Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
#* No, our project is not raising any of these safety issues.<br />
# Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience?<br />
#* No, our BioBrick parts or devices are not going to raise any safety issues.<br />
# Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?<br />
#* In Germany, work with genetically modified organisms is regulated by the ‘Law on Gene Technology’ (Gesetz zur Regelung der Gentechnik, GenTG). According to these rules, the responsible governmental authorities of the state of Brandenburg have been notified about our work. Our work was classified as biosafety level 1.<br />
# Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br />
#* So far biosafety assessment is primarily based on the characterization of wild-type devices or systems. For synthetic biology these rules should be extended to better represent the variations made by synthetic biology approaches. In addition to the continuous evaluation of safety and security, a section on technological impact assessment should be added.<br />
<br><br><br />
<br />
=== Ethics Seminar===<br />
<br />
<!--Taken out<br />
=== Why Ethics?===<br />
Synthetic Biology is not only a current topic in the science community. A look through published articles in papers e.g. the Guardian [1], abstracts from governmental<br />
institutions [2] and a large amount of webblogs [3] regarding the latest developments in this new field, strengthen the impression of a general interest.<br />
<br><br>The reasons for this development are sure a combination of different factors. With the possibilities of the modern web and media people have the chance to<br />
understand science and communicate about new results. A general interest for information is forming with a new generation. The typical image of a researcher in a<br />
ivory tower has been replaced by lighthouses of knowledge, which interact with the society.<br />
<br><br>Former mistakes, where few decided in which amount and time manner new results in research are used e.g. the usage of nuclear power, are not imaginable in the<br />
age of information. These social trends forces science to take on a dialogue with society about the use of new research results, application and commercialization.<br />
We tried to think about these certain issues by holding a lecture on this topic, resulting into a field of forces for synthetic biology and a survey to reflect the opinion<br />
of the German politics.<br />
--><br />
<br />
====Seminar: "From engineer to creator: a controversy"====<br />
[[File:UP_Stoecker_profile.jpg|left|150px]] <br />
<br />
On the 5. July 2011 the iGEM Team Potsdam hosted a lecture about ethics in the field of synthetic biology. As guest speaker we were able to welcome Prof. Dr. Ralf Stoecker, professor of philosophy at the University of Potsdam and also member of the board of the Academy for Ethics in Medicine. This lecture was not only for the iGEM Team itself, but for all students with different fields of study. Through advertisement in different departments a audience with different background was the result. Starting with a general overview in the history of philosophy regarding the metaphysics of morals and the example of Immanuel Kant, Prof. Dr. Stoecker tried to explain the difficulty of ethics in modern times.<br />
<br><br><br>Kant started to think about general ideas concerning actions of humans a priori [4]. Philosopher often try to<br />
adapt these moral principles to certain specific cases. Nowadays they are confronted with the problem of a<br />
fast development in science and new applications. It is not any more possible to think first about a general<br />
principle and then adapt these to the new results. Today the society often needs an answer to a moral<br />
problem in short time, because the applications are needed or will be commercialize anyway. One can then<br />
try, to extract from the concrete problem e.g. synthetic biology in medicine general principles. This way is part<br />
of the applied philosophy.<br />
<br><br>To represent this current problem in finding a ethic view on synthetic biology, Prof. Dr. Stoecker suggested a<br />
field of forces, where different positive and negative effects pull at the decision to use synthetic biology in<br />
research for new applications. In conclusion, the ethic or moral decision for a new case in not determined by<br />
higher general principles. It is a combination and weighing of different positive and negative aspects.<br />
<br> <br />
[[File:UP_ethics_map.jpg|right|600px|thumb|'''Figure 1:''' Force-field map on synthetic biology]]<br />
We tried to find relative clear examples for advantages and disadvantages of synthetic biology. One should<br />
always remind himself, that the different aspects have different weight for each individual. While the iGEM<br />
competition is a good example for advantages of synthetic biology e.g. new applications and teaching of new<br />
knowledge, Miller and Selgelid [5] show in their paper, that new applications in biology also can used for military<br />
use . The „lone operator” scenario by Tucker and Zilinkas [6] is also a good example for possible biosafety<br />
problems. In this unlikely scenario one professional researcher follows his own terrorist goal. Still these<br />
examples show disadvantages which one has to consider, when trying to make his decision.<br />
<br />
====Literature====<br />
<br />
[1] McKie, R. (2003), 'Fluorescent fish' give the green light to GM pets, The Observer, Sunday 15 <br><br />
[2] Balmer, A. and Martin, P., (2008), Synthetic Biology Social and Ethical Challenges, Institute for Science and Society University of Nottingham <br><br />
[3] Maynard, A. (2008), Synthetic biology, ethics and the hacker culture, on http://2020science.org/2008/06/13/8613-synthetic-biology-ethics-and-the-hacker-culture/ (12.09.2011) <br><br />
[4] Kant, Immanuel. Foundations of the Metaphysics of Morals. Trans. Lewis White Beck Standard edition of Königliche Preussische Akademie der Wissenschaften. Berlin, 1902–38.<br><br />
[5] Miller S. and Selgelid, M., (2006), Ethical and philosophical consideration of the Dual-use dilemma in the biological sciences. Centre for Applied Philosophy and Public Ethics, Australian National University and Charles Sturt University, Canberra, Australia <br><br />
[6] Tucker, J.B., Zilinskas, R.A., (2006), The promise and perils of synthetic biology, The Atlantis news, spring 2006 <br><br />
[7] Habermas, J. (2003): The Future of Human Nature. Cambridge (UK), Malden/MA: Polity <br><br />
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== Survey ==<br />
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In Germany, synthetic biology is starting to gain more importance in academic research. However, the ethical issues and potential risks associated with it, are a major concern for the general public. So far, the German government did only fund the opinion-forming process but not synthetic biology projects in natural sciences.<br />
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In order to determine the general consensus on synthetic biology we generated a survey about the opinion of the members of the German parliament. The aim of this survey was to figure out what the members of the parliament think about “synthetic biology” and how the future of synthetic biology in Germany might look like.<br />
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This survey was sent to 637 members of the German parliament. 15 Members answered and 10 of them attended the survey. The participation was around 2.22 percent. Furthermore, Mr. René Röspel invited us to discuss about synthetic biology.<br />
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Survey: [[File:Survey.pdf]]<br />
*1. How would you assess your knowledge about Synthetic Biology?<br />
*2. Research in the field of nutrition:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*3. Research in the field of healthcare and medicine:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*4. Research in the field of energy:<br />
**a. How would you assess the potential of Synthetic Biology?<br />
**b. How would you assess the risk of Synthetic Biology?<br />
*5. Would you support an enhanced promotion of Synthetic Biology?<br />
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[[File:UP_survey1.png|center|400px]]<br />
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[[File:UP_survey2.png|center|600px]]<br />
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[[File:UP_survey3.png|center|600px]]<br />
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[[File:UP_survey4.png|center|600px]]<br />
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[[File:UP_survey5.png|center|500px]]<br />
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The survey said that the members of the parliament believe in a high potential of synthetic biology in the area of healthcare, medicine and energy research. In the field of nutrition, they do not expect to significant improvements through synthetic biology researches. The members of the parliament classified the risks of research in the field of synthetic biology as average.<br />
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This survey also shows a positive trend for the enhancement of research in the field of synthetic biology by the German government. Mainly the members of the parliament regard the field of healthcare, medicine and energy research as having great potential for the future of synthetic biology.<br />
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=== Potsdam_Bioware at the German parliament ===<br />
In our survey we asked politicians about the potential and risks of Synthetic Biology. Unfortunately we got only 15 answers. The disappointing quantity of answers we got is actually the answer itself. <br><br />
These results raised some questions: What should we expect from non-scientists? What do they know about Synthetic Biology!? Having this in mind, how can they judge about Synthetic Biology if they do not even know what it means? <br />
That is why we were really lucky that Mr. René Roespel, a member of the German Parliament and of the party SPD (Sozialdemokratische Partei Deutschland/ Social democratic party Germany) invited us to the Paul-Loebe House in Berlin on the 27th October as a response of the survey. We had a vivid discussion with him and his group about Synthetic Biology, its advantages and disadvantages for the community, its potential, as well as the differentiation of synthetic biology to gene- or bio- technology. <br><br />
We were able to present them our project and our perspective about Synthetic Biology.<br><br />
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Synthetic Biology is the approach to use and extend the principles and modules of the vitalized nature under technical aspects to achieve improved systems. We pointed out that Synthetic Biology is more than ONE research field. It is a connection of biology, chemistry, physics, informatics and engineering. There are no definable borders between synthetic biology, molecular biology, biochemistry and gene- or biotechnology. Synthetic biology links life sciences and engineering. This leads to an enormous potential. The confrontation of Mr. Roespel with these facts showed that synthetic biology is a difficult topic in Germany in contrast to other European countries and especially to the world-wide opinions. In Germany many people are frightened by topics including gene manipulation. We learned that it will be our task to dismantle these concerns and make people understand the potential of synthetic biology.<br><br />
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=== Statement of the German government ===<br />
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In Germany synthetic biology is considered as a new field of research in the biotechnology. It is based on biotechnology and genetic engineering and has the aim to generate new biological systems with accurately defined abilities. <br />
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In the near future, the resulting knowledge will be used in various research sectors such as biofuel or pharmaceutics. In spite of the possible potential of this field of research more and more people warn against potential risk of misusing of this new knowledge.<br />
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On this account, it is necessary to take care of this new technique in a social and political way, so that it would be possible to use the enormous prospects and potentials and to assess the possible risks of misusing. <br />
In this context, the German parliament required the government for a statement on this topic.<br />
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In response to the German parliament question on the position of the German government to “perspective and position of the government of synthetic biology”, the German government gave a statement to this topic : <br />
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(cited from “Antwort der Bundesregierung auf die Kleine Anfrage der Abgeordneten René Röspel, Dr. Ernst Dieter Rossmann, Dr. Hans-Peter Bartels, weiterer Abgeordneter und der Fraktion der SPD – Drucksache 17/4898 – Stand und Perspektiven der Synthetischen Biologie“, 22.03.2011, translated by iGEM-Team Potsdam)<br />
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The government’s expectations of this new field of research primarily relate to the benefit of environmental protection, energy research, development of new chemical products and renewals in biomedical science and pharmaceutical industry. However, the synthetic biology is still in its infancy. For this reason, it is not possible to evaluate the date when the first renewals will be established. <br />
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The military research in the field in relation to the protection against infectious agents will not be excluded in the future. However the risks which are associated with the abuse of this technology for terrorist acts were classified as low, because of the existence of naturally occurring pathogen. The research and development work in the area of synthetic biology is subject to the gene technology law, the foreign trade law and the weapons of war control law, wherefore there are no additional need for legislative or regulatory action.<br />
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The government monitored the opinion-forming process related to this field and established a temporary position at the ZKBS (Zentrale Kommission für die Biologische Sicherheit) to monitor the development of synthetic biology and support the ZKBS for risk assessment in this field. On international level the EU established the Knowledgebased Bio-Economy Collaborative Working Group (KBBE-NET CWG) which analyzes the possible potential of synthetic biology. <br />
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In the last 5 years, the German government invests in this field of research 709.000 € for social and ethical accompanying research and 450.000 € in innovation and technology analysis. But until now, the government didn’t promote a specific research and development project in the field of synthetic biology. By comparison, in the last 5 years the USA provides 430 Mio $ for this field of research. The same applies to the government of Great Britain who spending 30 Mio € for public research in this field.<br />
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== Outreach ==<br />
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===Poster Presentation at the Strategic Process Biotechnology 2020+===<br />
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[[File:UP_Poster_Biotechnologie2020.png|left|300px|thumb|'''Figure 2:''' [[Media:UP_iGEM_Poster_BMBF_2020+_20110706_final_version.pdf|Poster]] ]] <br />
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The Strategic Process Biotechnology 2020+ was hosted by the German Ministry of Education and Research in July 2011. This strategic meeting is organized every year. The purpose is to bring research facilities, companies and politicians together to discuss the development of biotechnology and synthetic biology in Germany in the next ten years.<br><br />
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The day started with an introduction from State Secretary Dr. Helge Braun and talks from different industry representatives. In the late morning we participated with a poster presentation together with this year's iGEM Bielefeld Team as well as the last year's Freiburg and Weimar-Heidelberg Arts Teams.<br><br />
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Throughout the poster presentation we had many interesting talks about iGEM. We discussed our idea with various people in terms of potential and application. Different thoughts were proposed about the project and about the Microviridin cluster. The feedback was great and encouraging. We also gave an interview to biotechnology.tv to promote our concept to a wide range of people (see youtube video below).<br> <br />
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The lunch break was followed by a podium discussion about the scientific advantages which have been made throughout the past year. Highlights were presented and discussed by the speakers. In the afternoon several workshops took place. In the relaxed atmosphere novel applications and innovations were designed in role plays. It seemed that the search for novel natural therapeutics is a main focus in the arising decade of biotechnology in Germany.<br />
This poster presentation on the Strategic Process Biotechnology 2020+ was a great opportunity and we are very thankful for all the ideas and new impressions. We would like to thank everybody for this experience.<br />
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=== biotechnologie.tv ===<br />
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<iframe width="560" height="315" src="http://www.youtube.com/embed/MN82qjx2-ic" frameborder="0" allowfullscreen></iframe><br />
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<center>biotechnologie.tv: interview with Stefan (German) (Time 04:58)</center><br />
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<iframe width="560" height="315" src="http://www.youtube.com/embed/9kkm0vpZpzk" frameborder="0" allowfullscreen></iframe><br />
<center>Deutsche Welle: interview with Kristian on synthetic biology</center><br />
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=== Press Report=== <br />
====Young scientists from University of Potsdam participate in an international synthetic biology competition====<br />
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For the first time an interdisciplinary group of students from University of Potsdam takes part in the international <b>iGEM</b> competition (<b>i</b>nternational <b>G</b>enetically <b>E</b>ngineered <b>M</b>achine). This competition is arranged by Massachusetts Institute of Technology in Cambridge for international teams every year since 2005.<br><br />
The junior scientists from Potsdam develop a system for the detection of novel peptide based agents for therapeutical applications in human. In the process they use the properties of bacteria, in that case of cyanobacteria. These bacteria are capable of linking short protein parts in an unusual way. These properties are transferred to bacterial laboratory strains and optimized. As a result the students hope to find inhibitors, which e.g. can be used in regulation of blood coagulation.<br><br />
The 15 students from 4th to 8th semester are guided by Dr. Kristian Müller and Prof. Katja Arndt, whose former groups were able to win gold-medals and special awards. The biochemistry students plan and perform the lab work. They are supported by physic students, contributing to the design of models, and informatics students, developing an application for smart phones. The work is aided by Dr. Elke Dittmann, Professor of microbiology. Moreover the students discuss the possibilities and borders of synthetic biology with the publicity and work on financing the project.<br><br />
In the beginning of October all results and projects will be presented during the European Jamboree in Amsterdam and evaluated by a jury. The final round takes place in November in Boston.<br><br />
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University of Potsdam (German)<br><br />
http://www.uni-potsdam.de/pm/news/archiv/up/date/2011/07/04/2011-135.html</div>UP Stefan