http://2011.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=Siegeljb2011.igem.org - User contributions [en]2024-03-29T05:03:12ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Washington/Alkanes/ResultsTeam:Washington/Alkanes/Results2011-10-29T00:38:24Z<p>Siegeljb: /* The FabBrick, a module for even chain length alkane production */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''GCMS confirms The PetroBrick enables diesel production from sugar using ''E. coli'''''=<br />
<br />
We performed GC-MS analysis on extracts from cell cultures expressing both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) (this composite part is designated [http://partsregistry.org/Part:BBa_K590025 The PetroBrick]). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.<br />
<br />
<br />
[[File:Washington PetroBrick.png|300px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
[[Image:Washinton_AARADCGC-MS.png|center|600px|thumb|GCMS confirms PetroBrick diesel production from sugar using E. coli]]<br />
<br />
<br><br />
<br />
'''No significant hydrocarbons peaks in samples extracted from cells expressing only ADC.''' The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.<br />
<br />
'''C16 alcohols are observed when AAR is expressed on its own.''' The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.<br />
<br />
'''The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.''' <br />
[[File:Washington2011 Spectra.png|right|550px|thumb|Comparasion of the NIST reference C15 alkane spectrum( in blue) to the C15 peak spectrum taken from a PetroBrick culture (in red). Note the strong m/z peak at 212, corresponding to the parent ion of the C15 alkane. For spectra of the C13 alkane and C17 alkene, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]]]<br />
The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C14 alcohol and the C17 alkene. The fact that the C17 alkene peak overlaps with the C14 alcohol peak makes exact quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the earlier section of the peak has an MS spectra consistent with the C16 alcohol, and the later section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra(for these spectra, as well as the C17 alkene spectrum, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]). In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane. Using the PetroBrick, we can turn simple sugars into diesel, a fuel fully compatible with modern infrastructure.<br />
<br />
<br />
----<br />
<br />
='''Initial Quantization of Alkane Production'''=<br />
In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute yield. To determine how much alkane was being produced by the Petrobrick, we grew up 3 MG1655 cell cultures transformed with the PetroBrick in M9 production media ([https://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.<br />
<br />
[[File:Washington_alkanestandardcurve.png|right|450px|thumb|Standard curves for converting peak area to an absolute amount. Note that these curves is almost perfectly linear. In addition, the curve generated from each alkane is nearly identical, allowing us to use 1, average curve for all 3 different alkanes.]]<br />
[[File:Washinton 2011 Pre-Optimization Quant.png|left|450px|thumb|Diagram showing yields of the C13 and C15 alkanes. Note: The C17 alkene is not included due to inability to quantify.]]<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Optimized Production'''=<br />
By optimizing the method of growth, media, vectors, and cell line we were able to successfully increase alkane yield over 80-fold. Note that this improvement doesn't include C17 alkene production, as we were unable to quantify the C17 alkene peak due to co-elution with the C14 alcohol. The parameters we adjusted for optimization are discussed in detail at [https://2011.igem.org/Team:Washington/Alkanes/Future/Vector our systems optimization] page under future directions, as this is an on going process.<br />
<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
='''The FabBrick, a module for even chain length alkane production'''=<br />
In order to more closely match the composition of diesel, we wanted a way to make the C14 and C16 alkanes, something that has never been reported before in the literature. For a detailed account of how we achieved this please see our [https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 future directions page]. But a brief summary of our results is as follows:<br />
<br />
We found an enzyme, FabH2 from ''Bacillus subtilis'' that has been hypothesized to start fatty acid biosynthesis with a 3 carbon unit instead of the normal 2 carbon unit. We thought that if we could express FabH2 together with the PetroBrick, we could make even chain length alkanes. Ww cloned FabH2 into a alc inducible promoter to form [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 the FabBrick], an add-on module to teh PetroBrick system. The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When the FabBrick is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels. The FabBrick system shows the modularity and expandability of the PetroBrick system, and is a first proof of concept of the expansion of PetroBricks to produce a wider range of products. For more information on FabH2, refer to our [https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 future directions page ].<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]</div>Siegeljbhttp://2011.igem.org/Team:Washington/Alkanes/ResultsTeam:Washington/Alkanes/Results2011-10-29T00:35:51Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''GCMS confirms The PetroBrick enables diesel production from sugar using ''E. coli'''''=<br />
<br />
We performed GC-MS analysis on extracts from cell cultures expressing both [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Acyl-ACP Reductase] (AAR) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 Aldehyde Decarbonylase] (ADC) (this composite part is designated [http://partsregistry.org/Part:BBa_K590025 The PetroBrick]). To act as controls and in order to show that the alkane production system is working as expected, we also analyzed cell cultures expressing either only AAR, or only ADC.<br />
<br />
<br />
[[File:Washington PetroBrick.png|300px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
[[Image:Washinton_AARADCGC-MS.png|center|600px|thumb|GCMS confirms PetroBrick diesel production from sugar using E. coli]]<br />
<br />
<br><br />
<br />
'''No significant hydrocarbons peaks in samples extracted from cells expressing only ADC.''' The blue GCMS chromatagram trace on the bottom show no significant peaks are found on the ADC only control within the time range that we expect alkane signal based upon GC-MS runs on chemical standards (8-10.5 minutes). This is expected, as ''E. coli'' does not normally produce any long chain length aldehyde substrates.<br />
<br />
'''C16 alcohols are observed when AAR is expressed on its own.''' The red GCMS chromatagram trace in the middle has a significant peak at 10.2 minutes corresponding to the C16 alcohol (as confirmed by comparison of the peak's MS spectra to a reference library) is observed when AAR is expressed in MG1655 E. coli. The production of C16 alcohols in cells expressing AAR (but not in cells expressing only ADC) is consistent with AAR reducing even chain length Acyl-ACPs into even chain length fatty aldehydes, which are further reduced by aldehyde dehydrogenases to the alcohol.<br />
<br />
'''The PetroBrick Works! C13, C15, and C17 alkanes are produced when both AAR and ADC are expressed.''' <br />
[[File:Washington2011 Spectra.png|right|550px|thumb|Comparasion of the NIST reference C15 alkane spectrum( in blue) to the C15 peak spectrum taken from a PetroBrick culture (in red). Note the strong m/z peak at 212, corresponding to the parent ion of the C15 alkane. For spectra of the C13 alkane and C17 alkene, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]]]<br />
The green GCMS chromatagram trace in the middle has a significant new peaks corresponding to the C13 (8.2min) and C15 (9.2min) alkanes, as well as a peak at 10.2 minutes that contains both the C14 alcohol and the C17 alkene. The fact that the C17 alkene peak overlaps with the C14 alcohol peak makes exact quantification of C17 akene yields impossible, but we have sufficient evidence to determine that both molecules are present in the 10.2 minute peak( the earlier section of the peak has an MS spectra consistent with the C16 alcohol, and the later section is consistent with the C17 alkene). The C13 and C15 alkane peaks showed MS spectra highly consistent with C13 and C15 alkane reference spectra(for these spectra, as well as the C17 alkene spectrum, refer to our [https://2011.igem.org/Team:Washington/Alkanes/spectra Spectra Page.]). In addition, both alkane molecules eluted off of the GC at the same time as C13 and C15 alkane standards, further increasing confidence that the 8.2 min and 9.2 min peaks do correspond to the C13 and C15 alkanes. Odd chain length alkane production is what is expected from our system, as ADC removes a carbonyl group from even chain length aldehydes produced by AAR, yielding an odd chain length alkane. Using the PetroBrick, we can turn simple sugars into diesel, a fuel fully compatible with modern infrastructure.<br />
<br />
<br />
----<br />
<br />
='''Initial Quantization of Alkane Production'''=<br />
In order to be able to know how much alkane was being produced by our ''E. coli'', we spiked known amounts of alkane into cell cultures known to not produce alkanes. We then extracted using ethyl acetate, and analyzed extracts using GC-MS. Since peak area corresponds to the amount of each substance present, we used these GC plots to make a standard curve that allows us to convert peak area into an absolute yield. To determine how much alkane was being produced by the Petrobrick, we grew up 3 MG1655 cell cultures transformed with the PetroBrick in M9 production media ([https://2011.igem.org/Team:Washington/alkanebiosynthesis link]), and analyzed using GC-MS.<br />
<br />
[[File:Washington_alkanestandardcurve.png|right|450px|thumb|Standard curves for converting peak area to an absolute amount. Note that these curves is almost perfectly linear. In addition, the curve generated from each alkane is nearly identical, allowing us to use 1, average curve for all 3 different alkanes.]]<br />
[[File:Washinton 2011 Pre-Optimization Quant.png|left|450px|thumb|Diagram showing yields of the C13 and C15 alkanes. Note: The C17 alkene is not included due to inability to quantify.]]<br />
<br />
<br />
<br />
<br />
<br />
----<br />
<br />
='''Optimized Production'''=<br />
By optimizing the method of growth, media, vectors, and cell line we were able to successfully increase alkane yield over 80-fold. Note that this improvement doesn't include C17 alkene production, as we were unable to quantify the C17 alkene peak due to co-elution with the C14 alcohol. The parameters we adjusted for optimization are discussed in detail at [https://2011.igem.org/Team:Washington/Alkanes/Future/Vector our systems optimization] page under future directions, as this is an on going process.<br />
<br />
[[File:Washinton 2011 Optimization Quant.png|center|550px|thumb|Our optimized growth conditions resulted in an 80 fold increase in total alkane yield.]]<br />
='''The FabBrick, a module for even chain length alkane production'''=<br />
In order to more closely match the composition of diesel, we wanted a way to make the C14 and C16 alkanes. We found an enzyme, FabH2 cfrom ''Bacillus subtilis'' that has been hypothesized to start fatty acid biosynthesis with a 3 carbon unit instead of the normal 2 carbon unit. We thought that if we could express FabH2 together with the PetroBrick, we could make even chain length alkanes. WE cloned FabH2 into a alc inducible promoter to form [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590064 the FabBrick], an add-on module to teh PetroBrick system. The FabBrick was co-transformed with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] in XL1-Blue ''E. coli''. cells were grown up in rich TB media+ 5uM IPTG, and innocultated to OD10 in M9 production media + 5uM IPTG. Alkanes were extracted after 24 hours. In addition, GC runs were performed on uninduced FabH2/PetroBrick cultures, and on cells expressing only [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick].<br />
<br />
There was no significant difference between the GC peaks in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] extract and in the uninduced FabH2/PetroBrick Extract. All of the peaks in these two extracts that fall within the expected elution time of the C16 alkane( 9.5-10 min) correspond to trace amounts of compounds that we cannot identify. When the FabBrick is induced, we see a new peak at approximetly 9.75 minutes. The MS spectra of this peak is highly consistent with C16 alkane. The overall ion fragmentation fingerprint is identical to that of alkane. Identification as a C16 alkane is confirmed by the presence of a strong parent ion at a mass of 226, exactly the mass of the C16 alkane. This confirms production of C16 alkane. In addition, some C14 alkane production was observed. This is the first time that even chain length alkanes have been produced recombinantly, and expands [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025 the PetroBrick] system to be able to produce all of the alkanes in the range C13-C17. Note that this peak corresponds to only trace levels of alkane( approximately 4mg/L), and future optimization is needed to increase even chain length production levels. The FabBrick system shows the modularity and expandability of the PetroBrick system, and is a first proof of concept of the expansion of PetroBricks to produce a wider range of products. For more information on FabH2, refer to our [https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 future directions page ].<br />
<br />
[[Image:FabBrickGCMS.png|left|400px|thumb|GCMS trace confirming C16 alkane produced only upon FabBrick induction. ]]<br />
<br />
<br />
<br />
<br />
[[Image:FabBrickSpectrum.png|middle|400px|thumb|MS spectrum verifies peak contains C16 alkane. ]]</div>Siegeljbhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-10-28T22:33:14Z<p>Siegeljb: /* Calculating the catalytic efficiency */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''Testing Kumamolisin-As against SC-PEP'''=<br />
<br />
After identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin has a much higher activity level than SC-PEP, in addition to being amenable to engineering and effective at gastric pH.]]<br />
<br />
----<br />
<br />
='''Testing mutants for activity on breaking down PQLP'''=<br />
<br />
=='''Using a whole cell lysate assay to screen a large number of mutants for good activity'''==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we screened each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed and the assay was performed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<br />
<br />
=='''Purifying and characterizing promising mutants for accurate rate comparison'''==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
<br />
='''Combining Mutations for the Construction of a Gluten Hydrolase'''=<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them together to make combinatorial variants.<br />
<br />
=='''Successful mutations were combined to construct a second library for screening'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP has been achieved.]]<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<br />
<br />
By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance.<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]<br />
<br />
<br />
<br />
=='''Calculating the catalytic efficiency'''==<br />
<br />
[[File:Washington_Linear_Michaelis_Celiac.png|right|450px|thumb|Activity vs. substrate graph shows assays were done at substrate levels where the Michaelis-Menten curve is linear. R<sup>2</sup> values were greater 0.9 for both lines of best fit.]]<br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Mutation(s)'''<br />
| align="center" style="background:#f0f0f0;"|'''''k<sub>cat</sub>''/''K<sub>M</sub>'' (M<sup>-1</sup> s<sup>-1</sup>)'''<br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023 '''N291D''']||2.40 x 10<sup>6</sup><br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590024 '''S354N, D358G, D368H''']||1.13 x 10<sup>6</sup> <br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590022 '''G319S, D358G, D368H''']||9.30 x 10<sup>5</sup><br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 '''G319S, D358G, D368H, N291D (KumaMax)''']||1.78 x 10<sup>7</sup><br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590021 '''wt-Kumamolisin''']||1.74 x 10<sup>5</sup><br />
|-<br />
| SC-PEP ||2.28 x 10<sup>4</sup><br />
|-<br />
|} <br />
<br />
<br />
<br />
Since we were able to measure the activity of these mutants at substrate and enzyme concentrations corresponding to the linear portion of a Michaelis-Menten curve, we were also able to calculate the catalytic efficiency (''k<sub>cat</sub>''/''K<sub>M</sub>'') values for wt-Kumamolisin, SC-PEP, and a few of our best mutants. This was done by first converting the raw activity in milli-Fluorescent Units (mFU)/min to the observed velocity (V<sub>obs</sub>) in M/min using a conversion constant with units of mFU/M. Since the concentration of total substrate and enzyme in each case was known, we were then able to use the equation (''k<sub>cat</sub>''/''K<sub>M</sub>'') = V<sub>obs</sub>/([E][S]), derived from the Michaelis-Menten equation, to calculate the catalytic efficiency of each mutant enzyme of interest. We were not, however, able to measure the independent catalytic (''k<sub>cat</sub>'') and binding constants (''K<sub>M</sub>'') using this methodology. Thus, one of our priorities moving forward will be to develop a mass spectroscopy assay to determine these constants independently.</div>Siegeljbhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-10-28T22:27:33Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''Testing Kumamolisin-As against SC-PEP'''=<br />
<br />
After identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin has a much higher activity level than SC-PEP, in addition to being amenable to engineering and effective at gastric pH.]]<br />
<br />
----<br />
<br />
='''Testing mutants for activity on breaking down PQLP'''=<br />
<br />
=='''Using a whole cell lysate assay to screen a large number of mutants for good activity'''==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we screened each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed and the assay was performed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<br />
<br />
=='''Purifying and characterizing promising mutants for accurate rate comparison'''==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
<br />
='''Combining Mutations for the Construction of a Gluten Hydrolase'''=<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them together to make combinatorial variants.<br />
<br />
=='''Successful mutations were combined to construct a second library for screening'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP has been achieved.]]<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<br />
<br />
By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance.<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]<br />
<br />
<br />
<br />
=='''Calculating the catalytic efficiency'''==<br />
<br />
[[File:Washington_Linear_Michaelis_Celiac.png|right|450px|thumb|Activity vs. substrate graph shows assays were done at substrate levels where the Michaelis-Menten curve is linear. R<sup>2</sup> values were greater 0.9 for both lines of best fit.]]<br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Mutation(s)'''<br />
| align="center" style="background:#f0f0f0;"|'''''k<sub>cat</sub>''/''K<sub>M</sub>'' (M<sup>-1</sup> s<sup>-1</sup>)'''<br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023 '''N291D''']||(5.21 ± 0.10) x 10<sup>8</sup><br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590024 '''S354N, D358G, D368H''']||(2.45 ± 0.02) x 10<sup>8</sup> <br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590022 '''G319S, D358G, D368H''']||(2.02 ± 0.02) x 10<sup>8</sup><br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 '''G319S, D358G, D368H, N291D (KumaMax)''']||(3.86 ± 0.04) x 10<sup>9</sup><br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590021 '''wt-Kumamolisin''']||(3.25 ± 0.05) x 10<sup>7</sup><br />
|-<br />
| SC-PEP ||(4.96 ± 0.75) x 10<sup>6</sup><br />
|-<br />
|} <br />
<br />
<br />
<br />
Since we were able to measure the activity of these mutants at substrate and enzyme concentrations corresponding to the linear portion of a Michaelis-Menten curve, we were also able to calculate the catalytic efficiency (''k<sub>cat</sub>''/''K<sub>M</sub>'') values for wt-Kumamolisin, SC-PEP, and a few of our best mutants. This was done by first converting the raw activity in milli-Fluorescent Units (mFU)/min to the observed velocity (V<sub>obs</sub>) in M/min using a conversion constant with units of mFU/M. Since the concentration of total substrate and enzyme in each case was known, we were then able to use the equation (''k<sub>cat</sub>''/''K<sub>M</sub>'') = V<sub>obs</sub>/([E][S]), derived from the Michaelis-Menten equation, to calculate the catalytic efficiency of each mutant enzyme of interest. We were not, however, able to measure the independent catalytic (''k<sub>cat</sub>'') and binding constants (''K<sub>M</sub>'') using this methodology. Thus, one of our priorities moving forward will be to develop a mass spectroscopy assay to determine these constants independently.</div>Siegeljbhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-10-28T22:27:19Z<p>Siegeljb: /* Calculating the catalytic efficiency */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''Testing Kumamolisin-As against SC-PEP'''=<br />
<br />
After identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolisin has a much higher activity level than SC-PEP, in addition to being amenable to engineering and effective at gastric pH.]]<br />
<br />
----<br />
<br />
='''Testing mutants for activity on breaking down PQLP'''=<br />
<br />
=='''Using a whole cell lysate assay to screen a large number of mutants for good activity'''==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we screened each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed and the assay was performed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate. *"deg" in the data labels indicates use of a degenerate primer. Data for these points is representative of a group of variants, each with different substitutions at one residue. This accounts for the <100 data points on this graph, despite testing >100 novel mutants in total.]]<br />
<br />
=='''Purifying and characterizing promising mutants for accurate rate comparison'''==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|Concentration controlled rate data relative to native Kumamolisin for three of our most active mutants.]]<br />
<br />
<br />
----<br />
<br />
<br />
='''Combining Mutations for the Construction of a Gluten Hydrolase'''=<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them together to make combinatorial variants.<br />
<br />
=='''Successful mutations were combined to construct a second library for screening'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP has been achieved.]]<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<br />
<br />
By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance.<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]<br />
<br />
=='''Calculating the catalytic efficiency'''==<br />
<br />
[[File:Washington_Linear_Michaelis_Celiac.png|right|450px|thumb|Activity vs. substrate graph shows assays were done at substrate levels where the Michaelis-Menten curve is linear. R<sup>2</sup> values were greater 0.9 for both lines of best fit.]]<br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Mutation(s)'''<br />
| align="center" style="background:#f0f0f0;"|'''''k<sub>cat</sub>''/''K<sub>M</sub>'' (M<sup>-1</sup> s<sup>-1</sup>)'''<br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023 '''N291D''']||(5.21 ± 0.10) x 10<sup>8</sup><br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590024 '''S354N, D358G, D368H''']||(2.45 ± 0.02) x 10<sup>8</sup> <br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590022 '''G319S, D358G, D368H''']||(2.02 ± 0.02) x 10<sup>8</sup><br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 '''G319S, D358G, D368H, N291D (KumaMax)''']||(3.86 ± 0.04) x 10<sup>9</sup><br />
|-<br />
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590021 '''wt-Kumamolisin''']||(3.25 ± 0.05) x 10<sup>7</sup><br />
|-<br />
| SC-PEP ||(4.96 ± 0.75) x 10<sup>6</sup><br />
|-<br />
|} <br />
<br />
<br />
<br />
Since we were able to measure the activity of these mutants at substrate and enzyme concentrations corresponding to the linear portion of a Michaelis-Menten curve, we were also able to calculate the catalytic efficiency (''k<sub>cat</sub>''/''K<sub>M</sub>'') values for wt-Kumamolisin, SC-PEP, and a few of our best mutants. This was done by first converting the raw activity in milli-Fluorescent Units (mFU)/min to the observed velocity (V<sub>obs</sub>) in M/min using a conversion constant with units of mFU/M. Since the concentration of total substrate and enzyme in each case was known, we were then able to use the equation (''k<sub>cat</sub>''/''K<sub>M</sub>'') = V<sub>obs</sub>/([E][S]), derived from the Michaelis-Menten equation, to calculate the catalytic efficiency of each mutant enzyme of interest. We were not, however, able to measure the independent catalytic (''k<sub>cat</sub>'') and binding constants (''K<sub>M</sub>'') using this methodology. Thus, one of our priorities moving forward will be to develop a mass spectroscopy assay to determine these constants independently.</div>Siegeljbhttp://2011.igem.org/Team:Washington/Alkanes/FutureTeam:Washington/Alkanes/Future2011-10-28T22:17:23Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
Our current ''in vivo'' alkane production system efficiently makes C15 alkanes. However, to be efficient enough for factory production, there are two broad goals to be done: <br />
# Increase production efficiency <br />
# Increase the diversity of the range of alkanes that can be produced. <br />
# Increase the scale of the system for industrial processes. <br />
We have already begun efforts to expand the efficiency and scope of alkane production, '''check them out below!'''<br />
<br />
[[Image:UW_2011_Alkane_Future_Work_Image.png|7px||right]]<br />
<br />
[[Image:Washington system optimization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Vector]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Vector '''System Optimization''']<br />
:<nowiki> The efficiency of this system was reported to be much higher than our initial system, but the growth conditions of the assay and the DNA was not available to us. We increased production efficiency by altering the initial environmental conditions in the assay.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_ADC_Redesign.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign '''Decarbonylase Redesign''']<br />
:<nowiki>One way to diversify the kind of alkanes produced is to alter the substrate specificity of the proteins involved. Since an alternative alkane-producing enzyme has not been found, we decided to mutate the aldehyde decarbonylase to produce shorter-chain alkanes.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_LuxCDE.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE '''Alternative Aldehyde''']<br />
:<nowiki>Another way to diversify our system is to use alternative proteins. Our current system uses acyl-ACP reductase, and we've identified an hypothetical alternative system that produces aldehydes: LuxCDE, made from parts from the 2010 competition.</nowiki><br />
<br><br><br><br />
<br />
[[Image:Washington_2011_fabh2_branch.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 '''Branched Alkane Biosynthesis''']<br />
:<nowiki> Our system is only capable of producing unbranched alkanes, as the cell mainly utilizes straight chained fatty acids. However, fuel we use are also composed largely of branched alkanes that affect very important properties of the fuel such as flash point and freezing point. If our fuels are truly intended to be synthesized in bacteria, we need to work on methods of making those crucial branched chained alkanes. We explored FabH2, a protein that when involved in fatty acid synthesis makes branched fatty acids. </nowiki><br />
<br><br />
<br />
[[Image:Washington 2011 Protein Localization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Localization]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Localization '''Enzyme Localization''']<br />
:<nowiki> The easiest way to increase yield is to increase the concentration; if the number of molecules remained the same but the volume decreased, the reaction speed increases with occurrences of molecular collisions. However, this is only easily done with purified protein assays. However, there are ways to "increase the concentration" of reactions in cells. We cannot make the cells literally denser, but we can localize the enzymes involved in the reaction, which either decreases the volume of the cell in which the enzymes reside and/or limit the number of reaction steps.</nowiki><br />
<br />
[[Image:Washington_2011_Alternate_Chassis.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast '''Alternative Chassis''']<br />
:<nowiki> By synthesizing alkanes in organisms, alkanes can become a useful renewable energy source; CO2 emitted from burning the alkanes are converted to glucose through photosynthesis, and then the glucose can be fed to alkane-synthesizing organisms to restart the cycle. This cycle can be drastically improved if there were fewer steps in the cycle. We explored the possibility of optimizing the alkane biosynthesis system in different micro-organisms, with the idea that perhaps with autotrophic organisms, we do not need to obtain glucose for the reaction.</nowiki><br />
<br />
<br />
<br />
[[Image:Washington_FB.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling '''System Modeling''']<br />
:<nowiki> We have begun Flux Balance Modeling the alkane production system in order to better understand the carbon flux through the pathway. We believe that by understanding the curent flux through the pathway we can intelligently reengineer the platform in order to maximize flux to alkanes. </nowiki></div>Siegeljbhttp://2011.igem.org/File:Washington_FB.pngFile:Washington FB.png2011-10-28T22:14:54Z<p>Siegeljb: </p>
<hr />
<div></div>Siegeljbhttp://2011.igem.org/Team:Washington/Alkanes/Future/ModelingTeam:Washington/Alkanes/Future/Modeling2011-10-27T18:36:34Z<p>Siegeljb: Created page with "{{Template:Team:Washington/Templates/Top}} __NOTOC__ <center><big><big><big><big>'''Diesel Production: System Modeling, Flux Balance Analysis'''</big></big></big></big></center>..."</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: System Modeling, Flux Balance Analysis'''</big></big></big></big></center><br><br><br />
<br />
FILL ME IN</div>Siegeljbhttp://2011.igem.org/Team:Washington/Alkanes/FutureTeam:Washington/Alkanes/Future2011-10-27T18:36:02Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Future Directions'''</big></big></big></big></center><br><br><br />
<br />
Our current ''in vivo'' alkane production system efficiently makes C15 alkanes. However, to be efficient enough for factory production, there are two broad goals to be done: <br />
# Increase production efficiency <br />
# Increase the diversity of the range of alkanes that can be produced. <br />
# Increase the scale of the system for industrial processes. <br />
We have already begun efforts to expand the efficiency and scope of alkane production, '''check them out below!'''<br />
<br />
[[Image:UW_2011_Alkane_Future_Work_Image.png|7px||right]]<br />
<br />
[[Image:Washington system optimization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Vector]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Vector '''System Optimization''']<br />
:<nowiki> The efficiency of this system was reported to be much higher than our initial system, but the growth conditions of the assay and the DNA was not available to us. We increased production efficiency by altering the initial environmental conditions in the assay.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_ADC_Redesign.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign]]<br />
<br><br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/DecarbDesign '''Decarbonylase Redesign''']<br />
:<nowiki>One way to diversify the kind of alkanes produced is to alter the substrate specificity of the proteins involved. Since an alternative alkane-producing enzyme has not been found, we decided to mutate the aldehyde decarbonylase to produce shorter-chain alkanes.</nowiki><br />
<br><br><br />
<br />
[[Image:Washington_2011_LuxCDE.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/LuxCDE '''Alternative Aldehyde''']<br />
:<nowiki>Another way to diversify our system is to use alternative proteins. Our current system uses acyl-ACP reductase, and we've identified an hypothetical alternative system that produces aldehydes: LuxCDE, made from parts from the 2010 competition.</nowiki><br />
<br><br><br><br />
<br />
[[Image:Washington_2011_fabh2_branch.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/FabH2 '''Branched Alkane Biosynthesis''']<br />
:<nowiki> Our system is only capable of producing unbranched alkanes, as the cell mainly utilizes straight chained fatty acids. However, fuel we use are also composed largely of branched alkanes that affect very important properties of the fuel such as flash point and freezing point. If our fuels are truly intended to be synthesized in bacteria, we need to work on methods of making those crucial branched chained alkanes. We explored FabH2, a protein that when involved in fatty acid synthesis makes branched fatty acids. </nowiki><br />
<br><br />
<br />
[[Image:Washington 2011 Protein Localization.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Localization]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Localization '''Enzyme Localization''']<br />
:<nowiki> The easiest way to increase yield is to increase the concentration; if the number of molecules remained the same but the volume decreased, the reaction speed increases with occurrences of molecular collisions. However, this is only easily done with purified protein assays. However, there are ways to "increase the concentration" of reactions in cells. We cannot make the cells literally denser, but we can localize the enzymes involved in the reaction, which either decreases the volume of the cell in which the enzymes reside and/or limit the number of reaction steps.</nowiki><br />
<br />
[[Image:Washington_2011_Alternate_Chassis.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast '''Alternative Chassis''']<br />
:<nowiki> By synthesizing alkanes in organisms, alkanes can become a useful renewable energy source; CO2 emitted from burning the alkanes are converted to glucose through photosynthesis, and then the glucose can be fed to alkane-synthesizing organisms to restart the cycle. This cycle can be drastically improved if there were fewer steps in the cycle. We explored the possibility of optimizing the alkane biosynthesis system in different micro-organisms, with the idea that perhaps with autotrophic organisms, we do not need to obtain glucose for the reaction.</nowiki><br />
<br />
[[Image:Washington_2011_Alternate_Chassis.png|140px||left|link=https://2011.igem.org/Team:Washington/Alkanes/Future/Yeast]]<br />
<br><br />
[https://2011.igem.org/Team:Washington/Alkanes/Future/Modeling '''System Modeling''']<br />
:<nowiki> FILL ME IN </nowiki></div>Siegeljbhttp://2011.igem.org/Team:Washington/Team/AmericasTeam:Washington/Team/Americas2011-10-22T06:15:09Z<p>Siegeljb: Created page with "{{Template:Team:Washington/Templates/Top}} <center><big><big><big><big>'''Americas Regional Jamboree'''</big></big></big></big></center><br><br>"</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
<center><big><big><big><big>'''Americas Regional Jamboree'''</big></big></big></big></center><br><br></div>Siegeljbhttp://2011.igem.org/Template:Team:Washington/Templates/TopTemplate:Team:Washington/Templates/Top2011-10-22T06:14:05Z<p>Siegeljb: </p>
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<div id="ddnav" align="center"><br />
<ul><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington">Home</a><br />
<div><br />
<a href="https://2011.igem.org/Main_Page"> iGEM Homepage </a><br />
<a href="https://2011.igem.org/Team:Washington">UW 2011</a><br />
<a href="https://2010.igem.org/Team:Washington"> UW 2010 </a><br />
<a href="https://2009.igem.org/Team:Washington"> UW 2009 </a><br />
<a href="https://2008.igem.org/Team:University_of_Washington"> UW 2008 </a><br />
<a href="http://synbio.washington.edu/"> UW SynBio </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">About Us</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">The team</a><br />
<a href="https://2011.igem.org/Team:Washington/Team/Sponsors"> Sponsors </a><br />
<a href="https://2011.igem.org/Team:Washington/Team/Americas"> Americas Regional Jamboree </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Diesel Production</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Results"> Results Summary </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Gluten Destruction</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Results"> Results Summary</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background">iGEM Toolkits</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background"> Background </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors">Gibson Assembly Toolkit </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults">Gibson Toolkit Results</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit">Magnetosome Toolkit</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results">Magnetosome Results</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Parts"> Parts Submitted </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Future"> Future Directions </a><br />
<br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2011&group=Washington"> Parts Registry </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Protocols">Protocols</a><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Outreach</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Local Outreach</a><br />
<br />
<a href="https://2011.igem.org/Team:Washington/Outreach/iGEM_Collaborations">iGEM Collaborations</a></div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Safety">Safety</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</html></div>Siegeljbhttp://2011.igem.org/Team:Washington/Protocols/Gib_RxnTeam:Washington/Protocols/Gib Rxn2011-10-13T19:55:44Z<p>Siegeljb: /* Gibson Cloning Protocol */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
__NOTOC__<br />
<br />
=Gibson Cloning Protocol=<br />
<br />
<br />
==Materials Needed==<br />
#5X isothermal (ISO) reaction buffer (25% PEG-8000, 500 mM Tris-HCl pH 7.5, 50 mM MgCl2, 50 mM DTT, 1 mM each of the 4 dNTPs, and 5 mM NAD). This is prepared as described below.<br />
#T5 exonuclease (Epicentre)<br />
#Phusion DNA polymerase (New England Biolabs)<br />
#Taq DNA ligase (New England Biolabs)<br />
<br />
<br />
==Equipment==<br />
#Heat block or thermocycler with PCR tubes.<br />
<br />
<br />
==Procedure==<br />
#Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:<br />
##3 ml of 1 M Tris-HCl pH 7.5<br />
##150 μl of 2 M MgCl2<br />
##60 μl of 100 mM dGTP<br />
##60 μl of 100 mM dATP<br />
##60 μl of 100 mM dTTP<br />
##60 μl of 100 mM dCTP<br />
##300 μl of 1 M DTT<br />
##1.5 g PEG-8000<br />
##300 μl of 100 mM NAD<br />
##Add water to 6 ml<br />
##Aliquot 100 μl and store at -20 °C<br />
#Prepare an assembly master mixture. This can be prepared by combining the following:<br />
##320 μl 5X ISO buffer<br />
##0.64 μl of 10 U/ μl T5 exo<br />
##20 μl of 2 U/μl Phusion pol<br />
##160 μl of 40 U/μl Taq lig<br />
##Add water to 1.2 ml<br />
##Aliquot 15 μl and store at -20 °C. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles.<br />
###This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.<br />
#Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.<br />
#Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).<br />
#Incubate at 50 °C for 15 to 60 min (60 min is optimal).<br />
#If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli.<br />
<br />
<br />
<br />
===Based on Methods described in [http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Enzymatic assembly of DNA molecules up to several hundred kilobases], modified by Rob Egbert===</div>Siegeljbhttp://2011.igem.org/Team:Washington/Protocols/Gib_RxnTeam:Washington/Protocols/Gib Rxn2011-10-13T19:55:21Z<p>Siegeljb: /* Gibson Cloning/Assembly */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
<br />
__NOTOC__<br />
<br />
=Gibson Cloning Protocol=<br />
<br />
==Materials Needed==<br />
#5X isothermal (ISO) reaction buffer (25% PEG-8000, 500 mM Tris-HCl pH 7.5, 50 mM MgCl2, 50 mM DTT, 1 mM each of the 4 dNTPs, and 5 mM NAD). This is prepared as described below.<br />
#T5 exonuclease (Epicentre)<br />
#Phusion DNA polymerase (New England Biolabs)<br />
#Taq DNA ligase (New England Biolabs)<br />
<br />
==Equipment==<br />
1. Heat block or thermocycler with PCR tubes.<br />
<br />
<br />
==Procedure==<br />
#Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:<br />
##3 ml of 1 M Tris-HCl pH 7.5<br />
##150 μl of 2 M MgCl2<br />
##60 μl of 100 mM dGTP<br />
##60 μl of 100 mM dATP<br />
##60 μl of 100 mM dTTP<br />
##60 μl of 100 mM dCTP<br />
##300 μl of 1 M DTT<br />
##1.5 g PEG-8000<br />
##300 μl of 100 mM NAD<br />
##Add water to 6 ml<br />
##Aliquot 100 μl and store at -20 °C<br />
#Prepare an assembly master mixture. This can be prepared by combining the following:<br />
##320 μl 5X ISO buffer<br />
##0.64 μl of 10 U/ μl T5 exo<br />
##20 μl of 2 U/μl Phusion pol<br />
##160 μl of 40 U/μl Taq lig<br />
##Add water to 1.2 ml<br />
##Aliquot 15 μl and store at -20 °C. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles.<br />
###This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.<br />
#Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.<br />
#Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).<br />
#Incubate at 50 °C for 15 to 60 min (60 min is optimal).<br />
#If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli.<br />
<br />
<br />
<br />
===Based on Methods described in [http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Enzymatic assembly of DNA molecules up to several hundred kilobases], modified by Rob Egbert===</div>Siegeljbhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-28T07:13:56Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
=<center>'''Make It or Break It: <br/> Diesel Production and Gluten Destruction, the Synthetic Biology Way'''</center>=<br />
<br />
Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. <br />
<br />
<br />
[[Image:Washington_Fire.jpg|right|320px|borderless]]<br />
[[Image:Washington_Bottle.jpg|left|200px|borderless]]<br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to bring to the Parts Registry the Magnetosome Toolkit: a set of 18 genes from an essential operon in magnetotactic bacteria which we are characterizing to create magnetic ''E. coli''.<br />
<br />
<br />
[[File:Washington_Spacer.jpg|15px]]<br />
[[Image:UW Diesel Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|10px]]<br />
<br />
<br/><br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]</div>Siegeljbhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-26T22:04:58Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
=<center>Make It or Break It: Diesel Production and Gluten Destruction, the Synthetic Biology Way</center>=<br />
Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. <br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to bring to the Parts Registry the Magnetosome Toolkit: a set of genes for biofabrication of magnetic particles.<br />
<br />
<br />
<br />
[[File:Washington_Spacer.jpg|15px]]<br />
[[Image:Diesel Production for Wiki front page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
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[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
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[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
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[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]</div>Siegeljbhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-26T22:04:43Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
=<center>Make It or Break It: Diesel Production and Gluten Destruction, the Synthetic Biology Way</center>=<br />
Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. <br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to bring to the Parts Registry the Magnetosome Toolkit: a set of genes for biofabrication of magnetic particles.<br />
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[[File:Washington_Spacer.jpg|15px]]<br />
[[Image:Diesel Production for Wiki front page.png|300px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
[[Image:UW Gluten Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|5px]]<br />
[[Image:UW Toolkits Front Page.png|300px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
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[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]</div>Siegeljbhttp://2011.igem.org/Team:WashingtonTeam:Washington2011-09-26T03:23:13Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
=<center>Make It or Break It: Diesel Production and Gluten Destruction, the Synthetic Biology Way</center>=<br />
Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. This summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. <br />
<br />
[https://2011.igem.org/Team:Washington/Alkanes/Background '''Make It: Diesel Production'''] We constructed a strain of ''Escherichia coli'' that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. <br />
<br />
[https://2011.igem.org/Team:Washington/Celiacs/Background '''Break It: Gluten Destruction'''] We identified a protease with gluten-degradation potential, and then reengineered it to have greatly increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. <br />
<br />
[https://2011.igem.org/Team:Washington/Magnetosomes/Background '''iGEM Toolkits'''] To enable next-generation cloning of standard biological parts, we built BioBrick vectors optimized for Gibson assembly and used them to bring to the Parts Registry the Magnetosome Toolkit: a set of genes for biofabrication of magnetic particles.<br />
<br />
<br />
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[[File:Washington_Spacer.jpg|35px]]<br />
[[Image:Diesel Production for Wiki.png|280px|link=https://2011.igem.org/Team:Washington/Alkanes/Background]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[Image:Gluten Destruction for Wiki.png|280px|link=https://2011.igem.org/Team:Washington/Celiacs/Background]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[Image:Gibson Assembly and Magnetosomes for Wiki.png|280px|link=https://2011.igem.org/Team:Washington/Magnetosomes/Background]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
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[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_OSLI.png|frameless|border|link=http://www.osli.ca|Oil Sands Leadership Intiative]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_UniversitySeal.gif|frameless|border|110px|link=http://http://www.washington.edu|University of Washington]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_Anaspec.gif|frameless|border|120px|link=http://www.anaspec.com|Anaspec]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington_ARPA-E_Logo.png|frameless|border|link=http://arpa-e.energy.gov/ProgramsProjects/Electrofuels.aspx|Advanced Research Projects Agency - Energy]]<br />
[[File:Washington_Spacer.jpg|35px]]<br />
[[File:Washington2011_Hhmi_362_72.jpg|link=http://www.hhmi.org/]]</div>Siegeljbhttp://2011.igem.org/Team:Washington/Outreach/iGEM_CollaborationsTeam:Washington/Outreach/iGEM Collaborations2011-09-23T20:31:49Z<p>Siegeljb: </p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
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<center><big><big><big><big>'''Community Outreach: iGEM Collaborations'''</big></big></big></big></center><br><br><br />
<br />
='''Primer Design Tool with LMU-Munich'''=<br />
<br />
We worked with [https://2011.igem.org/Team:LMU-Munich LMU-Munich 2011] iGEM team this summer on the testing and development of their PrimerDesign tool. To learn more about it check out their [https://2011.igem.org/Team:LMU-Munich/Primer_Design/Design_Primers website].<br />
<br />
<br />
----<br />
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<br />
[[Image:Washington_collab_test.png|right|550px|thumb|An example of the output from the tool based off of input from our luxC biobrick, multiple primers with various melting temperatures are given]]<br />
<br />
=='''Software Testing'''==<br />
<br />
We helped LMU-Munich test their primer design software by sending our primer data and comparing it with the output of their automated designer. To do this, we compared the sequences of the primers used to amplify out parts of the LuxBrick with the primers that their software designed and concluded that their software works as expected. Unfortunately, we did not have enough time to order the primers that the software designed to test it ourselves. Overall, we found the tool easy to use and potentially very useful for future primer design.<br />
<br />
<br />
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<br />
=='''Implemented Bug Fixes & Features'''==<br />
#Added cyanobacteria to available genomes<br />
#Switch coding sequences in the primer designer to allow for the user to incude a stop codon in a coding sequence. Before, software insisted that coding sequences not include the stop codon<br />
#Made primer designer default to only check for biobrick restriction enzymes. Before, Primer Designer defaulted to checking for all restriction sites, making the user scroll to the bottom of the page for results<br />
#Added support for FASTA files with headers<br />
#Added descriptions for each column of the Primer Designer output.<br />
<br />
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<center><gallery caption="Original vs. Revised Output" widths="425px" heights="250px" perrow="2"><br />
Image:Washington_OldPrimerDesignerdefault.png|<center>'''Original Output'''</center><br> The original output identified every commercially available enzyme recognition site by default. This made it annoying to reach the bottom of the page where the primer info was.<br />
Image:Washington_primernewdefault.png|<center>'''Revised Output'''</center> <br> New, more useful default output with only searching for BioBrick enzymes, and providing labels in the output<br />
</gallery></center><br />
<br />
<gallery><br />
[[File:Washington_OldPrimerDesignerdefault.png|left|550px|thumb|Old Default Primer Designer Output]]<br />
[[File:Washington_primernewdefault.png|left|550px|thumb|New, more useful default output with only searching for BioBrick enzymes, and providing labels in the output]]<br />
<gallery/></div>Siegeljbhttp://2011.igem.org/Team:Washington/Outreach/iGEM_CollaborationsTeam:Washington/Outreach/iGEM Collaborations2011-09-23T20:29:44Z<p>Siegeljb: /* Implemented Bug Fixes & Features */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Community Outreach: iGEM Collaborations'''</big></big></big></big></center><br><br><br />
<br />
='''Primer Design Tool with LMU-Munich'''=<br />
<br />
We worked with [https://2011.igem.org/Team:LMU-Munich LMU-Munich 2011] iGEM team this summer on the testing and development of their PrimerDesign tool. To learn more about it check out their [https://2011.igem.org/Team:LMU-Munich/Primer_Design/Design_Primers website].<br />
<br />
=='''Software Testing'''==<br />
<br />
We helped LMU-Munich test their primer design software by sending our primer data and comparing it with the output of their automated designer. To do this, we compared the sequences of the primers used to amplify out parts of the LuxBrick with the primers that their software designed and concluded that their software works as expected. Unfortunately, we did not have enough time to order the primers that the software designed to test it ourselves. Overall, we found the tool easy to use and potentially very useful for future primer design.<br />
<br />
[[File:Washington_collab_test.png|left|550px|thumb|An example of the output from the tool based off of input from our luxC biobrick, multiple primers with various melting temperatures are given]]<br />
<br />
=='''Implemented Bug Fixes & Features'''==<br />
#Added cyanobacteria to available genomes<br />
#Switch coding sequences in the primer designer to allow for the user to incude a stop codon in a coding sequence. Before, software insisted that coding sequences not include the stop codon<br />
#Made primer designer default to only check for biobrick restriction enzymes. Before, Primer Designer defaulted to checking for all restriction sites, making the user scroll to the bottom of the page for results<br />
#Added support for FASTA files with headers<br />
#Added descriptions for each column of the Primer Designer output.<br />
<br />
<br />
<center><gallery caption="Original vs. Revised Output" widths="425px" heights="250px" perrow="2"><br />
Image:Washington_OldPrimerDesignerdefault.png|<center>'''Original Output'''</center><br> The original output identified every commercially available enzyme recognition site by default. This made it annoying to reach the bottom of the page where the primer info was.<br />
Image:Washington_primernewdefault.png|<center>'''Revised Output'''</center> <br> New, more useful default output with only searching for BioBrick enzymes, and providing labels in the output<br />
</gallery></center><br />
<br />
<gallery><br />
[[File:Washington_OldPrimerDesignerdefault.png|left|550px|thumb|Old Default Primer Designer Output]]<br />
[[File:Washington_primernewdefault.png|left|550px|thumb|New, more useful default output with only searching for BioBrick enzymes, and providing labels in the output]]<br />
<gallery/></div>Siegeljbhttp://2011.igem.org/Team:Washington/Celiacs/PartsTeam:Washington/Celiacs/Parts2011-09-23T20:15:22Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Parts Submitted'''</big></big></big></big></center><br><br><br />
<br />
Gluten Destruction submitted four parts to the registry: Wild-type Kumamolisin-As and three of our promising mutants. A short description for each part is provided below.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590021 BBa_K590021: '''Kumamolisin-As''']<br />
<br />
An enzyme from the sedolisin family native to ''Alicyclobacillus sendaiensis'' with known collagenase activity at low pH and elevated temperatures.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590022 BBa_K590022: '''Kumamolisin-As_G319S, D358G, D368H''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has point mutations at residues 319, 358, and 368 from Glycine to Serine, Aspartate to Glycine, and Aspartate to Histidine, respectively.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023 BBa_K590023: '''Kumamolisin-As_N291D''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has a point mutation at residue 291 from Asparagine to Aspartate.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590024 BBa_K590024: '''Kumamolisin-As_S354N, D358G, D368H''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has point mutations at residue 354, 358, and 368 from Serine to Asparagine, Aspartate to Glycine, and Aspartate to Histidine, respectively.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 BBa_K590087: '''KumaMax (aka, Kumamolisin-As_G319S, D358G, D368H + N291D)''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant is the best combinatorial mutant found and has point mutations at residues 291, 319, 358, and 368 from Asparagine to Aspartate, Glycine to Serine, Aspartate to Glycine, and Aspartate to Histidine, respectively.</div>Siegeljbhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-09-23T07:40:38Z<p>Siegeljb: /* Gluten Destruction */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br><br />
<br />
<center><gallery caption="An Overview of the 2011 UW iGEM Team's Summer Projects" widths="250px" heights="400px" perrow="3"><br />
Image:Diesel Production for Wiki.png|'''Make It: Diesel Production'''<br>We designed and constructed a modular alkane production BioBrick, the PetroBrick, to generate alkanes, the main constituent of diesel. By expressing this BioBrick in ''E. coli'', we were able to produce alkanes of yields over 100 mg/mL.<br />
Image:Gluten Destruction for Wiki.png|'''Break It: Gluten Destruction'''<br>Gluten intolerance stems from an inappropriate immune response to PQLP, the most common motif in the immunogenic peptide. We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP. <br />
Image:Gibson Assembly and Magnetosomes for Wiki.png|'''The GibsonBricks ToolKit'''<br>We completed two iGEM toolkits. In the first we created five Gibson Cloning versions of traditional biobrick vectors, and in the second we cloned the genes essential for magnetosome formation and transformed two into ''E. coli''<br />
<br />
</gallery></center><br />
<br />
<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==''Data for Favorite New Parts''==<br />
<br />
<br />
==='''Diesel Production'''===<br />
<br />
:: '''1, 2.''' [http://partsregistry.org/Part:BBa_K590025 BBa_K590025: '''The PetroBrick'''] - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC], each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.<br />
<br />
==='''Gluten Destruction'''===<br />
<br />
:: '''3.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 BBa_K590087: '''KumamaMax''']- A modified version of the enzyme Kumamolisin, a protease ofthe sedolisin family native to ''Alicyclobacillus sendaiensis'' known to be active at low pH and elevated temperatures. To Kumamolisin, the mutations N291D, G319S D358G, D368H increase activity to the PQLP peptide, an antigenic epitope in gliadin, 118-fold.<br />
<br />
==='''Gibson Assembly Toolkit'''===<br />
<br />
::'''4.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010: '''pGA1A3_pLacGFP'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011: '''pGA1C3_pLacGFP'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012: '''pGA4C5_pLacGFP'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013: '''pGA4A5_pLacGFP'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014: '''pGA3K3_pLacGFP'''] - These are plasmid backbones optimized for use in Gibson cloning, with a variety of copy numbers and antibiotic resistances.<br />
<br />
==='''Magnetosome Toolkit'''===<br />
<br />
:: '''5.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015: '''sfGFP_mamK_pGA1C3'''] - This part consists of the ''mamK'' gene from ''Magnetospirillum magneticum'' strain AMB-1, fused to sfGFP, in the backbone of pGA1C3. MamK was previously reported to be required for proper alignment of magnetosomes in a chain in magnetic bacteria.<br />
<br />
:: '''6.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3'''] - This part consists of ''mamI'' gene from ''Magnetospirillum magneticum'' strain AMB-1, sfGFP in the backbone of pGA1C3. MamI is a membrane-localized protein that localizes magnetic vesicles to the surface of cells, thus forming characteristic magnetosome chains.<br />
<br />
==''Data for Existing Parts''==<br />
<br />
::'''7.''' [http://partsregistry.org/Part:BBa_K314100:Experience K314100: '''High Constitutive Expression Cassette'''] (Washington, iGEM 2010) - We used this part to express our Petrobrick.<br />
<br />
==''Improved Parts''==<br />
<br />
::'''8.''' [http://partsregistry.org/Part:BBa_K590059 BBa_K590059], [http://partsregistry.org/Part:BBa_K590060 BBa_K590060], [http://partsregistry.org/Part:BBa_K590061 BBa_K590061: '''LuxC, D, and E'''] (Cambridge, iGEM 2010) - Formerly part of the [http://partsregistry.org/Part:BBa_K325909 LuxBrick] the genes ''luxC, D,'' and ''E'' were not separated or codon-optimized. We codon-optimized these genes and put them under the control of standard Elowitz RBS's. This was accomplished by the Alternate Aldehyde branch of the Alkane Production team.<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<center><groupparts>iGEM011 Washington</groupparts></center></div>Siegeljbhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-09-23T07:38:37Z<p>Siegeljb: /* Improved Parts */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Data Page'''</big></big></big></big></center><br><br><br />
<br />
<center><gallery caption="An Overview of the 2011 UW iGEM Team's Summer Projects" widths="250px" heights="400px" perrow="3"><br />
Image:Diesel Production for Wiki.png|'''Make It: Diesel Production'''<br>We designed and constructed a modular alkane production BioBrick, the PetroBrick, to generate alkanes, the main constituent of diesel. By expressing this BioBrick in ''E. coli'', we were able to produce alkanes of yields over 100 mg/mL.<br />
Image:Gluten Destruction for Wiki.png|'''Break It: Gluten Destruction'''<br>Gluten intolerance stems from an inappropriate immune response to PQLP, the most common motif in the immunogenic peptide. We reengineered a protease, active at low pH, for strongly enhanced activity against PQLP. <br />
Image:Gibson Assembly and Magnetosomes for Wiki.png|'''The GibsonBricks ToolKit'''<br>We completed two iGEM toolkits. In the first we created five Gibson Cloning versions of traditional biobrick vectors, and in the second we cloned the genes essential for magnetosome formation and transformed two into ''E. coli''<br />
<br />
</gallery></center><br />
<br />
<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==''Data for Favorite New Parts''==<br />
<br />
<br />
==='''Diesel Production'''===<br />
<br />
:: '''1, 2.''' [http://partsregistry.org/Part:BBa_K590025 BBa_K590025: '''The PetroBrick'''] - A modular and open platform for the biological production of diesel fuel. The PetroBrick consists of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC], each behind a standard Elowitz RBS. All of this is under regulation by a high constitutive promoter in pSB1C3.<br />
<br />
==='''Gluten Destruction'''===<br />
<br />
:: '''3.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023 BBa_K590023: '''KumamaMax''']- A modified version of the enzyme Kumamolisin, a protease ofthe sedolisin family native to ''Alicyclobacillus sendaiensis'' known to be active at low pH and elevated temperatures. To Kumamolisin, the mutations N291D, G319S D358G, D368H increase activity to the PQLP peptide, an antigenic epitope in gliadin, 118-fold.<br />
<br />
==='''Gibson Assembly Toolkit'''===<br />
<br />
::'''4.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590010 BBa_K590010: '''pGA1A3_pLacGFP'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590011 BBa_K590011: '''pGA1C3_pLacGFP'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590012 BBa_K590012: '''pGA4C5_pLacGFP'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590013 BBa_K590013: '''pGA4A5_pLacGFP'''], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590014 BBa_K590014: '''pGA3K3_pLacGFP'''] - These are plasmid backbones optimized for use in Gibson cloning, with a variety of copy numbers and antibiotic resistances.<br />
<br />
==='''Magnetosome Toolkit'''===<br />
<br />
:: '''5.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590015 BBa_K590015: '''sfGFP_mamK_pGA1C3'''] - This part consists of the ''mamK'' gene from ''Magnetospirillum magneticum'' strain AMB-1, fused to sfGFP, in the backbone of pGA1C3. MamK was previously reported to be required for proper alignment of magnetosomes in a chain in magnetic bacteria.<br />
<br />
:: '''6.''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590016 BBa_K590016 '''sfGFP_mamI_pGA1C3'''] - This part consists of ''mamI'' gene from ''Magnetospirillum magneticum'' strain AMB-1, sfGFP in the backbone of pGA1C3. MamI is a membrane-localized protein that localizes magnetic vesicles to the surface of cells, thus forming characteristic magnetosome chains.<br />
<br />
==''Data for Existing Parts''==<br />
<br />
::'''7.''' [http://partsregistry.org/Part:BBa_K314100:Experience K314100: '''High Constitutive Expression Cassette'''] (Washington, iGEM 2010) - We used this part to express our Petrobrick.<br />
<br />
==''Improved Parts''==<br />
<br />
::'''8.''' [http://partsregistry.org/Part:BBa_K590059 BBa_K590059], [http://partsregistry.org/Part:BBa_K590060 BBa_K590060], [http://partsregistry.org/Part:BBa_K590061 BBa_K590061: '''LuxC, D, and E'''] (Cambridge, iGEM 2010) - Formerly part of the [http://partsregistry.org/Part:BBa_K325909 LuxBrick] the genes ''luxC, D,'' and ''E'' were not separated or codon-optimized. We codon-optimized these genes and put them under the control of standard Elowitz RBS's. This was accomplished by the Alternate Aldehyde branch of the Alkane Production team.<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<center><groupparts>iGEM011 Washington</groupparts></center></div>Siegeljbhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-23T05:42:48Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Magnetosome Toolkit: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
== Magnetosome gene-protein Fusions==<br />
<br />
Using our two genes of interest, we created C-terminal sfGFP fusions so we could track the localization of each gene separately within ''E.coli.'' <br />
<br />
[[File:Igem2011_mamK_and_I.png|700px|center]]<br />
<br />
The results we obtained with our sfGFP fusions inside ''E.coli'' were comparable to those done through other studies in the host organism ''Magnetospirillum magneticum''. In the image of mamK, a filament is seen running through the length of many bacteria. For mamI, the gene product is seen to fluoresce around the cell membrane of the bacteria but mostly concentrated at the ends. Similarly, the graph shows that as the arrow cross the cell membrane, the fluorescent peaks are at a maximum, and through the center of the cell, the level of fluorescence decreases.<br />
<br />
==Construction of the R5 region of the Magnetosome Island in ''E.coli''==<br />
<br />
After identifying that the construction of the scaffold had worked, we proceeded to work on the final assembly in three parts: mamHIEJKK, mamMNOPA, and mamQRBSTUV. The PCR products of the first, and the third part of the assembly are shown below. Both fragments of the assembly have been partially sequenced confirmed, and we are currently working on designing primers to fill in the gap sequences. Despite these gaps, when this samples were imaged, filaments in the first part (mamHIEJKL)were still apparent. <br/><br />
<center>[[File:Washington_iGEM2011_magentosome_HIEJKL3k3.png|400px|middle]][[File:Washington_iGEM2011_magentosome_MNOPA.png|100px|middle]][[File:Washington_iGEM2011_magentosome_QRBSTUV.png|100px|middle]]<br />
</center></div>Siegeljbhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ToolkitTeam:Washington/Magnetosomes/Magnet Toolkit2011-09-23T05:42:36Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Magnetosomes'''</big></big></big></big></center><br><br><br />
<br />
===What are magnetosomes? Where do they come from?===<br />
<br />
<br />
<br />
[[File:Magnetosome_chain.png|thumb|Fig. 1: A Chain of Magnetosomes within ''Magnetospirillum magneticum'' AMB-1]]<br />
<br />
Magnetotactic bacteria are prokaryotic organisms that possess the unique ability to align themselves along a magnetic field. This form of taxis is made possible by the formation of a magnetosome. Magnetosomes are small invaginations of the bacterial inner membrane that contain magnetite particles.<br />
<br />
These particles range in size between 20 and several hundred nanometers and are aligned in one or several chains along the long axis of the bacteria. These particles act together to form a magnetic dipole across the bacteria, allowing it to sense the earth’s magnetic field. Magnetotactic bacteria are microaerophilic; therefore, magnetosomes are thought to help aid the organism in its search for the optimal oxygen level from a search in three dimensional space (in all directions) to a one dimensional space along a single path.<br />
<br />
===A Closer look at Magnetosome Formation ===<br />
<br />
The formation of the magnetosome organelle is a highly regulated, step-wise process requiring a cascade of essential genes. The process is generally hypothesized as four stages: i) membrane invagination, ii) acquiring minerals for magnetite formation, iii) iron-oxidation and reduction, iv) magnetite nucleation and morphology regulation. Earlier gene products must be present for later gene products to be formed as shown in the diagram below: [http://www.pnas.org/content/107/12/5593.full.pdf+html]:<br />
[[File:F6.medium.png|center|350px|thumb|Fig. 2: Diagram of stepwise magnetosome construction within AMB-1.]]<br />
<br />
===What did the UW iGEM team do with Magnetotactic Bacteria?===<br />
<br />
It is thought that many of the essential genes associated with magnetosome formation are located within a well-conserved region known as the magnetosome island (MAI). The MAI consists of 14 gene clusters labeled R1-R14 (see diagram below).Our team focused on the genes of the mamAB gene cluster (R5), as they were previously shown to be the only cluster essential for magnetosome membrane biogenesis in AMB-1 (diagram show below).[http://www.pnas.org/content/107/12/5593/F1.expansion.html].<br />
<br />
The goal of our project was to extract all the essential genes from (R5) required for magnetosome formation and express them in ''E. coli''. We are doing this to learn more about magnetosome formation and the magnet synthesis mechanism, because many of the genes' functions are still unknown in the host species. Using the information we have gained, we have organized a '''Magnetosome Toolkit''' containing most of the essential genes for proper magnetosome formation. Ultimately, we would like to continue expanding the magnetosome toolkit to have enough parts to show complete magnetosome formation in ''E.coli''.<br />
[[File:MamAB.png|center|500px|thumb|Fig. 3: The mamAB operon (R5) located in the magnetosome island (MAI).]]<br />
<br />
===About the Magnetosome Toolkit:===<br />
<br />
Using standard synthetic biology protocols and the vectors we created in our Gibson Assembly Toolkit, our team created the '''"Magnetosome Toolkit"''' which contains many of the genes required for magnetosome formation. Providing this toolkit to the Parts Registry will help allow future iGEM teams to manipulate and further understand magnetosome formation to eventually synthesize magnets in multiple organisms. <br />
<br />
<br />
<br />
=== Toolkit construction and mamAB assembly in E.coli ===<br />
<br />
== Individual Magnetosome (mam) genes==<br />
<br />
Before piecing together the 16 kb genome of the mamAB gene cluster within the magnetosome island (MAI), we extracted out the genes in the following groups: <br />
<br />
[[File:Washington_iGEM2011_magentosome_all_gel.png|right|thumb|700px|Gel Extracts of Individual Magnetosome Genes]]<br />
{| class="wikitable"<br />
|-<br />
! Gene groups<br />
! Length (bp)<br />
|-<br />
| mamHI<br />
| 1541<br />
|-<br />
| mamE<br />
| 2172<br />
|-<br />
| mamJ<br />
| 1538<br />
|-<br />
| mamKL<br />
| 1336<br />
|- <br />
| mamMN<br />
| 2323 <br />
|-<br />
| mamO<br />
| 1914<br />
|-<br />
| mamPA<br />
| 1493<br />
|-<br />
| mamQRB<br />
| 2029<br />
|- <br />
| mamSTU<br />
| 2030<br />
|-<br />
| mamV<br />
| 1002<br />
|-<br />
|}. <br />
<br />
[[File:Washington Methode image.jpg|right|thumb|500px]]<br />
<br />
Using standard protocols and our high-copy pGA vectors pGA1C3 and pGA1A3, these genes were extracted from the host genome via PCR and sequenced to confirm their accuracy. <br />
<br />
As previously noted, magnetosome formation within the host-organism, ''Magnetospirillium magneticum'', strain AMB-1, is a highly regulated step-wise process. As shown in Fig. 2, some genes encode proteins that form an invagination of the inner membrane, other genes which help align the magnetosomes into their characteristics chains, and others which regulate the biomineralization of magnetic particles. Our team chose to focus on genes specifically related to magnetosome scaffolding/alignment since they are the essential foundation for magnetosome development. In addition, the creation of a scaffold to which other genes localize is highly applicable to systems in synthetic biology. (for more information, please see our Future Directions page)<br />
<br />
Our genes of interest were mamK and mamI as they have functions related to localization of the magnetosome. Specifically, mamK is a bacterial actin-like cytoskeleton protein required for proper alignment of the magnetosomes in a chain. mamK is also shown to localize the mamI, which is loss inhibits membrane formation. <br />
(for other gene functions, please see the iGEM Toolkits parts submitted page):<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br/><br />
=References:=<br />
<br />
<br />
# Matsunaga, T., Okamura, Y., Fukuda, Y., Wahyudi, A.T., Murase, Y., Takeyama, H. (2005). Complete genome sequence of the facultative anaerobic Magnetotactic bacterium Magnetospirillum sp. strain AMB-1. ''DNA research''; 12: 157-166. Doi:10.1093/dnares/dsi002. <br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Murat, D., Quinlan, A., Vali, H., Komeili, A. (2010). Supporting Information. ''PNAS''; 107 (12): 5593-5598. Doi:10.1073/pnas.0914439107.<br />
# Quinlan, A., Murat, D., Vali, H., Komeili, A. (2011).The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization. ''Molecular Microbiology''; 80 (4): 855-1131. Doi:10.1111/j.1365-2958.2011.07631.x.<br />
# Richter, M., Kube, M., Bazylinski, D.A., Lombardot, T.,Glockner, F.O., Reinhardt, R., Shuler, D. (2007). Comparative genome analysis of four Magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function. ''Journal of Bacteriology''; 189(13): 4899-4910. Doi:10.1128/JB.00119-07.<br />
# Rioux, J.B., Philippe, N., Pereia, S., Pignol, D., Wu, L.F., Ginet, N. (2010). A second actin-like mamK protein in Magnetospirillum magneticum AMB-1 encoded outside the genomic magnetosome island. ''PLoS ONE''; 5(2): e9151. Doi:10.1371/journal.pone.0009151.</div>Siegeljbhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-23T05:26:06Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
='''Testing Kumamolisin-As against SC-PEP'''=<br />
<br />
After identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, in addition to being amenable to engineering and effective at gastric pH.]]<br />
<br />
----<br />
<br />
='''Testing mutants for activity on breaking down PQLP'''=<br />
<br />
=='''Using a whole cell lysate assay to screen a large number of mutants for good activity'''==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we screened each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed and the assay was performed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
<br />
<br />
=='''Purifying and characterizing promising mutants for accurate rate comparison'''==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
<br />
----<br />
<br />
='''Combining Mutants for the Construction of a Gluten Hydrolase'''=<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them together to make combinatorial variants.<br />
<br />
=='''A second library based on the first round of mutagensis was constructed and tested'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP.]]<br />
<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<br />
<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]<br />
<br />
By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!</div>Siegeljbhttp://2011.igem.org/Team:Washington/Celiacs/MethodsTeam:Washington/Celiacs/Methods2011-09-23T05:25:40Z<p>Siegeljb: /* Assay */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Redesigning Kumamolisin to Have Higher Activity at Low pH'''=<br />
<br />
<br />
[[File:Washington Foldit.png|600px|thumb|right|A Sample Mutation in Foldit Showing a Change from Glycine to Serine]]<br />
<br />
=='''Using Foldit to Design Mutations'''==<br />
In order to design mutations to wild-type Kumamolisin that would increase the enzyme’s proteolytic activity on gluten, we used a computational enzyme editing program called Foldit, which allows the user to hypothetically modify the amino acid sequence of a protein by creating point mutations at any location within the protein’s crystal structure. <br />
<br />
Within Foldit, we loaded Kumamolisin’s crystal structure in complex with a model PQLP peptide that recurs frequently in gluten, thus mimicking gluten as a substrate. We then modified the amino acid residues around the active site of Kumamolisin in the crystal structure, attempting to decrease the free energy of, and thus stabilize, the system. Estimations of free energy were based on algorithms run by Foldit.<br />
<br />
Using this method, we designed over 100 novel mutants, each of which could potentially increase Kumamolisin’s proteolytic activity on gluten.<br />
<br />
<br />
<br />
<br />
=='''Mutagenizing Kumamolisin'''==<br />
<br />
Kunkel mutagenesis is a classic procedure for incorporating targeted mutations into a piece of DNA, so it was ideal for changing our wild-type Kumamolisin gene to code instead for specifically designed variant enzymes.<br />
<br />
[[File:Washington Kunkels.png|500px|thumb|left|Overview of how Kunkel Mutagenesis works]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
==='''Kunkel Mutagenesis'''===<br />
<br />
The first step to producing our specially designed enzymes was to change the wild-type gene that codes for Kumamolisin to code instead for variant enzymes with our desired amino acid substitutions. <br />
<br />
We designed mutagenic oligonucleotide primers that would anneal to the wild-type Kumamolisin gene and incorporate point mutations that, when expressed, would result in a variant of Kumamolisin with the desired amino acid shift. <br />
<br />
To incorporate these mutations, we first isolated single stranded DNA (ssDNA) of our vector harboring the wild-type Kumamolisin gene. To do this we infected cells with bacteriophage M13, which packages its own ssDNA genome identified by length, and so in tandem packaged our vector in single stranded form. We then harvested the phage from the lysed culture of E. coli, and extracted our single stranded vector DNA.<br />
<br />
Next, we annealed and extended our mutagenic oligos to incorporate the specified mutations into the newly synthesized antisense strand. This hybrid vector was transformed into E. coli that degraded the original uracil-containing DNA and replaced it with sections complementary to the mutagenized strand.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''Using a Whole Cell Lysate Assay to Test Activity of Mutants'''==<br />
To test our designs, we developed a whole cell lysate assay that allowed us to perform a rough screen of a large number of mutants. In this assay, we expressed our mutant enzymes in <i>E. coli</i>, lysed the cells and separated the enzymes from large cell particulate. We then performed the assay at pH 4, mimicking the gastric environment. We added our model PQLP peptide, conjugated to both a fluorophore and a quencher so that no fluorescence would be achieved until after the peptide had been enzymatically cleaved. We then measured the fluorescence of each reaction at 30 second intervals, and were thereby able to estimate relative activity on breaking down PQLP by increase in fluorescence of the system.<br />
<br />
[[File:Washington Whole Cell Lysate Assay.jpg|center|General Overview of the Whole Cell Lysate]]<br />
<br />
=='''Testing Purified Mutants to Accurately Assess Activity'''==<br />
==='''Purification'''===<br />
After compiling a set of mutants which showed a relative increase in activity we proceeded to purify our mutant proteins. This step is crucial because it allows us to determine how our mutant compares with the wild-type on a quantitative level, as high activity without purification could simply be the result of high protein concentration. Growth, induction, and lysation of single colonies allowed the enzymes to be released from the cells, followed by collection of the purified proteins.<br />
<br />
==='''Assay'''===<br />
Concentrations were taken of the purified proteins, and diluted to the same concentration, to produce an assay resulting in accurate data representing which mutants had higher activity than kumamolisin and by how much their activity was greater.<br />
<br />
[[File:Washington First Raw Data.png|center|500px|thumb|We measured fluorescence of each reaction at 30 second intervals to see the rate at which each mutant cleaved PQLP.]]</div>Siegeljbhttp://2011.igem.org/Team:Washington/Celiacs/ResultsTeam:Washington/Celiacs/Results2011-09-23T05:25:22Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Results Summary'''</big></big></big></big></center><br><br><br />
<br />
<br />
<br />
<br />
='''Testing Kumamolisin-As against SC-PEP'''=<br />
<br />
After identifying Kumamolisin as a good candidate for activity at low pH, we tested its activity on breaking down PQLP at pH 4 against the activity of SC PEP, the enzyme currently in clinical trials for breaking down gluten. Kumamolisin had never been tested for its ability to breakdown gluten, and so we began novel experimentation into the enzyme's activity on our gluten model. From tests using the fluorescent PQLP system described in our methods section, Kumamolisin showed about 7 times better activity on breaking down PQLP at pH 4 when compared to the activity of SC PEP.<br />
<br />
[[File:Washington InitialKumavSC.png|center|500px|thumb|Initial screenings revealed that Kumamolysin has a much higher activity level than SC-PEP, in addition to being amenable to engineering and effective at gastric pH.]]<br />
<br />
----<br />
<br />
='''Testing mutants for activity on breaking down PQLP'''=<br />
<br />
=='''Using a whole cell lysate assay to screen a large number of mutants for good activity'''==<br />
<br />
In order to determine whether our proposed mutations to the wild-type Kumamolisin improved the ability of the enzyme to break down PQLP, we screened each mutant with a whole cell lysate fluorescence assay. Cells harboring the expressed mutants were lysed and the assay was performed at pH 4, mimicking the gastric environment. The released enzymes, after being roughly separated from cell material, were added to a fluorescent PQLP that had been conjugated to a quencher. Thus, no fluorescence was achieved until the peptide had been cleaved and the fluorophore had been released from the quencher. This allowed a relative assessment of rate of enzyme activity by measuring increase in fluorescence of the system.<br />
<br />
As one might expect, our first screen of mutants showed some mutants with a decrease in activity from the wild-type, some showed no change, and some actually showed great increase in activity. One single point mutant showed over 10-fold increase in activity from wild-type Kumamolisin!<br />
<br />
[[File:Washington Vertical Initial Screen.png|center|700px|thumb|Over 100 unique mutants were screened with a whole cell lysate assay for improved activity on the PQLP model substrate.]]<br />
<br />
<br />
<br />
=='''Purifying and characterizing promising mutants for accurate rate comparison'''==<br />
<br />
Once we had identified mutants that showed a promising increase in activity from the wild-type, we purified and characterized activity in concentration controlled fluorescence assays, identical to the fluorescence system used for the whole cell lysate assay. Our best mutant demonstrated an 11-fold increase in activity from the native enzyme.<br />
<br />
<br />
[[File:Washington Best Mutants.png|center|500px|thumb|We narrowed this down to a few of our best mutants.]]<br />
<br />
<br />
----<br />
<br />
='''Combining Mutants for the Construction of a Gluten Hydrolase'''=<br />
<br />
In order to achieve even more rate improvement from the native, we repeated our mutagenesis, this time taking successful mutations and adding them together to make combinatorial variants.<br />
<br />
=='''A second library based on the first round of mutagensis was constructed and tested'''==<br />
<br />
After designing a collection of combinatorial mutants, drawing from successful mutations discovered in the first round, we again performed a rough screen to identify promising combinations of mutations. From the initial screen on our combinatorial mutants, it appeared that we had achieved around 50 times better activity than native Kumamolisin on breaking down PQLP.<br />
<br />
[[File:Washington Comb Fold Change.png|center|500px|thumb|From initial whole cell lysate screens on combinatorial mutants, it appears that about 50-fold improvement over native Kumamolisin activity on PQLP.]]<br />
<br />
<br />
=='''One of the combinatorial mutants resulted in over a 100-fold increase in activity'''==<br />
<br />
<br />
[[File:Washington BestCombMutant.png|center|500px|thumb|Our final engineered enzyme showed activity over 100 fold higher than wild type Kumamolisin, and ~700 fold higher than SC-PEP.]]<br />
<br />
By combining two of our top groups of mutations from the first round, we achieved an over 100-fold increase in activity on breaking down PQLP from the wild-type enzyme. This variant enzyme is ultimately 784 times better at breaking down PQLP than SC PEP, the enzyme currently in clinical trials for treating gluten intolerance!</div>Siegeljbhttp://2011.igem.org/Team:Washington/Magnetosomes/Magnet_ResultsTeam:Washington/Magnetosomes/Magnet Results2011-09-23T05:21:50Z<p>Siegeljb: Created page with "{{Template:Team:Washington/Templates/Top}} __NOTOC__ <center><big><big><big><big>'''iGEM Toolkits: Background'''</big></big></big></big></center><br><br>"</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Background'''</big></big></big></big></center><br><br></div>Siegeljbhttp://2011.igem.org/Team:Washington/Magnetosomes/GibsonResultsTeam:Washington/Magnetosomes/GibsonResults2011-09-23T05:21:39Z<p>Siegeljb: Created page with "{{Template:Team:Washington/Templates/Top}} __NOTOC__ <center><big><big><big><big>'''iGEM Toolkits: Background'''</big></big></big></big></center><br><br>"</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''iGEM Toolkits: Background'''</big></big></big></big></center><br><br></div>Siegeljbhttp://2011.igem.org/Template:Team:Washington/Templates/TopTemplate:Team:Washington/Templates/Top2011-09-23T05:21:10Z<p>Siegeljb: </p>
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<ul><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington">Home</a><br />
<div><br />
<a href="https://2011.igem.org/Main_Page"> iGEM Homepage </a><br />
<a href="https://2011.igem.org/Team:Washington">UW 2011</a><br />
<a href="https://2010.igem.org/Team:Washington"> UW 2010 </a><br />
<a href="https://2009.igem.org/Team:Washington"> UW 2009 </a><br />
<a href="https://2008.igem.org/Team:University_of_Washington"> UW 2008 </a><br />
<a href="http://synbio.washington.edu/"> UW SynBio </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">About Us</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">The team</a><br />
<a href="https://2011.igem.org/Team:Washington/Team/Sponsors"> Sponsors </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Diesel Production</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Results"> Results Summary </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Gluten Destruction</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Results"> Results Summary</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background">iGEM Toolkits</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background"> Background </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors">Gibson Assembly Toolkit </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonResults">Gibson Results Summary </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit">Magnetosome Toolkit</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Results">Magnetosome Results Summary</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Parts"> Parts Submitted </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Future"> Future Directions </a><br />
<br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2011&group=Washington"> Parts Registry </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Protocols">Protocols</a><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Community Outreach</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Local Outreach</a><br />
<br />
<a href="https://2011.igem.org/Team:Washington/Outreach/iGEM_Collaborations">iGEM Collaborations</a></div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Safety">Safety</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</html></div>Siegeljbhttp://2011.igem.org/Team:Washington/Alkanes/MethodsTeam:Washington/Alkanes/Methods2011-09-23T05:15:54Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Introducing the PetroBrick'''=<br />
[[Image:Washington_2011_PetroBrick.png|220px|frameless|border="2"|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025|left]]<br />
<br />
<br />
:::::::::<p>In order to produce alkanes, we need both <partinfo>BBa_K590031</partinfo> acyl-ACP reductase ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC]) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Part:BBa_K590031] aldehyde decarbonylase ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR]) to work together in the cell. In order to achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick that contained both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product, and thus we have created a new modular alkane-producing platform:</p><br />
<br />
<br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;'''the <font size="5" weight=bold>PetroBrick.'''</font><br />
<br />
<br><br />
<br />
[[Image:Washington2011_PetroBrick_Construct.png|730px|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br><br />
<br><br />
<br />
----<br />
<br />
='''Alkane Production & Extraction'''=<br />
[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis.</p><br />
<br />
<br />
----<br />
<br />
<br />
='''Alkane Detection'''=<br />
==Gas Chromatography and Mass Spectroscopy==<br />
<br />
We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on it's boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. <br />
<br />
Inside the mass spectrometer each compound is then broken down into it's individual molecular components through electron stream ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.<br />
<br />
<br />
<center><gallery widths="400px" heights="300px" perrow="2"><br />
Image:Washington2011_Chrom.png|'''Gas chromatography:''' A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve<br />
<br />
Image:Washington2011 Spectra.png|'''Mass Spectroscopy:''' Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.<br />
<br />
</gallery></center></div>Siegeljbhttp://2011.igem.org/Team:Washington/Alkanes/MethodsTeam:Washington/Alkanes/Methods2011-09-23T05:11:27Z<p>Siegeljb: /* Alkane Detection */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Diesel Production: Methods'''</big></big></big></big></center><br><br><br />
<br />
='''Introducing the PetroBrick'''=<br />
[[Image:Washington_2011_PetroBrick.png|220px|frameless|border="2"|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025|left]]<br />
<br />
<br />
:::::::::<p>In order to produce alkanes, we need both <partinfo>BBa_K590031</partinfo> acyl-ACP reductase ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K590031 ADC]) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 Part:BBa_K590031] aldehyde decarbonylase ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K590032 AAR]) to work together in the cell. In order to achieve this goal, we used standard cloning methods combining both to construct the [http://partsregistry.org/Part:BBa_K590025 BBa_K590025] Biobrick that contained both AAR and ADC under a high constitutive promoter, each with its own Elowitz standard RBS. This construct successfully synthesized our target product, and thus we have created a new modular alkane-producing platform:</p><br />
<br />
<br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;'''the <font size="5" weight=bold>PetroBrick.'''</font><br />
<br />
<br><br />
<br />
[[Image:Washington2011_PetroBrick_Construct.png|730px|frameless|border|bottom|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
<br><br />
<br><br />
<br />
----<br />
<br />
='''Alkane Production & Extraction'''=<br />
[[File:Washington_Alkane_extraction.png|right|400px|thumb|Diagram showing the process of extraction.]]<p>After we had the complete gene assembly in our hands, the next step was to transform it into cells and start them growing for alkane production. We let them grow in 37 degree shaker for 48-72 hours, in sealed glass tubes. After the cells have gone through the alkane production process, the next step is to extract the alkanes out of the cell broth. We add acyl acetate directly into the glass test tube for cell growth. Then we vortex until to everything is well mixed, to make sure all of the alkanes go directly into the ethyl acetate solvent. Next, we spin down the mixture by using a centrifuge at full speed to form three layers (cell pellet, media, and ethyl acetate supernatant). We use only the ethyl acetate layer to send for GCMS analysis.</p><br />
<br />
<br />
----<br />
<br />
<br />
='''Alkane Detection'''=<br />
==Gas Chromatography and Mass Spectroscopy==<br />
<br />
We utilized a Gas Chromatograph / Mass Spectrometer (GCMS) to analyze alkane production concentrations. The GCMS is considered a "specific" test, because it identifies compounds specifically, not just to a category of compounds. It works by separating the individual components of a sample through a capillary column based mainly on it's boiling point, similar to fractional distillation. The separated compounds generally elute from the column at different retention times, and are passed to the mass spectrometer. <br />
<br />
Inside the mass spectrometer each compound is then broken down into it's individual molecular components through electron stream ionization. These ions differ in mass-to-charge (m/z) ratios, creating a unique ion de-composition profile for each compound that can be used to identify it through comparison to known chemical standards. Because compounds occasionally have similar elution times or mass spec fingerprints, the combination of analyses results in reducing the chances for overlap.<br />
<br />
[[Image:Washington2011 Spectra.png|480px|thumb|right| '''Mass Spectroscopy:''' Molecules exiting gas chromatography enter an electron impact mass spectrometer. The molecules are ionized and fragmented. The resulting spectra is compared to a database of molecules in order to predict its chemical identity. On the top (red) is an experimental spectra of our biologically produced C15 alkane, on the bottom (blue) is the NIST standard spectra for a C15 alkane. The fragmentation pattern and parent ion (blue arrow, bottom right) match perfectly.]]<br />
<br />
[[Image:Washington2011_Chrom.png|425px|thumb|left| '''Gas chromatography:''' A method used to separate molecules from the media extraction based on boiling point. In this image the temperature is increasing over time, and molecules with a higher boiling point are being eluted and detected by a mass spectrometer. The ion abundance is concentration dependent and can be converted if using a standard curve]]</div>Siegeljbhttp://2011.igem.org/File:Washington2011_Chrom.pngFile:Washington2011 Chrom.png2011-09-23T04:55:19Z<p>Siegeljb: </p>
<hr />
<div></div>Siegeljbhttp://2011.igem.org/File:Washington2011_Spectra.pngFile:Washington2011 Spectra.png2011-09-23T04:54:57Z<p>Siegeljb: </p>
<hr />
<div></div>Siegeljbhttp://2011.igem.org/Team:Washington/Celiacs/PartsTeam:Washington/Celiacs/Parts2011-09-23T04:45:41Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<center><big><big><big><big>'''Gluten Destruction: Parts Submitted'''</big></big></big></big></center><br><br><br />
<br />
Gluten Destruction submitted four parts to the registry: Wild-type Kumamolisin-As and three of our promising mutants. A short description for each part is provided below.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590021 BBa_K590021: '''Kumamolisin-As''']<br />
<br />
An enzyme from the sedolisin family native to ''Alicyclobacillus sendaiensis'' with known collagenase activity at low pH and elevated temperatures.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590022 BBa_K590022: '''Kumamolisin-As_G319S, D358G, D368H''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has point mutations at residues 319, 358, and 368 from Glycine to Serine, Aspartate to Glycine, and Aspartate to Histidine, respectively.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590023 BBa_K590023: '''Kumamolisin-As_N291D''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has a point mutation at residue 291 from Asparagine to Aspartate.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590024 BBa_K590024: '''Kumamolisin-As_S354N, D358G, D368H''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant has point mutations at residue 354, 358, and 368 from Serine to Asparagine, Aspartate to Glycine, and Aspartate to Histidine, respectively.<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K590087 BBa_K590087: '''Kumamolisin-As_G319S, D358G, D368H + N291D''']<br />
<br />
A mutated Kumamolisin-As enzyme aimed to combat gluten intolerance by increased activity with the PQLP peptide, an antigenic epitope in gliadin. This mutant is the best combinatorial mutant found and has point mutations at residues 291, 319, 358, and 368 from Asparagine to Aspartate, Glycine to Serine, Aspartate to Glycine, and Aspartate to Histidine, respectively.</div>Siegeljbhttp://2011.igem.org/Team:Washington/Alkanes/Future/YeastTeam:Washington/Alkanes/Future/Yeast2011-09-23T04:00:16Z<p>Siegeljb: </p>
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By synthesizing alkanes in organisms, alkanes can become a useful renewable energy source; CO2 emitted from burning the alkanes are converted to glucose through photosynthesis, and then the glucose can be fed to alkane-synthesizing organisms to restart the cycle. This cycle can be drastically improved if there were fewer steps in the cycle. We explored the possibility of optimizing the alkane biosynthesis system in different micro-organisms, with the idea that perhaps with autotrophic organisms, we do not need to obtain glucose for the reaction.</div>Siegeljbhttp://2011.igem.org/Team:Washington/Team/MembersTeam:Washington/Team/Members2011-09-23T02:59:56Z<p>Siegeljb: </p>
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<br />
== '''Who we are''' ==<br />
<br />
<gallery caption="Undergraduate Team Members" widths="180px" heights="120px" perrow="4"><br />
Image:Washington Casey Ager Profile1.jpg|<center>Casey Ager</center><br />
Image:Washington Photo-0088A.png|<center>Juhye An</center><br />
Image: Profile_Michael_Brasino.jpg|<center>Michael Brasino</center><br />
Image:Washington_.jpg|<center>Marika Cheng</center><br />
Image:Washington_choe_chris.png|<center>Chris Choe</center><br />
Image:Washington_.jpg|<center>Justin De Leon</center><br />
File:Princess Peachy.JPG|<center>Sydney Gordon</center><br />
Image:CIMG0021.jpg|<center>Daniel Hadidi</center><br />
Image:Washington_Matthew_Harger.JPG|<center>Matthew Harger</center><br />
Image:Washington_Elaine.jpg|<center>Elaine Lai</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Benjamin Mo</center><br />
Image:Washington 2011 CIMG0015-2.jpg|<center>Austin Moon</center><br />
Image:Washington_.jpg|<center>Rashmi Ravichandran</center><br />
Image:Washington_.jpg|<center>Seth Sagulo</center><br />
File:Washington_Liz.png|<center>Liz Stanley</center><br />
Image:Washington_.jpg|<center>Angus Toland</center><br />
Image:Washington_.jpg|<center>Sarah Wolf</center><br />
Image:Washington_.jpg|<center>Alicia Wong</center><br />
Image:Washington_Cindy.jpg|<center>Cindy Wu</center><br />
Image:Washington_Sean_Wu.jpg|<center>Sean Wu</center><br />
Image:Washington_2011_BATMAN.jpg|<center>Lei Zheng</center><br />
Image:Washington_david_zong.jpg|<center>David Zong</center><br />
<br />
</gallery><br />
<br />
<br />
<gallery caption="Advisors" widths="180px" heights="120px" perrow="4"><br />
Image:Washington_.jpg|<center>Aaron Chevalier <br/> Bioengineering </center><br />
Image:Washington_.jpg|<center>Rob Egbert <br/> Electrical Engineering </center><br />
Image:Washington_.jpg|<center>Chris Eiben <br/> Biochemistry </center><br />
Image:Washington_.jpg|<center>Jeremy Mills <br/> Biochemistry </center><br />
Image:Siegel_UW_2011.jpg|<center>Justin Siegel <br/> Biomolecular Structure and Design</center><br />
Image:Washington2011_Matthew-d-smith.jpg|<center>Matt Smith <br/> Molecular and Cellular Biology</center><br />
Image:Washington_.jpg|<center>Ingrid Swanson <br/> Microbiology </center><br />
</gallery><br />
<br />
<br />
<gallery caption="Faculty" widths="180px" heights="120px" perrow="5"><br />
image:Washington_David_Baker.jpg|<center>David Baker <br/> Biochemistry</center><br />
Image:Washington_Klavins.jpg|<center>Eric Klavins <br/> Electrical Engineering</center><br />
</gallery><br />
<br />
<br />
<gallery caption="Collaborators and Support" widths="140px" heights="100px" perrow="5"><br />
<br />
Image:Washington_OSLI.png|<center><b>Oil Sand Leadership Initiative</b> <br/> Funding to support Travel and Registration Costs</center><br />
<br />
Image:Washington_UniversitySeal.gif|<center><b>Biochemistry</b> <br/> Lab space</center><br />
<br />
Image:Washington_Anaspec.gif|<center>Anaspec <br/> <b>Peptide Discounts</b></center><br />
<br />
Image:Washington_ARPA-E_Logo.png|<center><b>Advanced Research Projects Agency - Energy </b><br/> Registration Support</center><br />
<br />
Image:Washington2011_Hhmi_362_72.jpg|<center><b>Howard Hughes Medical Institute</b> <br/> Lab materials and supplies</center><br />
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<br />
</gallery><br />
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<br />
<br />
<br />
== '''Who did what''' ==<br />
The teams were assembled during our winter quarter. During this term we went around to classes and had weekly meetings to introduce students to synthetic biology through a series of guest lectures. During the spring, students participated in brainstorming projects related to the sponsoring Faculty advisors labs (Baker and Klavins) and came up with the project they wanted to carry out. Students also planned and partcipated in community outreach events where we taught our community about synthetic biology. Over the summer, students completed all of the experimental work, but worked closely with both graduate students and faculty advisors to plan the most pertinent experiments and make the most of our limited time.<br />
<br />
=== Alkanes Production ===<br />
After producing promising results, in future directions <br />
<br />
Casey Ager, Austin Moon, and Seth Sagulo worked on Enzyme Localization via Direct Fusion and Zinc Finger Fusion methods.<br />
<br />
Juhye An, Elaine Lai, and Benjamin Mo worked on Decarbonylase Redesign<br />
<br />
Marika Cheng and Justin De Leon worked on Alternative Chassis<br />
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Chris Choe and David Zong worked on Alternate Aldehyde Production<br />
<br />
Matthew Harger worked on Branched Alkanes Production<br />
<br />
Matthew Harger and Lei Zheng worked on System Optimization<br />
<br />
=== Gluten Degradation ===<br />
Sydney Gordon, Daniel Hadidi, Liz Stanley, Angus Toland, Sarah Wolf, and Sean Wu designed, built, and tested Kumamolisin-As and over 100 mutants to combat gluten intolerance by increasing the activity on the PQLP antigenic peptide.<br />
<br />
=== iGEM Toolkits ===<br />
Michael Brasino, Rashmi Ravichandran, and Alicia Wong<br />
<br />
=== Community Outreach ===<br />
Cindy Wu headed and organized all iGEM 2011 Community Outreach events.</div>Siegeljbhttp://2011.igem.org/Team:Washington/Alkanes/BackgroundTeam:Washington/Alkanes/Background2011-09-23T02:00:39Z<p>Siegeljb: </p>
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<center><big><big><big><big>Diesel Production: Background</big></big></big></big></center><br><br><br />
<br />
=Petroleum, an Unfortunate Necessity=<br />
[[Image:Washington2011_PetroUsage.png|border|450px|right|thumb|Petroleum may be a necessity for modern civilization, but current extraction methods are unsustainable and non-cyclical in the long run.]]<br />
<br />
Modern society is completely dependent on petroleum based fuels. Automobiles are slowly transitioning towards electric power. However, for the foreseeable future, batteries will not be able to hold the energy needed for applications that require long range (e.g. jet planes, maritime shipping, and long range trucking) or high horsepower (e.g. agriculture, construction, industry). Without the use of petroleum, society as we know it would crumble. Petroleum is not a viable long term fuel due because it a non-renewable resource. When petroleum based fuels are combusted, CO<sub>2</sub> is released into the atmosphere. Using current technology, it is impossible to turn this carbon dioxide back into fuel, meaning that the amount of petroleum based fuel is a finite commodity. In addition, this excess carbon dioxide is a potent greenhouse gas that contributes to global warming.<br />
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<br />
=Todays biofuels are ''renewable'', but do not work as "drop-in" replacements=<br />
<br />
<br />
[[Image:Washington2011_BiofuelsAreRenewable.png|right|450px|frameless|thumb|Since carbon in biofuels can come from cellular respiration, biofuels may be carbon neutral. However, current biofuels have clear and problematic limits concerning energy production.]]<br />
<br />
Many different attempts have been made to produce a renewable, biologically derived fuel that would alleviate both the limited supply and emissions issues presented by petroleum based fuels. These efforts include alcohols (ethanol, butanol and other, higher alcohols), and biodiesel . Like petroleum based fuels, biofuels consist of combustible molecules that emit carbon dioxide. However, unlike petroleum based fuels, biofuels are renewable. CO<sub>2</sub> can be converted into more biofuel by feeding biofuel producing microbes (bacteria, yeast) photosynthetically derived plant biomass. Since the amount of CO<sub>2</sub> produced by burning a biofuel cannot exceed the CO<sub>2</sub> incorporated into plant biomass, a biofuel can be used indefinitely without any net carbon emissions.<br />
However, current biofuels consist of drastically different compounds from those found in petroleum. Petroleum consists of mostly long-chain length alkanes consisting of long hydrocarbon chains. Current biofuels contain either alcohols or long chain esters (biodiesel). Both of these molecules contain oxygen, which dramatically changes chemical properties. Both alcohols and biodiesel are more corrosive than unreactive alkanes. Alcohols are highly corrosive, both in pipelines ( [[#References | [1]]] ), and in engines not designed for the use of alcohols, even at concentrations as low as 20%( [[#References | [2]]] ). The corrosive property of alcohols in pipelines means that ethanol (the main alcohol in widespread use) is transported in vehicles (mostly by train)(cite), as opposed to by cheaper and less energy intensive pipelines( [[#References | [1]]] ). Transport of alcohols by pipeline would require retrofitting the entire fuel distribution infrastructure. The Esters in biodiesel are not directly as corrosive as alcohols, but can be biodegraded by anaerobic bacteria, producing hydrogen sulfide and other acids( [[#References | [3]]] ). Biodiesel has a higher freezing point than diesel, causing engine fuel filer clogging at low temperatures( [[#References | [4]]] ). Ethanol suffers from a much lower energy density than diesel(21.27-23.56 MJ/L vs 32.36-34.66 MJ/L)( [[#References | [5]]] ), resulting in lower gas mileage. The table below shows selected chemical properties of diesel, JP-8 (jet fuel), as well as the common biofuels ethanol, butanol, and biodiesel( data from [[#References | [5]]] and [[#References | [6]]]). <br />
{| {{table}}<br />
| align="center" style="background:#f0f0f0;"|'''Property'''<br />
| align="center" style="background:#f0f0f0;"|'''Diesel'''<br />
| align="center" style="background:#f0f0f0;"|'''Ethanol'''<br />
| align="center" style="background:#f0f0f0;"|'''Biodiesel'''<br />
|-<br />
| Specific gravity @ 15.5°C||0.85||0.794||0.88<br />
|-<br />
| Density @ 15.5°C(g/L)||848.25||792.05||878.09<br />
|-<br />
| Energy Density(MJ/L)||32.36-34.66 ||21.27-23.56 ||33.32-35.66<br />
|-<br />
| Cetane number||40-55||0-54||48-65<br />
|-<br />
| Freezing point(°C)||-40 - -1||-114||-3 -19<br />
|-<br />
| Viscosity( @ 20°C)(mm<sup>2</sup>/s)||2.8-5.0||1.5||6.4-6.6<br />
|-<br />
| Flash point(°C)||60-80||12.8||100-170<br />
|-<br />
| <br />
|} <br />
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<br />
=The Ideal Fuel is Diesel=<br />
<br />
[[Image:Washington2011_PetroBiofuel.png|frameless|left|400px]]<br />
:::::::::::::::::<p>The ideal fuel would be compatible with modern engines and infrastructure, and also be able to be produced in a renewable manner. No current biofuel has the same identical enough to that of diesel to be able to fully integrate with current engines and infrastructure. No known alternative fuel is able to match the chemical properties of diesel. Currently, the only way to renewably produce a fuel with the chemical properties and compatibility of diesel would be to make a biofuel with a composition identical to that of diesel. This would require a biological pathway that is able to produce alkanes, the main class of compounds in diesel. Alkanes are simple chains of carbon and hydrogen. The majority of the alkanes found in diesel have a carbon chain of 10 to 20 carbons long. Alkanes make up approximately 62% of jet diesel (a fairly representative diesel fuel)([[#References | [7]]]). This 62% includes 34% straight chain alkanes that contain only one linear chain, and 28% branched chain alkanes that contain 1 or more carbon branches. The remaining 38% consists mostly of cyclic and aromatic hydrocarbons. If long (10+) chain length alkanes could be biologically produced, it would allow for the production of a fuel that is both renewable and fully compatible with current engines and infrastructure.</p><br />
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<br />
= The Solution: a Microbial Alkane Production Pathway=<br />
[[Image:Washington2011_AlkaneAndBackCycle.png|right|400px|frameless|thumb|The use of AAR and ADC converts the fatty acid intermediate Acl-ACP into an aldehyde, then an alkane--biofuel. Acyl-ACP is naturally produced by all organisms, which increases host choice considerably]]<br />
A recent study([[#References | [8]]]),has shown the production of long chain length alkanes in ''E. coli'' using two genes found in many cyanobacteria species. The first gene codes for Acyl-ACP Reductase (AAR) which reduces long chain length acyl-ACPs into the corresponding fatty aldehydes. Acyl-ACPs are essential intermediates in fatty acid biosynthesis in every known organism, meaning that this system can work in a wide range of organisms. This long chain fatty acid acts as a substrate for Aldehyde Decarbonylase (ADC), the enzyme that removes the carbonyl group (C=O) from the fatty aldehyde, yielding an alkane one carbon shorter than the original Acyl-ACP and a molecule of formate. Since the vast majority of the fatty acyl-ACPs produced by ''E. coli'' have an even chain length, this system produces detectable amounts of only odd chain length alkanes. This study reported production of the C13, C15, and C17 alkanes, as well as the C17 alkene (unsaturated hydrocarbon). This chain length range fall well within the range of those found in diesel, so this system is theoretically able to make the alkane portion of a fuel compatible with current engines and infrastructure.<br />
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<br />
=The PetroBrick: A modular and open platform for the biological production of diesel fuel=<br />
<br />
[[Image:Washington_2011_PetroBrick.png|200px|frameless|border|left|link=http://partsregistry.org/wiki/index.php?title=Part:BBa_K590025]]<br />
The goal of our "'''''Make It: Diesel Production'''''" portion of this summer's iGEM project is to turn convert this recently discovered set of enzymes (AAR and ADC) for microbial alkane production into an open and modular platform for iGEM teams to develop into a robust replacement for petrochemical fuels. Our alkane production system is specifically designed to be easily improved upon, and we have started work on improving this open system, both by increasing alkane yields and by changing the product produced. In addition, we have started to move this system into an alternative chassis, yeast.<br />
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<br />
==References:==<br />
1. http://ourenergypolicy.org/docs/2/biofuels-taskforce.pdf<br />
<br />
2. http://www.environment.gov.au/atmosphere/fuelquality/publications/2000hours-vehicle-fleet/pubs/2000-hours-vehicles.pdf<br />
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3. Anaerobic Metabolism of Biodiesel and Its Impact on Metal Corrosion<br />
Deniz F. Aktas, Jason S. Lee, Brenda J. Little, Richard I. Ray, Irene A. Davidova, Christopher N. Lyles, Joseph M. Suflita Energy & Fuels 2010 24 (5), 2924-2928(http://pubs.acs.org/doi/full/10.1021/ef100084j)<br />
<br />
4. http://www.mda.state.mn.us/news/publications/renewable/biodiesel/biodieselcoldissues.pdf<br />
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5. http://www.afdc.energy.gov/afdc/pdfs/fueltable.pdf<br />
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6. The viscosities of three biodiesel fuels at temperatures up to 300°C<br />
R.E. Tate, K.C. Watts, C.A.W. Allen K.I. Wilkie Fuel 2006 85, 1010-1015 (https://netfiles.uiuc.edu/mccrady/shared/Biodiesel/The%20viscosities%20of%20three%20biodiesel%20fuels%20at%20temperatures%20up%20to%20300%208C.pdf)<br />
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7. http://www.dtic.mil/cgi-bin/GetTRDoc?AD=ADA317177&Location=U2&doc=GetTRDoc.pdf<br />
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8. Microbial Biosynthesis of Alkanes Andreas Schirmer, Mathew A. Rude, Xuezhi Li, Emanuela Popova and Stephen B. del Cardayre Science 30 July 2010: Vol. 329 no. 5991 pp. 559-562 http://www.sciencemag.org/content/329/5991/559</div>Siegeljbhttp://2011.igem.org/Team:Washington/alkanebiosynthesisTeam:Washington/alkanebiosynthesis2011-09-23T01:53:25Z<p>Siegeljb: </p>
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=Microbial Alkane Production Protocol=<br />
==Current Protocol for 100mL of Media==<br />
Adopted From Supplemental Information In [http://www.sciencemag.org/content/329/5991/559.full Microbial Biosynthesis of Alkanes Science Report]<br />
<br />
==='''Long Term Stocks to Prepare'''===<br />
<br />
Store at room temperature unless otherwise noted<br />
*1L of 1M Bis‐Tris (pH 7.25)<br />
*10mL of 1mg/mL Thiamine (store at -20 in 1mL aliquots)<br />
*10mL of 10% Triton X-100<br />
*1M MgSO4<br />
*0.1M FeCl3-6H2O<br />
<br />
<br />
<br />
==='''100mL M9 minGlucose Media Prep'''===<br />
'''ADD IN ORDER, make sure you have a sterilized Erlenmeyer flask for the initial mixing and a sterilized bottle to sterile filter into'''<br />
<br />
Constantly mix using a stir bar<br />
*75mL ddiH2O<br />
*3g glucose (Final 3%, 100% = 1g/mL)<br />
*0.6g Na2HPO4<br />
*0.3g KH2PO4<br />
*0.05g NaCl<br />
*0.2g NH4Cl<br />
*20mL of 1M Bis-Tris (pH 7.25)<br />
*1mL of 10% Triton<br />
*100uL of 1mg/mL thiamine<br />
*100uL of FeCl3-6H2O<br />
*100uL of MgSO4<br />
<br />
Once media is prepared sterile filter into a pre-sterilized glass bottle<br />
<br />
==='''General Production Protocol'''===<br />
<br />
*Grow 5mL of cells in Terrific Broth with Antibiotics overnight to saturation in 14mL culture tube<br />
*Measure OD (should be roughly 1.5)<br />
*Spin down cells at 4000rpm for 10 minutes<br />
* Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL eppindorf tube<br />
*Spin down cells at 4000rpm for 10 minutes<br />
*Resuspend in 1mL of sterile ddiH2O and transfer to a 1.5mL eppindorf tube<br />
*Spin down cells at 4000rpm for 10 minutes<br />
*Resuspend in 0.7mL of Production Media with Antibiotic<br />
*Transfer to a 24mL 13x250<br />
*Seal the top with alluminum foil and grow at 37degC for 48 hours<br />
*Extract by adding 0.7mL of EthylAcetate, vortex, transfer to an eppindorf tube, and spin at max speed for 1minute.<br />
*Remove 200uL of the top Ethyl Acetate layer into a glass vial with insert<br />
*Run sample on GC-MS and identify Alkanes<br />
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==='''GCMS Settings and Information'''===</div>Siegeljbhttp://2011.igem.org/Team:Washington/alkanebiosynthesisTeam:Washington/alkanebiosynthesis2011-09-23T01:45:22Z<p>Siegeljb: /* Long Term Stocks to Prepare */</p>
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=Microbial Alkane Production Protocol=<br />
==Current Protocol for 100mL of Media==<br />
Adopted From Supplemental Information In [http://www.sciencemag.org/content/329/5991/559.full Microbial Biosynthesis of Alkanes Science Report]<br />
<br />
==='''Long Term Stocks to Prepare'''===<br />
<br />
Store at room temperature unless otherwise noted<br />
*1L of 1M Bis‐Tris (pH 7.25)<br />
*10mL of 1mg/mL Thiamine (store at -20 in 1mL aliquots)<br />
*10mL of 10% Triton X-100<br />
*1M MgSO4<br />
*0.1M FeCl3-6H2O<br />
<br />
==='''100mL M9 minGlucose Media Prep'''===<br />
'''ADD IN ORDER, make sure you have a sterilized Erlenmeyer flask for the initial mixing and a sterilized bottle to sterile filter into'''<br />
<br />
Constantly mix using a stir bar<br />
*75mL ddiH2O<br />
*3g glucose (Final 3%, 100% = 1g/mL)<br />
*0.6g Na2HPO4<br />
*0.3g KH2PO4<br />
*0.05g NaCl<br />
*0.2g NH4Cl<br />
*20mL of 1M Bis-Tris (pH 7.25)<br />
*1mL of 10% Triton<br />
*100uL of 1mg/mL thiamine<br />
*100uL of FeCl3-6H2O<br />
*100uL of MgSO4<br />
*5uL of Each Trace Metal (do not mix together before, they crash out)<br />
<br />
<br />
Once media is prepared sterile filter into a pre-sterilized glass bottle<br />
<br />
Aliquot 0.7mL into culture tubes and add appropriate antibiotic (1microL of Kan or Chlor)<br />
<br />
Pick colonies and inoculate cultures, but DO NOT eject the tip into the tube<br />
<br />
Grow cultures for 40+ hrs at 37degC (if inducible add 1mM IPTG at and OD600 of ~1.0)<br />
<br />
Extract by adding 0.7mL of EthylAcetate, vortex, transfer to an eppindorf tube, and spin at max speed for 1minute.<br />
<br />
Remove 200uL of the top Ethyl Acetate layer into a glass vial with insert<br />
<br />
Run sample on GC-MS and identify Alkanes</div>Siegeljbhttp://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_AssayTeam:Washington/Protocols/Purified Enzyme Assay2011-09-23T01:44:00Z<p>Siegeljb: /* Substrate */</p>
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=Enzyme Assay=<br />
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=Substrate=<br />
The substrate was ordered at a 20% discount from Anasepc in support of Undergraduate education. The peptide Motif PQPQLP commonly found in gluten was synthesized with a fluorophore (5-FAM) or Quencher (QXL52) available from anaspec. It is denoted as the following<br />
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Ordered Peptide (95% Purity): QXL520-PQPQLP-K(5-FAM)-NH2<br />
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*'''Assay'''<br />
**Add 90uL of 5uM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate<br />
**Start reaction by adding 0.0125mg/mL enzyme (try to avoid bubbles and pippette quickly, but accurately)<br />
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!<br />
**Monitor the reaction with the SpectraMax<br />
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<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
=Substrate=<br />
The substrate was ordered at a 20% discount from Anasepc in support of Undergraduate education. The peptide Motif PQPQLP commonly found in gluten was synthesized with a fluorophore (5-FAM) or Quencher (QXL52) available from anaspec. It is denoted as the following<br />
<br />
Ordered Peptide (95% Purity): QXL520-PQPQLP-K(5-FAM)-NH2<br />
<br />
<br />
*'''Assay'''<br />
**Add 90uL of 5uM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate<br />
**Start reaction by adding 0.0125mg/mL enzyme (try to avoid bubbles and pippette quickly, but accurately)<br />
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!<br />
**Monitor the reaction with the SpectraMax<br />
***SpectraMax Settings:<br />
****Excitation:<br />
****Emission:<br />
**** Sensitivity Level:<br />
<br />
<br />
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------><br />
<div style="text-align:center"><br />
<br />
<br />
'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''<br />
&nbsp; &nbsp; &nbsp;<br />
</div></div>Siegeljbhttp://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_AssayTeam:Washington/Protocols/Purified Enzyme Assay2011-09-23T01:43:05Z<p>Siegeljb: /* Purified Enzyme Assay */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
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<!---------------------------------------PAGE CONTENT GOES BELOW THIS----------------------------------------><br />
<br />
=Substrate=<br />
The substrate was ordered at a 20% discount from Anasepc in support of Undergraduate education. The peptide Motif PQPQLP commonly found in gluten was synthesized with a fluorophore (5-FAM) or Quencher (QXL52) available from anaspec. It is denoted as the following<br />
<br />
Ordered Peptide (95% Purity): QXL520-PQPQLP-K(5-FAM)-NH2<br />
<br />
<br />
*'''Assay'''<br />
**Add 90uL of 5uM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate<br />
**Start reaction by adding 0.0125mg/mL enzyme (try to avoid bubbles and pippette quickly, but accurately)<br />
***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!<br />
**Monitor the reaction with the SpectraMax<br />
***SpectraMax Settings:<br />
****Excitation:<br />
****Emission:<br />
**** Sensitivity Level:<br />
<br />
<br />
<!---------------------------------------PAGE CONTENT GOES ABOVE THIS----------------------------------------><br />
<div style="text-align:center"><br />
<br />
<br />
'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''<br />
&nbsp; &nbsp; &nbsp;<br />
</div></div>Siegeljbhttp://2011.igem.org/Team:Washington/Celiacs/BackgroundTeam:Washington/Celiacs/Background2011-09-22T02:32:45Z<p>Siegeljb: /* Protein engineering of the peptidase Kumamolisin-As for use in treating gluten intolerance */</p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<br />
====What is Gluten Intolerance?====<br />
<br />
[[File:Washington CD Diagram.png|right|250px|thumb|Proline(P) and glutamine(Q) -rich peptide fragments of gluten provoke an immune response which causes painful inflammation in the digestive tract of gluten intolerant individuals.]]<br />
<br />
People who suffer from gluten intolerance have a adverse reaction to gluten proteins found in wheat, barley, and rye products. The glutens invoke an immune response in the digestive tract of genetically predisposed individuals resulting in inflammation of the gut, impeding the absorption of nutrients. Symptoms can appear in early childhood or later in life, and range widely in severity, from diarrhea, fatigue and weight loss to abdominal distension, anemia, and neurological symptoms. There are currently no effective therapies for this lifelong disease except the total elimination of glutens from the diet. Although celiac sprue remains largely underdiagnosed, its prevalence in the US and Europe is estimated at 0.5-1.0% of the population. With this in mind, we set out to design an enzyme therapeutic for gluten intolerance that could be taken in pill form.<br />
<br />
Proline (P)- and glutamine (Q)-rich components of gluten known as ‘gliadins’ appear to be responsible for the bulk of the immune response in most patients. Their high PQ content protects gliadin oligopeptides from degradation by gastrointenstinal endoproteases, but also presents a target for drug design. Any peptidase capable of cleaving at or near the P-Q bond while remaining active at the temperature and harsh pH of the stomach would have pharmacological potential as a therapy for celiac sprue.<br />
<br />
----<br />
<br />
====There is currently a protein therapeutic in clinical trials, but a second generation is needed====<br />
<br />
[[File:Washington_SC-PEP.png|left|250px|thumb|SC-PEP, a prolyl endopeptidase from ''Sphingomonas capsulata'']]<br />
<br />
One candidate enzyme, currently in clinical trials, utilizes a prolyl endopeptidase (PEP) from ''Sphingomonas capsulata'' (SC) to hydrolyze gliadins. This enzyme was a logical drug candidate as it has a native specificity for the proline rich gliadin peptides. Unfortunately, the enzyme’s optimal activity is at pH 7, and engineering attempts to enhance its activity at relevant gastric pH levels has not yet been met with significant success. It is therefore pertinent to identify new candidate enzymes that have both activity on the gliadin PQ structural motif, as well as optimal activity at gastric pH levels during digestion around pH 4. <br />
<br />
<br />
Here, we describe an alternative approach to identifying and engineering an enzyme therapeutic for celiac sprue. Rather than focusing primarily on substrate specificity when choosing our candidate enzyme, we identified an enzyme already capable of catalyzing peptide hydrolysis at gastric pH levels, regardless of peptide substrate specificity. Upon identification of such an enzyme we used computational tools to reengineer its substrate specificity for enhanced activity on immunogenic gliadin peptides with the common PQ structural motif.<br />
<br />
<br />
<br />
<br />
----<br />
<br />
[[File:Washington_Kumamolisin-As.png|right|200px|thumb|Kumamolisin-As, a protease from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'']]<br />
<br />
====We have identified an enzyme that could potentially act as a therapeutic for gluten intolerance====<br />
<br />
When searching for a protease with optimal activity at gastric pH levels that could be produced in a recombinant host, we identified an enzyme known as Kumamolisin-As. Kumamolisin-As, isolated from the thermoacidophilic bacterium ''Alicyclobacillus sendaiensis'' strain NTAP-1, has been shown to be produced in a recombinant host, and exhibits significant enzymatic activity at pH 2.5 and above. Its maximal activity is at about pH 4.0. It is this robust activity under acidic, gastric conditions that makes Kumamolisin-As so promising for the development of a pill for gluten intolerance.<br />
<br />
====A special set of catalytic residues enables high activity at gastric pH levels====<br />
<br />
One of the primary reasons that Kumamolisin-As is more active than SC-PEP at low pH is due its catalytic triad, the three amino acid residues most heavily involved in conducting the chemistry of cleaving peptides. SC-PEP is part of a large class of enzymes known as serine proteases, which make use of the catalytic triad Serine-Histidine-Aspartate. Acidic conditions can impede the histidine's ability to play its role in this triad, rendering most serine proteases inactive at low pH. Kumamolisin-As, on the other hand, is a member of the sedolisin family of serine-carboxyl peptidases, which utilize the key catalytic triad Serine-Glutamate-Aspartate to hydrolyze their substrates. It is the acidic Glutamate, as opposed to Histidine, in the triad that makes Kumamolisin-As optimal for catalyzing peptide cleavage at low pH. The native substrate for this enzyme in ''Alicyclobacillus sendaiensis'' is unknown. In addition, this enzyme has been shown to be thermostable, with an ideal temperature at 60&ordm;C, but still showing significant activity at 37&ordm;C. <br />
<br />
[[File:Kuma Bonded triad.png|left|250px|thumb|Kumamolisin-As' unusual catalytic triad Ser278-Glu78-Asp82 makes the enzyme well suited to a low pH environment.]]<br />
[[File:Serine protease triad2.png|left|250px|thumb|The traditional Ser-His-Asp catalytic triad found in serine proteases like SC-PEP does not function optimally at low pH.]]<br />
<br />
====Kumamolisin-As is already active for the dipeptide motif PR, we just need to change it to PQ====<br />
<br />
:::::::::::::::::::::::<p>Specificity studies have been conducted on the native enzyme, which is most efficient at hydrolyzing peptides at the carboxyl side after the PR dipeptide motif (where the P1 and P2 subsites are arginine and proline, respectively, in standard proteolysis nomenclature). The enzyme shows little preference for what is on the amino S1 and S2 subsites. The combination of Kumamolisin-As having the desired enzymatic activity under gastric pH levels, and the P2 subsite already having the specificity for the proline from the desired PQ motif makes it a highly attractive candidate for further engineering. In addition, several crystal structures of this enzyme have been solved, allowing for the use of computational design techniques.</p><br />
<br />
----<br />
<br />
====References====<br />
<br />
1.Shan, Lu, et al. "Structural Basis for Gluten Intolerance in Celiac Sprue." Science 297.5590 (2002): 2275-2279.<br />
<br />
2.Mustalahti, Kirsi, et al. "The prevalence of celiac disease in Europe: Results of a centralized, international mass screening project." Annals of Medicine 42.8 (2010): 587-595.<br />
<br />
3.Ehren, Jennifer, et al. "Protein engineering of improved prolyl endopeptidases for celiac sprue therapy." Protein Engineering, Design & Selection 21.12 (2008): 699-707.<br />
<br />
4.Wlodawer, Alexander, et al. "Crystallographic and Biochemical Investigations of Kumamolisin-As, a Serine-Carboxyl Peptidase with Collagenase Activity." The Journal of Biological Chemistry 279.20 (2004): 21500-21510.</div>Siegeljbhttp://2011.igem.org/Template:Team:Washington/Templates/TopTemplate:Team:Washington/Templates/Top2011-09-22T02:28:52Z<p>Siegeljb: </p>
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<ul><br />
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<a href="https://2011.igem.org/Team:Washington">Home</a><br />
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<a href="https://2011.igem.org/Main_Page"> iGEM Homepage </a><br />
<a href="https://2011.igem.org/Team:Washington">UW 2011</a><br />
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<a href="https://2011.igem.org/Team:Washington/Team/Members">About Us</a><br />
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<a href="https://2011.igem.org/Team:Washington/Alkanes/Results"> Results Summary </a><br />
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</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Gluten Destruction</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Results"> Results Summary</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background">iGEM Toolkits</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background"> Background </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors">Gibson Assembly Toolkit </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit">Magnetosomes Toolkit</a><br />
<br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Reference"> References </a> <a href="https://2011.igem.org/Team:Washington/Magnetosomes/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2011&group=Washington"> Parts Registry </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Protocols">Protocols</a><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Community Outreach</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Outreach/iGEM_Collaborations">iGEM Collaborations</a><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Local Outreach</a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Safety">Safety</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</html></div>Siegeljbhttp://2011.igem.org/Template:Team:Washington/Templates/TopTemplate:Team:Washington/Templates/Top2011-09-22T02:25:43Z<p>Siegeljb: </p>
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<a href="http://www.washington.edu/"><img src='https://static.igem.org/mediawiki/2011/1/10/UW_seal_desat.png' align="right" width= "50"/></a><br />
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<div id="ddnav" align="center"><br />
<ul><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington">Home</a><br />
<div><br />
<a href="https://2011.igem.org/Main_Page"> iGEM Homepage </a><br />
<a href="https://2011.igem.org/Team:Washington">UW 2011</a><br />
<a href="https://2010.igem.org/Team:Washington"> UW 2010 </a><br />
<a href="https://2009.igem.org/Team:Washington"> UW 2009 </a><br />
<a href="https://2008.igem.org/Team:University_of_Washington"> UW 2008 </a><br />
<a href="http://synbio.washington.edu/"> UW SynBio </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">About Us</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">The team</a><br />
<a href="https://2011.igem.org/Team:Washington/Team/Sponsors"> Sponsors </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Diesel Production</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Results"> Results Summary </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Gluten Destruction</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Results"> Results Summary</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background">iGEM Toolkits</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background"> Background </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors">Gibson Assembly Toolkit </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit">Magnetosomes Toolkit</a><br />
<br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Reference"> References </a> <a href="https://2011.igem.org/Team:Washington/Magnetosomes/Parts"> Parts Submitted </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<a href="http://partsregistry.org/Main_Page"> Parts Registry </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Protocols">Protocols</a><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Community Outreach</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Outreach/iGEM_Collaborations">iGEM Collaborations</a><br />
<a href="https://2011.igem.org/Team:Washington/Outreach">Local Outreach</a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Safety">Safety</a><br />
</li><br />
</ul><br />
</div><br />
</div><br />
</html></div>Siegeljbhttp://2011.igem.org/Team:Washington/PartsTeam:Washington/Parts2011-09-22T02:24:18Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<br />
<gallery caption="An Overview of the 2011 UW iGEM Teams Summer Projects" widths="435px" heights="300px" perrow="2"><br />
Image:Diesel Production for Wiki 2.png|<center>'''Make It: Diesel Production'''<br>Insert brief explanation of figure above here</center><br />
Image:Gluten Destruction for Wiki.png|<center>'''Break It: Gluten Destruction'''<br>Insert brief explanation of figure above here</center><br />
Image:Magnetosome Toolkit for Wiki.png|<center>'''The Magnetosome ToolKit'''<br>Insert brief explanation of figure above here</center><br />
Image:Gibson Toolkit for Wiki.png|<center>'''The GibsonBricks ToolKit'''<br>Insert brief explanation of figure above here</center><br />
</gallery><br />
<br />
<br />
<br />
<br />
='''Data Summary'''=<br />
<br />
==Data for Favorite New Parts==<br />
Fill Me In.. here is an example:<br />
# [http://partsregistry.org/Part:BBa_X0X0X Main Page] - '''BAR responsive promoter, BBa_X0X0X''': promoter can be induced to express a marker gene (GFP) when exposed to skunk smell (Butanethiol, C<sub>4</sub>H<sub>9</sub>SH), but not when exposed to pleasant scents<br />
# [http://partsregistry.org/Part:BBa_XXX00 Main Page] - '''Insulated Vector, BBa_XXX00''': un-induced leaky expression is blocked by transcriptional terminators<br />
# [http://partsregistry.org/Part:BBa_000XX Main Page] - '''BAR Bad Odor Receptor, BBa_000XX''': yellow fluorescent protein-tagged BAR (BBa_X00XX) shows that BAR is produced in E. coli<br />
<br />
<br />
==Data for Existing Parts==<br />
Fill Me In<br />
# [http://partsregistry.org/Part:BBa_J45119:Experience Experience] - '''Wintergreen odor enzyme generator, BBa_J45119''' (MIT, iGEM 2006): 98 out of 100 volunteer subjects standing up to 5 feet away from the bacterial cultures could distinguish wintergreen-producing bacteria from negative controls. <br />
# [http://partsregistry.org/Part:BBa_J61110:Experience Experience] - '''RBS, BBa_J61110''' (Arkin Lab, 2007): Of the 5 RBS Parts we tested, this RBS works best for expressing yellow fluorescent protein-tagged BAR<br />
<br />
<br />
==Improved Parts==<br />
Fill Me In<br />
# [http://partsregistry.org/Part:BBa_XXXXX Main Page] - '''Air Freshilizor, BBa_XXXXX''': Our mathematical model predicts that the threshold of activation is 10 parts per billion, the concentration of Butanethiol that humans can typically smell<br />
<br />
<br />
<br />
='''All Submitted Parts'''=<br />
<br />
<groupparts>iGEM011 Washington</groupparts></div>Siegeljbhttp://2011.igem.org/Team:Washington/Protocols/CompDesignTeam:Washington/Protocols/CompDesign2011-09-21T23:47:25Z<p>Siegeljb: </p>
<hr />
<div>{{Template:Team:Washington/Templates/Top}}<br />
<br />
=[[Image:Washington 2011 Uw foldit lofo.jpeg|250px|center]]=<br />
<br />
As can be seen below Foldit uses an easily learned user interface and uses a score board to show the current player who is the best folder. You can work alone or in teams, share puzzles, and test your ''protein design'' abilities against your friends. Below we depict a small set of the MANY options that are available to you in the program [http://www.fold.it Foldit].<br />
<br><br />
<div id="uw_foldit_div" style="text-align:center;font-size:100%;"><br />
'''[http://www.fold.it/ This accessible format has allowed over 100,000 users to help design proteins.<br/>Try Foldit Today]'''</div><br />
<html><br />
<br />
<table><tr><td><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/P-UR3G7TBb4&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
REPACK, a.k.a. Shake:<br>Here we see a video of Foldit, showing one of our biotin puzzles "Hold Me Tightly". The protein is represented as a cartoon model, showing off its secondary structure as well as key amino acid groups. Steric clashes of the amino acid side chains show up as red balls and can also be observed in the video. These steric clashes can be removed with the Shake Function. The Shake function in Foldit performs coarse sampling of the amino acid conformations, looking for a global-minima. <br />
</td></tr></table><br />
<br />
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<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/_Ugmw69_94g&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
</td><td><br />
REDESIGN, a.k.a. Mutate:<br>The function we see here is the Mutate function. This allows the user to sample many amino acids at a particular site, or the whole protein. Mutate looks for global-minima while sampling amino acids. As is seen here Alanine is mutated to Asparagine. The blue and white striped band indicates that a hydrogen bond has been formed, which is a favorable interaction between two polar residues.<br />
</td></tr></table><br />
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MINIMIZE, a.k.a. Wiggle:<br>Another nice feature of Foldit is the ability to select a sphere of amino acids around your ligand, and optimize these amino acids based off of a fine sampling of conformations. Here we see the amino acids surrounding the ligand being selected and having the Wiggle function performed on them. The Wiggle function in Foldit allows the user to fine tune the protein structure. Finding a local-minima for the amino acid conformations.<br />
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<br/></div>Siegeljbhttp://2011.igem.org/Team:Washington/Outreach/iGEM_CollaborationsTeam:Washington/Outreach/iGEM Collaborations2011-09-21T23:42:31Z<p>Siegeljb: /* Implemented Bug Fixes/Features */</p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<br />
=Primer Design Tool with LMU-Munich=<br />
<br />
We worked with [https://2011.igem.org/Team:LMU-Munich LMU-Munich 2011] iGEM team this summer on the testing and development of their PrimerDesign tool. To learn more about it check out their [https://2011.igem.org/Team:LMU-Munich/Primer_Design/Design_Primers website].<br />
<br />
==Software Testing==<br />
<br />
We helped LMU-Munich test their primer design software by sending our primer data and comparing it with the output of their automated designer. To do this, we compared the sequences of the primers used to amplify out parts of the LuxBrick with the primers that their software designed and concluded that their software works as expected. We also gave feedback on other possible features and spotted a few bugs to fix. Unfortunately, we did not have enough time to order the primers that the software designed to test it ourselves. Overall, we found the tool easy to use and potentially very useful for future primer design.<br />
<br />
[[File:Washington_collab_test.png|left|550px|thumb|An example of the output from the tool based off of input from our luxC biobrick, multiple primers with various melting temperatures are given]]<br />
==Implemented Bug Fixes & Features==<br />
# Added cyanobacteria to available genomes<br />
#Switch coding sequences in the primer designer to allow for the user to incude a stop codon in a coding sequence. Before, software insisted that coding sequences not include the stop codon<br />
#Made primer designer default to only check for biobrick restriction enzymes. Before, Primer Designer defaulted to checking for all restriction sites, making the user scroll to the bottom of the page for results<br />
#added support for FASTA files with headers<br />
#added desriptions for each column of the Primer Designer output.</div>Siegeljbhttp://2011.igem.org/Team:Washington/Outreach/iGEM_CollaborationsTeam:Washington/Outreach/iGEM Collaborations2011-09-19T19:09:58Z<p>Siegeljb: Created page with "{{Template:Team:Washington/Templates/Top}} __NOTOC__ =Primer Design Tool with LMU-Munich= We worked with [https://2011.igem.org/Team:LMU-Munich LMU-Munich 2011] iGEM team this ..."</p>
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<div>{{Template:Team:Washington/Templates/Top}}<br />
__NOTOC__<br />
<br />
<br />
=Primer Design Tool with LMU-Munich=<br />
<br />
We worked with [https://2011.igem.org/Team:LMU-Munich LMU-Munich 2011] iGEM team this summer on the testing and development of their PrimerDesign tool. To learn more about it check out their website: [https://2011.igem.org/Team:LMU-Munich/Primer_Design/Design_Primers Primer Design]<br />
<br />
===FILL IN MORE INFO ABOUT HOW WE HELPED===</div>Siegeljbhttp://2011.igem.org/Template:Team:Washington/Templates/TopTemplate:Team:Washington/Templates/Top2011-09-19T19:06:35Z<p>Siegeljb: </p>
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<ul><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington">Home</a><br />
<div><br />
<a href="https://2011.igem.org/Main_Page"> iGEM Homepage </a><br />
<a href="https://2011.igem.org/Team:Washington">UW 2011</a><br />
<a href="https://2010.igem.org/Team:Washington"> UW 2010 </a><br />
<a href="https://2009.igem.org/Team:Washington"> UW 2009 </a><br />
<a href="https://2008.igem.org/Team:University_of_Washington"> UW 2008 </a><br />
<a href="http://synbio.washington.edu/"> UW SynBio </a><br />
</div><br />
<br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">About Us</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Team/Members">The team</a><br />
<a href="https://2011.igem.org/Team:Washington/Team/Sponsors"> Sponsors </a><br />
</div><br />
</li><br />
<li><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Diesel Production</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Results"> Results Summary </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Alkanes/Parts"> Parts Submitted </a><br />
</div><br />
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<li><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Gluten Destruction</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Background">Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Methods"> Methods </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Results"> Results Summary</a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Celiacs/Parts"> Parts Submitted </a><br />
</div><br />
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<li><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background">iGEM Toolkits</a><br />
<div><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/GibsonVectors">Gibson Assembly Toolkit </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Background">Magnetosomes Background</a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Results">Magnetosome Toolkit (Methods and Results) </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Future"> Future Directions </a><br />
<a href="https://2011.igem.org/Team:Washington/Magnetosomes/Reference"> Reference </a> <a href="https://2011.igem.org/Team:Washington/Magnetosomes/Parts"> Parts Submitted </a><br />
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<li><br />
<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
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<a href="https://2011.igem.org/Team:Washington/Parts">Data Page</a><br />
<a href="https://2011.igem.org/Team:Washington/Primers">Our Primers</a><br />
<a href="http://partsregistry.org/Main_Page"> Parts Registry </a><br />
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<a href="https://2011.igem.org/Team:Washington/Protocols">Protocols</a><br />
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<a href="https://2011.igem.org/Team:Washington/Outreach">Getting Kids Into SynBio</a><br />
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