http://2011.igem.org/wiki/index.php?title=Special:Contributions/Nkruger&feed=atom&limit=50&target=Nkruger&year=&month=2011.igem.org - User contributions [en]2024-03-28T12:13:41ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:WITS-CSIR_SA/Project/MotilityTeam:WITS-CSIR SA/Project/Motility2011-10-27T20:25:51Z<p>Nkruger: </p>
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<h1><br />
Bacterial Chemotaxis</h1><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Introduction'"><br />
<p><br />
During the 17th century, the advent of the light microscope allowed scientists to<br />
observe “tiny particles” that were proposed to be living due to their seemingly<br />
purposeful motion (Baker, Wolanin et al. 2006). It was only in the 19th century<br />
when these directed bacterial movements were first characterised by Wilhelm Pfeffer<br />
(Baker, Wolanin et al. 2006). Pfeffer’s work described the ability of bacteria to<br />
navigate through complex environments, in response to changes in temperature (thermotaxis),<br />
osmolarity (osmotaxis), light (phototaxis) and chemical substrates (chemotaxis)<br />
(Baker, Wolanin et al. 2006).</p><br />
<p><br />
Bacterial chemotaxis is a regulated response that involves the processing of chemical<br />
substrates as input signals, into physical movements that result in bacterial motility<br />
(Topp and Gallivan 2007). This response allows for bacteria to selectively move<br />
along chemical gradients (Vladimirov, Lebiedz et al. 2010) (Fig 1), directing them<br />
towards substances that are favourable to their survival, and away from noxious<br />
substances (Adler 1975). Therefore, chemotaxis confers an important survival advantage<br />
to bacteria, particularly in their natural, non-mixed environment in which chemical<br />
gradients exist (Adler 1975; Vladimirov, Lebiedz et al. 2010).</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/7/78/Bacterial_chemotaxis_gradient.jpg"></center><br />
<p><br />
Fig 1: The movement of a bacterial cell in chemotaxis-stimulated conditions[1]</p><br />
</div><br />
<br /><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Motions of chemotaxis'"><br />
<p><br />
Motile bacteria are endowed with flagella, which are long, helical projections that<br />
are anchored to the cell surface (Adler 1975) (Fig 2). At the base of the flagella,<br />
there is a rotary motor that is powered by the electrochemical energy that is generated<br />
by a transmembrane ion flux (Berg 2003; Baker, Wolanin et al. 2006). This motor<br />
device induces reversible rotation of the flagella, which serves as the impetus<br />
for bacterial cell propulsion (Baker, Wolanin et al. 2006). In the well studied<br />
E. coli, there is an array of flagella at one pole of the cell that function collectively<br />
to induce cell motility (Berg 2003). During the course of its movement, two types<br />
of motions are exhibited: running and tumbling.<br />
Running is associated with the flagella rotating in a counter-clockwise direction,<br />
forming a bundle that works to propel the cell forward in a directed manner. Alternatively,<br />
tumbling is the result of the flagella rotating in a clockwise direction, disrupting<br />
the flagella bundle and causing the bacterial cell to fall in solution (Fig 3).<br />
This allows for the cell to re-orientate itself and for the direction of its runs<br />
to be changed, if deemed necessary. The frequency of each of these two motions varies,<br />
depending on the environmental signals that are transduced to the flagella motors.<br />
Chemotactic bacteria are able to make spatial as well as temporal comparisons of<br />
the concentration of the substance that they encounter, allowing them to regulate<br />
their motion in response to an increasing or decreasing concentration gradient (Adler<br />
1975). In the event that the bacterial cell is moving towards an attractant or away<br />
from a repellent, the movement is direct and is characterised by longer runs and<br />
fewer tumbles. However, in a uniform environment, the two motions alternate in such<br />
a way that the cell moves in a random walk (Baker, Wolanin et al. 2006).<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2011/5/51/Wits_Project_Chemotaxis_Figure_4.jpg"><br />
<p><br />
Fig 2: (A) The polar flagella of E. coli viewed by transmission electron microscopy<br />
(B) A schematic of the organisation of the componenets associated with the flagella<br />
in E. coli [1]<br />
</p><br />
</div><br />
<br /><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Molecular mechanism of chemotaxis'"><br />
<p><br />
Adler and colleagues were the first to describe the intracellular signal transduction<br />
network involved in chemotaxis that is responsible for the relay of information<br />
from the environment, through the cell and to the flagella motor proteins (Baker,<br />
Wolanin et al. 2006). This led on to the elucidation of the existence and the function<br />
of a group of proteins that are responsible for this intracellular signalling known<br />
as the Che proteins (Fig 4).</p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/9/9c/Chemotaxis.PNG" width="450"></center><br />
<p><br />
Fig 4: The components involved in the signalling pathway responsible for chemotaxis<br />
in E. coli 2</p><br />
<p><br />
This signalling cascade begins at the cell surface, where there is an interaction<br />
between a ligand and a bacterial chemoreceptor. E. coli has five types of transmembrane<br />
chemoreceptors that exist as large polar clusters (Baker, Wolanin et al. 2006; Topp<br />
and Gallivan 2007). These receptor proteins have highly variable periplasmic domains,<br />
allowing E. coli cells to respond to over 30 different compounds even with a minimal<br />
number of receptor types (Adler 1975; Baker, Wolanin et al. 2006). The intracellular<br />
domain, on the other hand, is highly conserved and provides the scaffolding for<br />
the intracellular membrane associated Che protein complex. CheW directly binds to<br />
these conserved regions of the chemoreceptor to serve as an adapter between the<br />
chemoreceptor and the CheA protein, to form the intracellular Che protein complex<br />
(Baker, Wolanin et al. 2006).<br />
</p><br />
<br><br />
<p><br />
CheA is a large and complex histidine kinase that is able to phosphorylate its own<br />
histidine residues, using ATP as its substrate. This autophosphorylation of CheA<br />
results in the phosphorylation of CheY, which is a monomeric protein that is usually<br />
bound to CheA. Phospho-CheY (p-CheY) has a decreased affinity for CheA, resulting<br />
in its dissociation from the membrane associated Che protein complex. In this unbound<br />
state, p-CheY rapidly diffuses to the flagella switch proteins (FliM) and functions<br />
as an allosteric regulator to induce clockwise rotation of the flagella. The overall<br />
affect is the tumbling of the bacterial cell (Baker, Wolanin et al. 2006; Topp and<br />
Gallivan 2007). This process is the native state of the system and occurs without<br />
chemoreceptor stimulation. However, when the bacterial cell encounters an attractant<br />
or a repellent that binds to a specific chemoreceptor, a ligand-induced conformational<br />
change occurs in the chemoreceptor. These changes may include piston-like movements<br />
and rotation of the receptor protein, which serves as the signal to the intracellular<br />
Che protein complex (Baker, Wolanin et al. 2006). This inhibits the kinase activity<br />
of the CheA protein, which will abrogate the production of p-CheY. Flagella motor<br />
proteins, in the absence of p-CheY regulation, produce counter clockwise flagella<br />
movements which result in directed bacterial swimming (Baker, Wolanin et al. 2006).</p><br />
</div><br />
<br /><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Regulation of Chemotaxis'"><br />
<p><br />
The complexity of the signal transduction network that is responsible for chemotaxis<br />
lies within the mechanisms of its regulation. Factors that regulate the intracellular<br />
p-CheY concentration dictate the motion of the flagella and ultimately, the movement<br />
of the bacterial cell (Baker, Wolanin et al. 2006). p-CheY concentrations are regulated<br />
by transmembrane signals that modulate the kinase activity of CheA. However, there<br />
are other Che proteins that are peripheral to the main signalling cascade that can<br />
also regulate the p-CheY concentration.<br />
</p><br />
<p><br />
CheR and CheB are methyl transferases and demethylases, respectively (Baker, Wolanin<br />
et al. 2006). They are responsible for the methylation state of the cytoplasmic<br />
domain of the chemoreceptors. Methylation of the chemoreceptor induces clockwise<br />
rotation of the flagella, whereas demethylation causes counter-clockwise rotation<br />
(Eisenbach 2001). This is thought to occur via conformational changes of the chemoreceptor<br />
as a result of methylation or demethylation (Eisenbach 2001). The state of methylation<br />
of the receptor is important as it modifies the sensitivity of the chemoreceptor,<br />
to prevent sensory saturation as the bacteria travel towards higher or lower concentrations<br />
(Sourjik 2004). Moreover, it provides a mechanism of short term memory, allowing<br />
for temporal comparisons of the stimulant concentration (Baker, Wolanin et al. 2006).</p><br />
<p><br />
Another protein that can regulate bacterial chemotaxis is CheZ. This is a phosphatase<br />
that dephosphorylates p-CheY, freeing it from the flagella FliM protein and restoring<br />
the counter clockwise rotation of the flagella to elicit running (Topp and Gallivan<br />
2007). Therefore, CheZ influences the intracellular concentration of p-CheY directly,<br />
and is capable of modulating the motion that the bacterial chemotaxis.</p><br />
</div><br />
<br /><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'References'"><br />
<p><br />
Adler, J. (1975). "Chemotaxis in Bacteria." Annual Review of Biochemistry 44(1):<br />
341-356.</p><br />
<p><br />
Baker, M. D., P. M. Wolanin, et al. (2006). "Signal transduction in bacterial chemotaxis."<br />
Bioessays 28(1): 9-22.</p><br />
<p><br />
Berg, H. C. (2003). "The rotary motor of bacterial flagella." Annual Review of Biochemistry<br />
72(1): 19-54.</p><br />
<p><br />
Eisenbach, M. (2001). Bacterial Chemotaxis. Enyclopedia of Life Sciences.</p><br />
<p><br />
Sourjik, V. (2004). "Receptor clustering and signal processing in E. coli chemotaxis."<br />
Trends Microbiol 12(12): 569-576.</p><br />
<p><br />
Topp, S. and J. P. Gallivan (2007). "Guiding bacteria with small molecules and RNA."<br />
J Am Chem Soc 129(21): 6807-6811.</p><br />
<p><br />
Vladimirov, N., D. Lebiedz, et al. (2010). "Predicted auxiliary navigation mechanism<br />
of peritrichously flagellated chemotactic bacteria." PLoS Comput Biol 6(3): e1000717.</p><br />
<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/AcknowledgementsTeam:WITS-CSIR SA/AboutUs/Acknowledgements2011-10-22T20:51:42Z<p>Nkruger: </p>
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<h1> Acknowledgements </h1><br />
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<p>The Wits-CSIR 2011 iGEM team was proudly sponsored by:</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a1/Wits_Home_Page_Sponsors.jpg"></center><br />
<p>The Wits-CSIR 2011 iGEM team would like to thank all the above sponsors for their generous financial contributions, which lightened the team’s financial burden thus allowing them to focus their attention on the project. Your generosity has inspired the team to help others and give back to the community, through human advances. </p> <br />
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<p>The Wits_CSIR 2011 iGEM team would like to thank the following advisors and supervisors for their moral and scientific support, especially; the supervisors for leading the team effectively and efficiently in this competitive, fast-paced and ever-changing world of synthetic biology; and Michelle Robinson for her tenacious effort to keep the team on track by assisting the team with implementing time management and organisational skills of the highest calibre. </p><br />
<h2>Supervisors </h2><br />
<p>Karl Rumbold: </p><br />
<p>Marco Weinberg: </p> <br />
<p>Musa Mhlanga: Continuous academic and financial assistance from the Synthetic Biology unit at the CSIR </p><br />
<p>Raymond Sparrow: </p><br />
<br />
<br />
<h2>Advisors </h2><br />
<p>Michelle Robinson </p><br />
<p>Laura Millroy </p><br />
<p>Youtaro Shibayama</p> <br />
<p>Scott Hazelhurst </p><br />
<p>Robyn Brackin </p><br />
<br />
<h2>We would also like to extend our gratitude to the following people:</h2><br />
<p>Prof Kramer from the Wits faculty of Health Science and Prof Crouch from the the Wits faculty of Science for their continued support of the iGEM team.</p><br />
<p>Prof Patrick Arbuthnot for access to of the Anti-viral Gene Therapy Research Unit (AGTRU) laboratory facilities.</p> <br />
<p>Prof Theresa Coetzer for the use of Malaria Research Unit (MRU) laboratory space.</p><br />
<p>Dr Sylvia Fanucchi and Prof Heini Dirr for the use of the Jasco Spectrofluorometer.</p><br />
<p>David Balchin for his assistance with the Jasco Spectrofluorometer.<br />
<p>Robyn Brackin for all her assistance with the Epi-Fluorescence Microscopy imaging.</p><br />
<p>Dr Clement Penny for all his help and advice with the Fluorescent microscopy imaging.</p><br />
<p>The Synthetic Biology Research Unit at the CSIR, for their academic assistance and use of equipment in their lab facilities.</p><br />
<p>Dr Sonja Lauterbach and Kubendran Naidoo for all their help, advice and care in the lab.</p><br />
<p> Dr Toru Nakayashiki and Prof Hirotada Mori from the Nara Institute of Science and Technology for their kind donation of the <i>E. coli</i> CheZ deletion mutant and parental strain.</p><br />
<p>Dr Joy Sinha and Prof Justin Gallivan from Emory University for their time and the invaluable advice on riboswitches and bacterial chemotaxis.</p> <br />
<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/AttributionsTeam:WITS-CSIR SA/AboutUs/Attributions2011-10-22T20:50:19Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Applications">Potential applications</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Achievements">Achievements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Parts">Parts submitted</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Data</a></li><br />
<li><a href="./" class="dir">Outreach</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Forum">Synthetic biology forum</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Survey">Survey</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Safety">Safety</a></li><br />
<br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Diary/Gallery">Gallery</a></li><br />
<li><a href="./" class="dir">About us</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeam">Meet the team</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelations">Media attention</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/ContactUs">Contact us</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div data-dojo-type="dijit.layout.ContentPane" data-dojo-props="region: 'top'" class="top"><br />
<a href="https://2011.igem.org/Team:WITS-CSIR_SA"><br />
<img src="https://static.igem.org/mediawiki/2011/6/63/Wits_Logo_Biotweet_Rearrange.gif" alt="Biotweet" height="60px"<br />
style="float: left; margin-bottom: 5px; border: none;" /><br />
</a><a href="https://2011.igem.org/Main_Page" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/ICL_iGEM_Logo.png" alt="iGem" height="60px"<br />
style="float: right; margin-bottom: 5px; border: none;" /><br />
</a><br />
<div class="ui-helper-clearfix"><br />
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class="center"><br />
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style="height: 100%;"><br />
<div data-dojo-type="dijit.layout.ContentPane" data-dojo-props="region: 'center'"><br />
<div style="height: 100%; overflow-y: auto;"><br />
<h1>Attributions</h1><br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Division of work'"><br />
<h2> Lab work</h2> <br />
<p> All of the lab work presented on this wiki is the work of the team members. </p><br />
<p>- Cloning was done by all team members. </p><br />
<p>- Fluorometry experiments were conducted by Ezio and Gloria. </p><br />
<p>- Fluorescence microscopy was conducted by Sasha, with the help of Robyn Brackin and Dr. Clement Penny. </p><br />
<p>- Motility and Chemotaxis Assays were performed by Natasia, with some help from all the other team members. </p><br />
<br />
<h2> Modelling</h2> <p><br />
All modelling was completed by Brad, our information engineer.</p><br />
<h2> Logo</h2> <p>Our logo was also designed by Brad</p><br />
<h2> Animation</h2> <br />
<p>This task was performed by a professional animator, Jess Scholtz.</p><br />
<h2> Website</h2> <p><br />
Our site was built by a contracted web developer, Shaun Farrell.</p><br />
<p>All team members were involved in uploading, editing and updating the wiki content. </p><br />
<br />
<br />
</div><br />
<br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
</div><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelationsTeam:WITS-CSIR SA/Outreach/PublicRelations2011-10-22T20:44:36Z<p>Nkruger: </p>
<hr />
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA">Home</a></li><br />
<li><a href="#" class="dir">Project</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Concept">Overview</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Characterization</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Modelling">Modelling</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Collaboration/Index">Collaboration</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Notebook">Lab notebook</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Applications">Potential applications</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Achievements">Achievements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Parts">Parts submitted</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Data</a></li><br />
<li><a href="./" class="dir">Outreach</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Forum">Synthetic biology forum</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Survey">Survey</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Safety">Safety</a></li><br />
<br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Diary/Gallery">Gallery</a></li><br />
<li><a href="./" class="dir">About us</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeam">Meet the team</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelations">Media attention</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/ContactUs">Contact us</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div data-dojo-type="dijit.layout.ContentPane" data-dojo-props="region: 'top'" class="top"><br />
<a href="https://2011.igem.org/Team:WITS-CSIR_SA"><br />
<img src="https://static.igem.org/mediawiki/2011/6/63/Wits_Logo_Biotweet_Rearrange.gif" alt="Biotweet" height="60px"<br />
style="float: left; margin-bottom: 5px; border: none;" /><br />
</a><a href="https://2011.igem.org/Main_Page" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/ICL_iGEM_Logo.png" alt="iGem" height="60px"<br />
style="float: right; margin-bottom: 5px; border: none;" /><br />
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<h1>Media Attention</h1><br />
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<p>The South African press was very supportive of our entry into this competition. We had interviews on Cape Talk and Talk Radio 702. We also had an article featured in <i>The New Age</i> newspaper. Wits university published an article about Biotweet on their website home page. </p><br />
<img src="https://static.igem.org/mediawiki/2011/9/9a/Wits_PR_Newspaper.JPG" align="right" style="padding: 5px;" /><br />
<p>For the material, click the links below:</p><br />
<br />
<a href="https://static.igem.org/mediawiki/2011/e/e9/Wits_PR702_inteview_Part_1.mp3">702 interview with Sasha Part 1</a><br />
<br/><br />
<br/><br />
<a href="https://static.igem.org/mediawiki/2011/7/7b/Wits_702_interview_Part2_final_final.mp3">702 interview with Sasha Part 2</a><br />
<br/><br />
<br/><br />
<a href="https://static.igem.org/mediawiki/2011/2/2b/Wits_Cape_talk_interview.mp3">Cape talk interview with Ezio</a><br />
<br/><br />
<br/><br />
<a href="http://www.wits.ac.za/newsroom/newsitems/201109/13841/news_item_13841.html">Wits News article</a><br />
<br/><br />
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<a href="http://thenewage.co.za/29458-1008-53-Wits_students_put_social_networking_to_use">New Age news article</a><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/AttributionsTeam:WITS-CSIR SA/AboutUs/Attributions2011-10-22T20:43:23Z<p>Nkruger: </p>
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<h2> Lab work</h2> <br />
<p> All of the lab work presented on this wiki is the work of the team members. </p><br />
<p>- Cloning was done by all team members. </p><br />
<p>- Fluorometry experiments were conducted by Ezio and Gloria. </p><br />
<p>- Fluorescence microscopy was conducted by Sasha, with the help of Robyn Brackin and Dr. Clement Penny. </p><br />
<p>- Motility and Chemotaxis Assays were performed by Natasia, with some help from all the other team members. </p><br />
<br />
<h2> Modelling</h2> <p><br />
All modelling was completed by Brad, our information engineer.</p><br />
<h2> Logo</h2> <p>Our logo was also designed by Brad</p><br />
<h2> Animation</h2> <br />
<p>This task was performed by a professional animator, Jess Scholtz.</p><br />
<h2> Website</h2> <p><br />
Our site was built by a contracted web developer, Shaun Farrell.</p><br />
<p>All team members were involved in uploading, editing and updating the wiki content. </p><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelationsTeam:WITS-CSIR SA/Outreach/PublicRelations2011-10-22T20:42:37Z<p>Nkruger: </p>
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style="float: right; margin-bottom: 5px; border: none;" /><br />
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<p>The South African press was very supportive of our entry into this competition. We had interviews on Cape Talk and Talk Radio 702. We also had an article featured in <i>The New Age</i> newspaper. Wits university published an article about Biotweet on their website home page. </p><br />
<img src="https://static.igem.org/mediawiki/2011/9/9a/Wits_PR_Newspaper.JPG" align="right" style="padding: 5px;" /><br />
<p>For the material, click the links below:</p><br />
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<a href="https://static.igem.org/mediawiki/2011/e/e9/Wits_PR702_inteview_Part_1.mp3">702 interview with Sasha Part 1</a><br />
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<a href="https://static.igem.org/mediawiki/2011/7/7b/Wits_702_interview_Part2_final_final.mp3">702 interview with Sasha Part 2</a><br />
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<a href="https://static.igem.org/mediawiki/2011/2/2b/Wits_Cape_talk_interview.mp3">Cape talk interview with Ezio</a><br />
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<a href="http://www.wits.ac.za/newsroom/newsitems/201109/13841/news_item_13841.html">Wits News article</a><br />
<br/><br />
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<a href="http://thenewage.co.za/29458-1008-53-Wits_students_put_social_networking_to_use">New Age news article</a><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeamTeam:WITS-CSIR SA/AboutUs/MeetTheTeam2011-10-22T20:40:30Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA">Home</a></li><br />
<li><a href="#" class="dir">Project</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Concept">Overview</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Characterization</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Modelling">Modelling</a></li><br />
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Notebook">Lab notebook</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Applications">Potential applications</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Achievements">Achievements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Parts">Parts submitted</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Data</a></li><br />
<li><a href="./" class="dir">Outreach</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Forum">Synthetic biology forum</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Survey">Survey</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Safety">Safety</a></li><br />
<br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Diary/Gallery">Gallery</a></li><br />
<li><a href="./" class="dir">About us</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeam">Meet the team</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelations">Media attention</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/ContactUs">Contact us</a></li><br />
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<img src="https://static.igem.org/mediawiki/2011/6/63/Wits_Logo_Biotweet_Rearrange.gif" alt="Biotweet" height="60px"<br />
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<h1><br />
Meet the team</h1><br />
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Sasha Reznichenko<br />
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<td rowspan="2" valign="top" align="right"><br />
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<th><br />
Field of Study:<br />
</th><br />
<td><br />
Molecular Medicine<br />
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<tr><br />
<th><br />
About Sasha:<br />
</th><br />
<td colspan="2"><br />
<p>Sasha is the “MOM” of the team – making sure everyone eats on time and has a substantial meal. She is always the one that is most stressed out. She is very dedicated to her work – only arriving 10-15min late, like a true lady. While waiting for a PCR reaction, Sasha can be found in the common room painting her nails, talking and laughing (too loud for anyone else in the department to concentrate). Her primary interest is the field human genetics and her passion lies in the medical successes that can be achieved through developments such as synthetic biology and gene therapy. She has thoroughly enjoyed doing a project with a team of colleagues since she always has someone to talk to and there is never a dull moment with all the crazy conversations that arise in the lab. </p><br />
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Ezio Fok<br />
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Field of Study:<br />
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Molecular Medicine<br />
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<th><br />
About Ezio:<br />
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<p>Ezio has a keen interest in all things that are fun. He enjoys day dreaming and jamming. His drink of preference is Schweppes Dry Lemon. He claims that it is wildly delicious and provides him with a regular, prophylactic dose of the malaria drug quinine (30 mg/l). He is commonly known for his wisdom, which is demonstrated by his mane of grey hair. His hobbies involve sleeping, eating food and learning how to swim. He also enjoys things like iGEM and science.</p><br />
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Name:<br />
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<td width=45%><br />
Gloria Hlongwane<br />
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<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/b/b6/WITS_GLORIa_Picture2141.jpg" class="thumbnail" /><br />
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Field of Study:<br />
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Microbiology and biotechnology <br />
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<th><br />
About Gloria:<br />
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<p>Gloria is a super crazy stickler for perfection with a broad sense of humour. She likes organizing all her thoughts and ideas that occur throughout the busy day on paper towels, particularly serviettes. She talks non-stop, that is,every time, all day and every day. She believes that, she is probably the most affable and aloof person she has ever met. If not doing work, she is somewhere doing one of three things; (1)laughing, (2) thinking of what to eat or (3) eating like a horse. </p><br />
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Natasia Kruger<br />
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<td rowspan="2" valign="top"><br />
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Microbiology and biotechnology <br />
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<th><br />
About Natasia:<br />
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<p>Natasia is a workaholic, which means if you can't find her at any time of the day or night, you probably haven't checked the lab. Her passion lies with biotechnology and iGEM has narrowed this interest to the specific field of synthetic biology. She is the “listener” of the team and helps team members calm down when they are STRESSED! She has many late night lab activities including trips to the vending machine and inventing new games to pass time, such as the ability to play tetris manually o_O. She thoroughly enjoyed the iGEM process and would recommend it to engineering or biology undergrads! </p><br />
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Name:<br />
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<td width=45%><br />
Bradley Marques<br />
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<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/9/93/BRAD_-_WITS_cropped_Me_copy.png" class="thumbnail" /><br />
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Field of Study:<br />
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<td><br />
Information engineering </td><br />
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<th><br />
About Bradley:<br />
</th><br />
<td colspan="2"><br />
<p>As a biomedical engineer, Bradley likes to think of blood flow as an electrical current, and is occasionally known to model people as spheres. He can usually be found whittling the early hours of the morning away banging out an emergent behaviour simulation. His information engineering degree has piqued his interest in pervasive computing, the human-computer interface as well as an active interest in digital arts. When not contemplating his lack of existence after Ray Kurzweils’ singularity, he may found trolling game design forums with the judicious use of smileys.</p><br />
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Michelle Robinson<br />
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<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/e/ea/Wiki_Michelle.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
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<th><br />
Field of Study:<br />
</th><br />
<td><br />
Master's in Molecular Medicine<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Michelle:<br />
</th><br />
<td colspan="2"><br />
<p>After experiencing firsthand the fun and insanity of iGEM as a member of the first-ever Wits iGEM team, Michelle Robinson elected to become a student advisor to the 2011 team. In between working towards her Masters of Science in Medicine Michelle practices her nagging skills and makes sure that the team knows that ‘we only have 62 days, 13 hours, 42 minutes and 4 seconds until the Wiki Freeze!’ The iGEM competition inspired her to stay within synthetic biology and Michelle is currently working on a gene therapy-based project with a synthetic biology twist. When she isn’t slaving in the lab or slave-driving the iGEM team, Michelle pursues some of her other interests such as activism and advocacy - she is involved in various projects that advocate for public and rural health; as well as scientific literacy and the interface between science and the media.</p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Laura Millroy<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/2/23/Laura_image.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
PhD Molecular Medicine<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Laura:<br />
</th><br />
<td colspan="2"><br />
<p>Laura has a keen interest in synthetic biology and genetic engineering, especially in relation to advances in human health. Her PhD project involves the development of aptamer siRNA chimeras for the treatment of HIV. Beyond lab work and research, Laura spends her time baking, painting and editing iGEM documents! Laura is a student advisor to the team for a second year. She enjoys helping the team to realise their goals while understanding the importance of having fun.</p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
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<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Youtaro Shibayama<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/0/0c/CIMG6093.JPG" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
Genetics<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Youtaro:<br />
</th><br />
<td colspan="2"><br />
<p>During his postgraduate studies, Youtaro specialized in the genetics of<br />
bacteriophages of Gram positive pathogens and also in the structures of<br />
endogenous plasmids occurring in these bacteria. He is currently pursuing his<br />
interests in the nuclear architecture of both eukaryotic and prokaryotic cells as a<br />
postdoc in Synthetic Biology of the CSIR.</p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
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<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Robyn Brackin<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/2/25/WITS_ROBYN.JPG" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field:<br />
</th><br />
<td><br />
Gene Expression and Biophysics<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Robyn:<br />
</th><br />
<td colspan="2"><br />
<p>Robyn has always had a thrist for acquiring scientific knowledge, from her early years when<br />
<br />
she dissected stamens from the flowers in her mothers garden to later on in life when she<br />
<br />
acquired degrees in both Molecular Biology and Electrical Engineering. Now working as<br />
<br />
a Biomedical Engineer she gets to play not only with pipettes, eppies and solutions but<br />
<br />
also with soft polymers, resistors, wago controllers and really cool microscopes. Robyn<br />
<br />
is an advisor to team for a second year and enjoys helping the team with imaging on the<br />
<br />
microscope. Robyn and the team really enjoy seeing flourescently labelled bacteria! </p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Dr Karl Rumbold<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/5/52/Karl_image.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
Industrial <br> Biotechnology </br> </td><br />
</tr><br />
<tr><br />
<th><br />
About Karl:<br />
</th><br />
<td colspan="2"><br />
<p>Dr Rumbold is a microbiologist by training; he graduated from the University of Graz, Austria, and started an international academic career at the University of Stellenbosch, South Africa, where he received his PhD. He then held research positions at University College London, Ghent University and the Dutch research organization TNO. He returned to South Africa after accepting a faculty position at the University of the Witwatersrand, Johannesburg in 2009. He thinks iGEM is an awesome experience and that the team is so fortunate to be in it for the second time. </p> </td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Prof Marco Weinberg<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/1/1a/MarcoAdvisorProfileFinal4.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
RNA Biology and <br> Gene Therapy </br> </td><br />
</tr><br />
<tr><br />
<th><br />
About Marco:<br />
</th><br />
<td colspan="2"><br />
<p> Prof Marco Weinberg loves science…and good coffee…but mostly science. An associate professor of the University of the Witwatersrand, Marco works within the Antiviral Gene Therapy Research Unit and became entangled with the iGEM competition quite by accident. He has been one of the team faculty advisors for the past two years, working closely with the biology students in particular. He believes that it is important to push the team members to approach their project with scientific rigour and a strong work ethic, but also to remind them of the importance of being imaginative, and to inspire them to actively enjoy the experience of working in such a stimulating and creative environment. </p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Dr Raymond Sparrow<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/3/35/Raymond_image.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field:<br />
</th><br />
<td><br />
CSIR: Biosciences<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Raymond:<br />
</th><br />
<td colspan="2"><br />
<p> Dr Sparrow is from the UK and came over to South Africa in 2005 to the National Laser Centre then transferred to Biosciences in 2007. He is currently the Human Capital Development Manager for the Synthetic Biology ERA.<br />
<br />
In the UK he conducted research into ultra-fast photosynthetic light harvesting and energy transfer reactions.<br />
<br />
He was a Post-Doctoral Fellow at the University of Central Lancashire and conducted research at the Lasers For Science Facility at the Rutherford Appleton Laboratory and Daresbury Synchrotron Radiation Source. Then a Higher Scientific Officer at Daresbury on a Vacuum Ultra Violet Beamline.<br />
<br />
From 1992 - 2005 he was a lecturer in Life Sciences at Bromley College of F&HE.</p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Dr Musa Mhlanga<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/5/59/Musa_image2.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
Cell Biology and Biophysics </td><br />
</tr><br />
<tr><br />
<th><br />
About Musa:<br />
</th><br />
<td colspan="2"><br />
<p>Dr Musa Mhlanga is the research leader of the Synthetic Biology ERA of the CSIR’s gene expression and biophysics research group. He studied at the New York University Medical School and the Rockefeller University before heading off to France for postdoctoral research. He has research interests in gene expression, imaging and microscopy, nuclear architecture, single molecule biology and high-throughput screening, and has presented a number of papers internationally in these fields. Musa has been instrumental in setting-up synthetic biology in South Africa and has played a significant role in helping both the 2010 and 2011 iGEM teams. </p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
</p><br />
</div><br />
<br />
<br /><br />
<br />
<br />
<br />
</div><br />
<br />
<br />
<br />
</div><br />
</div><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Diary/GalleryTeam:WITS-CSIR SA/Diary/Gallery2011-10-22T20:39:32Z<p>Nkruger: </p>
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Project/SafetyTeam:WITS-CSIR SA/Project/Safety2011-10-22T20:37:05Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Characterization</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Modelling">Modelling</a></li><br />
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/ContactUs">Contact us</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div data-dojo-type="dijit.layout.ContentPane" data-dojo-props="region: 'top'" class="top"><br />
<a href="https://2011.igem.org/Team:WITS-CSIR_SA"><br />
<img src="https://static.igem.org/mediawiki/2011/6/63/Wits_Logo_Biotweet_Rearrange.gif" alt="Biotweet" height="60px"<br />
style="float: left; margin-bottom: 5px; border: none;" /><br />
</a><a href="https://2011.igem.org/Main_Page" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/ICL_iGEM_Logo.png" alt="iGem" height="60px"<br />
style="float: right; margin-bottom: 5px; border: none;" /><br />
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<div style="height: 100%; overflow-y: auto;"><br />
<h1><br />
Safety</h1><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Team safety policy'"><br />
<br />
<p> The South African Genetically Modified Organisms (GMO) act regulates all research and development with these organisms to ensure environmental, researcher and public safety. The legislation governs all activities with these microbes including their transport and work in the laboratory by researchers. The GMO Act has three main focuses guarding human, environmental and socioeconomic safety. As the Wits iGEM team, we work hard to ensure that our safety policy is aligned with our country’s GMO Act. </p><br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Researcher safety'"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bb/Safety_Researcher_safety.JPG" align="left" style="padding: 5px;" /><br />
<p>The organism used in this iGEM poject is a strain of <i>E.coli</i> which requires that biosafety level 1 guidelines are adhered to in the laboratory. Regulations of this biosafety level indicate that lab coats and gloves should be worn at all times while working with <i>E. coli</i>. The team implements these rules without exception. </p><br />
<br />
<p> The final machine produced in our project, since it is only responsive to theophylline, atrazine and IPTG will present no harm to any of the team members in the unlikely event that they come into contact with it. For safety measures, care should be taken by the team to ensure no medication administered contains any of the above mentioned substances.</p><br />
<br />
<p>Microbes are not the only dangers presented to our team when working in the laboratory. Some chemicals, such as ethidium bromide pose a risk to the biologists. When working with this chemical, gloves are worn and disposed of immediately after contact. The team also works with atrazine and theophylline (see "Usage of Harmful Substances").</p><br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Public safety'"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6e/Wits_Safety_Page_Bin.jpg" align="right" style="padding: 5px;" /><p>To prevent the public from coming into contact with our genetically modified organisms, all contaminated laboratory materials are disposed of in assigned bins for decontamination. The strict adherence of the team to this policy ensures that the public, and researchers, are protected from the potential pathogenicity of <i>E. coli</i>. Should individuals in the public come into contact with the genetically engineered bacteria, it will pose little, if any, risk since the bacteria contain no harmful genetically engineered parts and are responsive only to substances rarely present in nature. Issues may arise from the chloramphenicol resistance of the bacteria being horizontally transferred to other bacteria in the environment. This said, chloramphenicol is an antibiotic seldom administered to fight bacterial infections and as such, the acquisition of such resistance by a species will not affect the efficiency of antibiotic treatments.<br />
<br><br />
<br><br />
<br><br />
</p><br />
<br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Environmental safety'"><br />
<img src="https://static.igem.org/mediawiki/2011/a/ab/Wits_Safety_Environment.JPG" align="left" style="padding: 5px;" /><br />
<p>The <i>E. coli</i> strain we use in our laboratory work will not pose any environmental safety risks if it is accidently released. The biobricks which have been transformed into this species will not cause any negative effects to the environment since the biobricks are only responsive to atrazine, IPTG and theophylline which are rarely present in the environment. </p><br />
<br><br />
<br><br />
<br><br />
<br><br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Usage of harmful substances'"><br />
<img src="https://static.igem.org/mediawiki/2011/e/ea/Wits_Safety_At-th.jpg" align="right" style="padding: 5px;" /><br />
<p>A component of our project involves working with harmful substances which are not commonly used in a laboratory setting. We will be using two hazardous chemicals as chemoattractants for our bacteria, to direct chemotaxis – namely atrazine and theophylline. </p><br />
<br />
<p>Although its usage has been banned in the European Union, atrazine remains one of the most widely used herbicides in the rest of the world. An organic compound which is biodegradable through the action of microbes in soil, atrazine is also a suspected oestrogen disruptor and teratogen. Contamination of drinking water with atrazine concentrations above government regulations has previously been linked to birth defects and menstrual problems in the case of human consumption. It may even infer possible carcinogenic effects and lower sperm levels in men. </p><br />
<br />
<p>Theophylline is an antibiotic. It does not impose as many biohazardous risks as atrazine but must still be handled with care and the relevant protection in the lab. </p><br />
<br />
<p>For the purpose of our project, we will be using very low concentrations of both substances, and following the safety regulations stipulated in the Sigma MSDS documents for these chemicals – in terms of storage, handling and disposal. The correct disposal of these substances in crucial in protecting the public from health issues. Face dust masks, gloves, lab coats and safety glasses will be worn at all times when handling these substances. </p><br />
<br />
<p>Material Safety Data Sheets (MSDS) :</p><br />
<br />
<a href="http://www.sigmaaldrich.com/catalog/Lookup.do?N5=All&N3=mode+matchpartialmax&N4=45330-250MG-R&D7=0&D10=45330-250MG-R&N1=S_ID&ST=RS&N25=0&F=PR " target="_blank">ATRAZINE</a><br />
<br><br />
<br />
<a href=">http://www.sigmaaldrich.com/catalog/Lookup.do?N5=All&N3=mode+matchpartialmax&N4=T1633-50G&D7=0&D10=T1633-50G&N1=S_ID&ST=RS&N25=0&F=PR " target="_blank">THEOPHYLLINE</a><br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Security risk'"><br />
<p>Our machine does not pose any security risks as it does not secrete any harmful substances. The species of bacteria utilized for our project is also biosafety level 1. Should this bacteria be released for malicious intent, it will unlikely cause harm to the public or environment.</p><br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Biobrick safety'"><br />
<br />
<p>Even with lateral gene transfer between bacteria, the only advantage that will be acquired is directional movement towards either atrazine or theophylline and the ability to then fluoresce. It is very unlikely that microorganisms will even come across these two substances in a normal environmental setting. Taking this into account, our parts pose little, if any, risk to public or environmental safety. The vectors used in cloning contain antibiotic resistance which could be transferred between bacteria. This could provide selective advantage to bacteria who obtain these vectors. To prevent bacteria in nature coming into contact with our engineered <i>E. coli</i>, all microbiological waste will be disposed of only after autoclaving. </p><br />
<br />
<p>The main feature of our device ensures the bacteria return to the position of their release. This means, should our system be used, the bacteria will not remain in the environment and will always return to the start. This reduces the hazard of these GMOs in the context of public, researcher and environmental safety.</p><br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Our knowledge of safety'"><br />
<p>All of the team members have attended presentations on laboratory safety and use the knowledge learned to ensure correct procedures are followed in the lab. Two of the team members also have first aid training, enabling them to help should an emergency of medical nature occur in the lab.</p><br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Safety at our institution'"><br />
<br />
<a href="http://www.wits.ac.za/academic/research/7074/biosafety_considerations.html " target="_blank">Wits Biosafety Committee information</a><br />
</div><br />
<br />
<br />
<br />
</div><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SurveyTeam:WITS-CSIR SA/Outreach/Survey2011-10-22T20:35:48Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA">Home</a></li><br />
<li><a href="#" class="dir">Project</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Concept">Overview</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Characterization</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Modelling">Modelling</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Collaboration/Index">Collaboration</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Notebook">Lab notebook</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Applications">Potential applications</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Achievements">Achievements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Parts">Parts submitted</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Data</a></li><br />
<li><a href="./" class="dir">Outreach</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Forum">Synthetic biology forum</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Survey">Survey</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Safety">Safety</a></li><br />
<br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Diary/Gallery">Gallery</a></li><br />
<li><a href="./" class="dir">About us</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeam">Meet the team</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelations">Media attention</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/ContactUs">Contact us</a></li><br />
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<img src="https://static.igem.org/mediawiki/2011/6/63/Wits_Logo_Biotweet_Rearrange.gif" alt="Biotweet" height="60px"<br />
style="float: left; margin-bottom: 5px; border: none;" /><br />
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<br />
<h1><br />
Survey</h1><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Introduction'"><br />
<p>As an attempt to interact with the public and to promote a conversation around the topics of synthetic biology and genetic engineering, a simple survey was conducted.<br />
The online survey tool, <a href = "http://www.surveymonkey.com/">surveymonkey</a>, was used to design, distribute and collect responses from the survey.<br />
The survey, which can be found <a href = "http://www.surveymonkey.com/s/8QJ22BR">here</a>, attempts to glean an understanding of public opinion towards the field of synthetic biology.<br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'The survey'"><br />
<p>The survey consists of ten simple questions:<br />
<ol><br />
<p>(1) How old are you?</p><br />
<p>(2) What is your highest level of education?</p><br />
<p>(3) To what religious denomination do you belong?</p><br />
<p>(4) How would you rate your understanding of "synthetic biology"?</p><br />
<br />
<p> - I have no idea what it is</p><br />
<p> - I have some idea of what it may entail</p><br />
<p> - I know what it is</p><br />
<p> - I have a deep understanding of the field</p><br />
<br />
<br/><br />
<p>(5) Which of the following have led to your understanding of synthetic biology?</p><br />
<br />
<p> - Television news</p><br />
<p> - Newspaper articles</p><br />
<p> - Internet articles</p><br />
<p> - School or university</p><br />
<p> - Friends and family</p><br />
<br/><br />
<p>(6) In your opinion genetic engineering is:</p><br />
<br />
<p>- Unethical</p><br />
<p>- Ethical but necessary</p><br />
<p>- Ethical and very useful for humanity</p><br />
<p>- I have conflicting opinions</p><br />
<br/><br />
<p>(7) Would you ever use a medicine if it was a genetically engineered bacterium?</p><br />
<p>(8) As a member of the public do you feel that scientists, in general, approach technologies such as stem cell research, genetic engineering and synthetic biology:</p><br />
<br />
<p>- Ethically</p><br />
<p>- Neutrally</p><br />
<p>- Unethically</p><br />
<br/><br />
<p>(9) As a member of the public, do you feel that new advance in science are communicated to you in an understandable way?</p><br />
<p>(10) Which of the following areas do you consider most important for humanity?</p><br />
<br />
<p> - Agriculture, food and beverage</p><br />
<p> - Health and Medicine</p><br />
<p> - Computer science and mathematics</p><br />
<p> - Engineering and architecture</p><br />
<p> - Sociology and philosophy</p><br />
<p> - Education</p><br />
<p> - History and politics</p><br />
</ol><br />
</div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Analysis of results'"><br />
<br />
<center><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2011/a/a0/Q2.png" width="540" alt="" title="#q1" /></a><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2011/2/21/Q3.png" width="540" alt="" title="#q3" /></a><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2011/2/20/Q4.png" width="540" alt="" title="#q4" /></a><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2011/1/13/Q5.png" width="540" alt="" title="#q5" /></a><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2011/6/64/Q6.png" width="540" alt="" title="#q6" /></a><br />
<a href="#"><img src="https://static.igem.org/mediawiki/2011/4/4b/Q7.png" width="540" alt="" title="#q7" /></a><br />
<br />
</center><br />
<br />
<br />
<br />
<br />
<p><br />
Of the 168 responses to the survey, 46 % were undergraduate university students, and 30 % postgraduates.<br />
Indeed, almost 70 % of respondents were aged 19 – 25. This does not nullify the results of the survey, but<br />
it should thus be understood that any results gleaned from the survey are heavily biased toward to a younger, educated audience.<br />
</p><br />
<br />
<p><br />
20 % of respondents did not know what the field of synthetic biology involves, whilst 26 % did. 43 % did not know exactly, but <br />
felt they had some idea of what it may entail, whilst 11 % considered themselves possessing a deep knowledge of the field. The <br />
understanding of the field was found to originate mainly from school or university education (38 %), internet articles (25 %) <br />
and word of mouth by friends or family (22%).<br />
</p><br />
<br />
<p><br />
Almost 60 % of correspondents felt that the field of genetic engineering is ethical and useful for humanity, whilst 30 % had<br />
conflicting opinions on the matter. Only 7 % felt that genetic engineering was unethical. Correspondingly, 63 % of<br />
responding parties indicated that they would use a medicine in the form of a genetically engineered bacterium, whilst 30 %<br />
would only do so if they were suffering from a life-threatening illness.<br />
</p><br />
<p><br />
43 % of people felt that new advances in science are communicated to them in an understandable way, whilst 36 % felt that they are not.<br />
21 % felt that they are not communicated at all.<br />
</p><br />
<p><br />
The three main areas that respondents felt were of the greatest importance to humanity are the fields of health<br />
and medicine (34 %), education (27 %) and agriculture, food and beverage (26%).<br />
</p><br />
<br />
<br />
<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Outreach/ForumTeam:WITS-CSIR SA/Outreach/Forum2011-10-22T20:34:35Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Characterization</a></li><br />
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
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<img src="https://static.igem.org/mediawiki/2011/6/63/Wits_Logo_Biotweet_Rearrange.gif" alt="Biotweet" height="60px"<br />
style="float: left; margin-bottom: 5px; border: none;" /><br />
</a><a href="https://2011.igem.org/Main_Page" target="_blank"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/ICL_iGEM_Logo.png" alt="iGem" height="60px"<br />
style="float: right; margin-bottom: 5px; border: none;" /><br />
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<h1>First ever synthetic biology forum in South Africa</h1><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Wits CSIR South Africa iGEM team presenting at the forum'"><br />
<br />
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<br />
<p>The University of the Witwatersrand iGEM 2010 team was the first African team ever to enter this competition. This year, the Wits team is back with new team members who make it their mission to promote synthetic biology and the iGEM competition. To achieve this, the Wits 2011 team attended the first synthetic biology forum in South Africa hosted by their co-sponsor CSIR together with the African Centre for Gene Technologies (ACGT). The team supervisors, Dr Musa Mhlanga and Prof Marco Weinberg gave presentations on synthetic biology and the ethical issues for synthetic biology were addressed by Prof Julian Kinderlerer, a member of the European Group on Ethics in Science and New Technologies. The 2010 team members shared their experiences while the 2011 members gave a short presentation on their project followed by a fun Q & A session. Students from other South African Universities such as University of Pretoria and UNISA showed interest in entering the competition. Who knows, next year the University of the Witwatersrand might not be the only South African University to enter their undergraduate students in the iGEM competition. </p><br />
<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBonoTeam:WITS-CSIR SA/Outreach/SciBono2011-10-22T20:32:53Z<p>Nkruger: </p>
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<h1><br />
SciBono Experience</h1><br />
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<p>The Wits CSIR iGEM team feels it is not only important what is done in the lab but, moreover, to share our knowledge of science and synthetic biology with the broader public. One issue most often associated with synthetic biology is the ethics surrounding the production of genetically modified organisms. Another issue, prevalent is South Africa, is the significant lack of scientists and engineers. The team set out to address both these issues by approaching SciBono, a national science centre whose goal it is to act as an interface between scientists and the public. By focusing on underprivileged students, we feel we approached the human advances aspect of iGEM in a novel away, opening these children's eyes to the potential uses of synthetic biology in their own communities. </p><br />
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<p>The team participated in National Science Week (an initiative of the South African Department of Science and Technology) by addressing both primary and high school children educating them on the importance of science, with a particular focus on synthetic biology. Ezio and Sasha presented to the junior school children, sharing with them some interesting facts about science and making them aware of the presence of bacteria. Emphasis was placed on the fact that bacteria are not only disease causing agents but also act as the flora in the intestines, helping to digest food and prevent invasion by pathogens. Ezio and Sasha held a fun interactive ‘Q and A’ session where the children were encouraged to develop research questions. Some of the children stood up and asked their questions and the whole group discussed possible ways of solving the problems. The children were so enthusiastic and came up with some interesting ways of researching their problems. The children were also then exposed to the concept of synthetic biology and some of the exciting applications of this technology.</p><br />
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<p>The high school children, from resource poor environments, were exposed to a workshop where there was an informative presentation about synthetic biology with a particular focus on the iGEM competition. Following this, there was an activity in which the children were asked to create ‘super hero’ bacteria to solve problems they face in their own communities. They constructed plasmids with start and stop sites and genes in between which gave the bacteria super hero qualities. Some of the ideas were incredible and showed an understanding of the potential of this technology, particularly in their own communities.</p><br />
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<img src="https://static.igem.org/mediawiki/2011/1/16/Wits_SciBono_Workshop.jpg" alt="SciBono Workshop"/><br />
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<p>The team advisor, Laura Millroy organised on behalf of the team, an information stand at SciBono where the team handed out synthetic biology information packs. The synthetic biology information packs were kindly put together by Michelle Robinson (team advisor) and Laura and contained flyers from the different faculties at Wits University, CSIR Synthetic Biology research groups and the iGEM team. In addition to raising awareness of science as a career, the team highlighted the importance of mathematics and science high school subjects. As a relatively new field, initially synthetic biology appeared disconnected from the other general scientific fields of study. Overall, the team tried to bridge the gap between the scientific fields such as biotechnology and synthetic biology thus inspiring young learners to choose science as a career. </p><br />
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<p>Both the junior school presentations and the high school workshops were successful. The aim to create an interest in the field of synthetic biology and, particularly, to show how it could be beneficial in resource-poor settings was achieved. The children left the workshops knowing 'super hero' bacteria can be used to fix problems faced by their communities. This novel approach to outreach exposed the field of synthetic biology to children from resource poor environments, enabling them to welcome the use of this science in their communities and, we hope, to encourage them to pursue a career in science.</p><br />
<p>In South Africa, few young adults take science subjects at a tertiary level. We hope that our talk has inspired a few more to consider this path. We thoroughly enjoyed this aspect of the project and we intend to continue to contribute to our surrounding communities in this way. </p><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/CharacterizationTeam:WITS-CSIR SA/Characterization2011-10-22T20:31:56Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeam">Meet the team</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelations">Media attention</a></li><br />
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/ContactUs">Contact us</a></li><br />
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<p id="Top"> <br />
<h1><br />
Characterization of Parts</h1><br />
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<p>In order to characterize our final machines, we performed the following assays:</p><br />
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<a href="#Fluorometry">Fluorometry</a><br />
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<a href="#Microscopy1">Fluorescence microscopy: Liquid Culture</a><br />
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<a href="#Microscopy2">Fluorescence microscopy: Smeared Culture</a><br />
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<a href="#Motility">Motility</a><br />
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<a href="#Chemotaxis">Chemotaxis</a><br />
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<p id="Fluorometry"><br />
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<h2>Theophylline Riboswitches</h2><br />
<p>To characterise the theophylline riboswitches (1 and 2), we quantified their activation at different theophylline concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM and 2 mM) over a period of time using fluorometry. Competent <i>E. coli</i> (strain DH5a) cells were transformed with plasmid vectors containing the riboswitch and were cultured until the mid-log phase of growth. Thereafter, a different concentration of theophylline was added to each culture for induction. The activation of the riboswitch was detected as a fluorescent response as a result of increased translation of the fluorescent fusion protein CheZ-Venus, in the presence of the activator. A Jasco FP-6300 spectrofluorometer was used to excite the cultures at 514 nm and the intensity of the emission peak was detected at 528 nm. Twenty readings were taken every 15 minutes for each culture, for a total period of 135 minutes. An empty vector was used to correct for autofluorescence. Conversely, a construct that comprised of a Promoter-RBS-CheZ-Venus-double terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>) was used as a positive control. Fluorescent data for the 3D plots (Fig 1-4) were corrected for autofluorescence by subtracting the fluorescent values of the cultures transformed with the empty vector, from that of the riboswitches.<br />
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<td><p> <b>Figure 1:</b> The fluorescence of the CheZ-Venus fusion protein under the regulation of theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). <b>A)</b> The activation of the riboswitch over time at different concentrations of theophylline. The fluorescence increased over time, with an increasing concentration of theophylline, indicating the theophylline concentration-dependent expression of the CheZ-Venus fluorescent fusion protein. The fluorescence peaks 105 minutes after the addition of 1 mM theophylline. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 1 mM theophylline after 105 minutes incubation, and to that of baseline levels (0 minutes with 1 mM theophylline). 1 mM of theophylline induced the expression of CheZ-Venus to increase 4-fold after 105 minutes (fold change was calculated by dividing the fluorescence observed at 105 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values) (Click to enlarge)<br />
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<b>Figure 2:</b> The fluorescence of the CheZ-Venus fusion protein under the regulation of theophylline riboswitch 2 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>). <b>A)</b> The activation of the riboswitch over time at different concentrations of theophylline. The same trend of increased switch activation over time, with increasing theophylline concentrations was seen as with theophylline riboswitch 1. However, with the theophylline riboswitch 2, the fluorescence peaks 135 minutes after the addition of 1.5 mM theophylline. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 1.5 mM theophylline after 135 minutes incubation, and to that of baseline levels (0 minutes with 1.5 mM theophylline). 1.5 mM of theophylline induced a 37-fold increase in CheZ-Venus expression after 135 minutes (fold change was calculated by dividing the fluorescence observed at 135 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values). (Click to enlarge)</p> </td><br />
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<td><p> <b>Figure 3:</b> The fluorescence of the Venus reporter protein under the regulation of theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>). <b>A)</b> The fluorescent peak occurred prematurely after 75 minutes of incubation with 0.5 mM theophylline, compared to riboswitch 1, which contained the gene for the CheZ-Venus fusion protein. The maximum fluorescent intensity observed was also lower by a factor of 2 RFU. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 75 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 4.5-fold increase in protein expression was observed as the maximum (fold change was calculated by dividing the fluorescence observed at 75 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values). This fold change is comparable to that described in <b>Figure 1</b>. (Click to enlarge)<br />
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<td><p> <b>Figure 4:</b> The fluorescence of the Venus reporter protein under the regulation of the theophylline riboswitch 2 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>). <b>A)</b> The activation of the riboswitch was affected by the lack of the CheZ gene in the switch construct. The increase in fluorescence was minimal upon induction and incubation over time. Furthermore, the fluorescent peak occurred prematurely after 60 minutes of incubation with 0.5 mM theophylline, when compared to the theophylline riboswitch 2 that contained the gene for the CheZ-Venus fusion protein. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 60 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 2-fold increase in protein expression was observed as the maximum (fold change was calculated by dividing the fluorescence observed at 60 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values) . This is far less than the value described for the same switch in <b>Figure 2</b>.(Click to enlarge)<br />
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Both the theophylline riboswitches displayed activation in response to increasing theophylline concentrations and incubation times. Theophylline riboswitch 2 controlling CheZ-Venus expression (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>) clearly showed a superior level of activation compared to that theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). This was expected as it was selected by FACS for its optimised expression platform that allows for minimal leakiness at basal levels, and a strong activation in the presence of theophylline (Lynch and Gallivan, 2009). The activation shown by both the riboswitches regulating CheZ-Venus expression should be able to restore the motility of <i>E. coli</i> cells deficient in CheZ, in the presence of theophylline. More importantly, the theophylline concentration-dependent increase in CheZ-Venus expression alludes to the ability of directed cell movement, that is moving up concentration gradients of theophylline (Topp and Gallivan, 2007). Interestingly, the data suggests that the presence of the CheZ gene, as part of the CheZ-Venus fusion sequence, enhances the activation of the switches when compared to the constructs with venus alone. This may be due to some sort of structural stability conferred by the presence of the CheZ sequence.</P><br />
<h2> Atrazine Riboswitch</h2><br />
<p>Preliminary activation data was obtained for the atrazine riboswitch.Fluorometry was performed on <i>E. coli</i> (DH5a) cells transformed with a construct that contained a Promoter-atrazine riboswitch-mRFP-double terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a>). Similar to the theophylline riboswitches, different concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) of atrazine were added to the cultures for induction. The cultures were excited at 584 nm and detected at 607 nm using a Jasco FP-6300 spectrofluorometer. Readings were taken every 15 minutes for 120 minutes in total. The same empty vector was used as a negative control. The 3D plot data was normalised the same way as for the theophylline riboswitches.</p><br />
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<td><p> <b>Figure 5:</b> The fluorescence of the mRFP1 reporter protein under the regulation of the atrazine riboswitch(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a>). <b>A)</b> The activation of the atrazine riboswitch at different atrazine concentrations over time. There was an increase in fluorescence as the atrazine concentration and incubation time increased. The amount of fluorescence detected was very small, with a maximum of 0.1 RFU detected after 75 minutes of incubation with 2 mM atrazine. <b>B)</b> The maximum was compared to the negative control at 75 minutes, 2 mM atrazine, and to that of fluorescent levels detected at basal levels (0 minutes, 2 mM atrazine). A 1.7-fold increase in expression of mRFP1 was observed over the 75 minute period (values used for the calculation had the corresponding negative control value subtracted first).(Click to enlarge)<br />
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<h2>References</h2><br />
<p>Lynch, S. A. & Gallivan, J. P. 2009. A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Res, 37, 184-92.</p><br />
<p>Topp, S. & Gallivan, J. P. 2007. Guiding bacteria with small molecules and RNA. J Am Chem Soc, 129, 6807-11.</p><br />
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<p id="Microscopy1"><br />
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<h2>Epi-fluorescence Wide-Field Microscopy</h2><br />
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<p> Fluorescent imaging was performed for a qualitative assay of the engineered bacteria. <br />
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<p> Bacterial culture and Manipulation: <br />
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<p> 4 ml of LB broth with Ampicillin was inoculated from colonies off a master plate of positive clones and grown overnight at 37°C in a shaking incubator. 2 ml of each bacterial culture was then used to inoculate 25 ml of LB broth with Ampicillin and grown for ± 3 hours in a shaking incubator at 37°C until the cells reached an OD600 of 0.55 (mid-log phase). 2 x 5 ml of each sample culture was aliquoted into separate 15 ml falcon tubes. One tube was left untreated; 150 µl of 15 mM theophylline was added to the other (to make a final concentration of 1.5mM theophylline). These 5 ml aliquots were placed in a shaking incubator at 37°C for 30 min each to allow for activation. 200 µl of each treated and untreated sample was then used in a microscope plate for imaging. Imaging was done using Zeiss Axioscope (Andor EMCCD Camera, Till Photonics Polychrome V). <br />
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<p> Widefield fluorescence microscopy was used to assess the expression of the Venus protein. The bacterial samples were excited at 500 nm using the polychrome as an excitation source. The fluorescence was then observed by capturing light in the emission spectrum of Venus (528 nm). The figure below represents a schematic of the imaging setup. Corresponding bright-field images (using white light) were also captured for each experiment. <br />
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<p>Below are the images that were obtained for the parental strain (BW25113) of the <i>E. coli</i> CheZ-deletion mutant, which served as the negative, and the CheZ deletion mutants transformed with Promoter-theophylline riboswitch 1-venus (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) and Promoter-theophylline riboswitch 2-venus (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>).<br />
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<p>Please note: Bacteria which were transformed with constructs which contained the CheZ gene were not tested in this assay. This experiment served as a means of observing the activation of the theophylline riboswitches, hence only expression of the venus reporter protein was needed for detection of fluorescence. Activation of motility was not assessed in this case – it was examined in the motility assays on semi-solid agar plates. Differences in fluorescence intensities were not calculated from the images below since the fluorometry experiments served as the quantitative approach to measurement of riboswitch activation.<br />
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<b>Figure 6:</b> The parental strain (BW25113) of the <i>E. coli</i> CheZ deletion mutants were not transformed with any plasmid DNA. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In both the absence and presence of theophylline, the parental bacteria did not fluoresce – which was the expected result. (Click to enlarge)</p> </td><br />
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<b>Figure 7:</b> <i>E. coli</i> CheZ-deletion mutants which were transformed with the Promoter-theophylline riboswitch 1-venus (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) construct. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, almost no fluorescence occurred. Upon the addition of theophylline at a concentration of 1.5 mM, many of the cells emitted fluorescence showing activation of the theophylline riboswitch type 1.(Click to enlarge)</p> </td><br />
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<b>Figure 8:</b> <i>E.coli</i> CheZ-deletion mutants which were transformed with the Promoter-theophylline riboswitch 2-venus (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>) construct. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, some fluorescence was observed. This result showed the leakiness of the riboswitch. A substantial amount of venus translation is permitted because of the flexible conformation of this riboswitch. Upon the addition of theophylline at a concentration of 1.5 mM, much more fluorescence was detected. More venus was expressed in these cells due to the activation of the riboswitch via theophylline.(Click to enlarge)</p> </td><br />
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<p> Fluorescent imaging was performed again after the European Jamboree, using a different technique. The aim of this data is to depict the exact same brightfield and fluorescent static fields of view. <br />
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<p id="Motility"><br />
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<p> Semi-solid agar plates containing different concentrations of theophylline (0mM, 0.5mM, 1.0mM and 1.5mM) were made. Promoter-theophylline riboswitch 1-CheZ-Venus-double terminator (thRS1-Ch-V)(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>), Promoter-theophylline riboswitch 2-CheZ-Venus-double terminator (thRS2-Ch-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>), Promoter-theophylline riboswitch 1-Venus-double terminator (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) and Promoter-theophylline riboswitch 2-Venus-double terminator (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>) were transformed into CheZ deletion mutants and grown to mid-log phase (OD600 = 0.55). 1.5 µl of culture was plated into the centre of the plates containing the various concentrations of theophylline and incubated at 37°C for 24 hours. This experiment was performed in triplicate. The protocol for this experiment can be found here.<br />
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<b>Figure 9:</b> The distance travelled by CheZ deletion mutants (mm) over a 24 hour period at 37°C on semi-solid agar plates containing different theophylline concentrations (mM)(Click to enlarge)</p> </td><br />
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The thRS1-V and thRS2-V transformants did not travel from the point of inoculation for all concentrations of theophylline. The CheZ deletion mutants containing thRS1-Ch-V travelled 0 mm at 0 mM theophylline and reached a maximum travelling distance of 5 mm at 1.5 mM theophylline. The thRS2-Ch-V containing CheZ deletion mutants travelled 2 mm in the absence of theophylline and at a concentration of 1.5 mM theophylline, were able to travel 22mm from the point of inoculation </p><br />
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<b>Figure 10:</b> 85 mm semi-solid agar plates containing 1.0 mM theophylline showing the point of inoculation and distance travelled by the bacteria. <b>A)</b> Inoculation of 1.5 µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS1-Ch-V(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). There is evidence of movement of bacteria from the point of inoculation. This can be observed as a faint halo surrounding the point of inoculation.<b>B)</b> Inoculation of 1.5 µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS1-V. Converse to <b>A</b>, there is no evidence of movement of the bacteria from the point of inoculation <b>C)</b> Inoculation of 1.5µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS2-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>). There is evidence of movement of the bacteria from the point of inoculation. This can be observed as a faint halo surrounding the point of inoculation. <b>D)</b> Inoculation of 1.5µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS2-V. Converse to <b>C</b>, there is no evidence of movement of bacteria from the point of inoculation (Click to enlarge)</p> </td><br />
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<h3> Discussion and conclusion</h3><br />
<p>The construct thRS1-Ch-V transformed into the CheZ deletion mutants are non-motile at a concentration of 0 mM theophylline, since in the absence of the activator, the riboswitch is sequestering the RBS and thus inhibiting the translation of CheZ (Topps and Gallivan, 2007). On the semi-solid agar plates containing theophylline, the motility of E.coli is evident, as the interaction of theophylline with the riboswitch enables the exposure of the RBS and the translation of the downstream CheZ, allowing for bacterial motility. The distance travelled by the bacteria transformed with the thRS1-Ch-V construct, from the start position (mm) in the presence of different concentrations of theophylline, is not significantly different (P=0.005). This indicates that the CheZ expression at 0.5 mM-1.5 mM is sufficient to allow the bacteria to move a distance of 5 mm. The construct thRS2-Ch-V transformed into the CheZ deletion mutants are slightly motile in the absence of theophylline, travelling 2mm over a 24 hour period indicating that this riboswitch is slightly leaky. Upon addition of 0.5 mM-1.5 mM theophylline, the bacteria were able to travel 20mm over a 24 hour period.</p><br />
<p>The distance travelled from the start position by the CheZ deletion mutants containing the thRS2-Ch-V is significantly higher than that those with the thRS1-Ch-V construct. This can be explained by the high fold-increase in CheZ protein expression by this riboswitch, as shown by fluorometry. Theophylline riboswitch 1 has a lower fold increase in CheZ expression from 0 mM to 1.5 mM theophylline, compared to the increase shown by the theophylline riboswitch 2. Therefore, in order to achieve maximum motility under the control of theophylline, at concentrations of between 0.5 mM and 1.5 mM, theophylline riboswitch 2 should be used.</p><br />
<h2>References</h3><br />
Topp, S. & Gallivan, J. P. 2007.Guiding bacteria with small molecules and RNA. J Am ChemSoc, 129, 6807-11 <br />
<p>Both the t1-V and t2-V constructs transformed into the CheZ deletion mutants are non-motile, each having travelled a distance of 0mm at all concentrations of theophylline (See A and B). This indicates that in the absence of the CheZ protein, the bacteria are non-motile (Topps and Gallivan, 2007).</p><br />
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<p id="Chemotaxis"><br />
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<p>In order to determine whether we had successfully engineered the <i>E. coli</i> CheZ deletion mutants to chemotactically migrate towards theophylline, we performed a capillary assay. Bacteria transformed with thRS1-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>), thRS2-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>), RBS-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>), thRS1-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>) and thRS2-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>) were grown to mid-log phase. Activation of chemotaxis was achieved by incubating the cultures with 1 mM theophylline prior to the assay. A 50 µl drop of each culture was then placed onto separate sterile surfaces and two capillary tubes were placed into each drop. The first capillary tube was a control, containing only PBS and 20 µM EDTA. The second tube contained PBS, 20 µM and 2mM theophylline. The two capillary tubes were suspended into each drop of culture for 30 minutes. A dilution series of the solutions in the tubes were plated and colonies counted after 12 hours. Further details of this protocol can be found here.<br />
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<h3>Discussion and conclusion</h3><br />
<p>Cells transformed with thRS1-V, thRS2-V and RBS-Ch-V all displayed an index of approximately 100, indicating the same number of bacteria moved towards the control (0mM theophylline) and the experiment (2mM theophylline) capillary tube. This proves that none of these constructs induce chemotactic behaviour. In terms of both thRS1-V and thRS2-V, the lack of CheZ results in the inability of CheY dephosphorylation and the swimming motion observed in chemotaxis, which is a key driver in directed movement towards an attractant (Topps and Gallivan, 2007). Although the RBS-Ch-V construct constitutively expresses CheZ, and bacterial swimming is achieved through CheY dephosphorylation, the expression levels remain constant regardless of the presence or absence of theophylline. Random movement will occur as a result.<p/><br />
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<p>The thRS1-Ch-V and thRS2-Ch-V transformants, with chemotaxis indices of 180 and 227 respectively, both indicated a significantly higher number of bacteria that travelled to the 2mM theophylline solution in the capillary tube compared to that of the control. The chemotaxis index of the thRS2-Ch-V construct is significantly higher (p=0.005) than that of thRS1-Ch-V, indicating that cells transformed with thRS2-Ch-V is more attracted to theophylline than thRS1-Ch-V. Should maximum chemotaxis towards theophylline be desired, thRS2-Ch-V would be the more suitable construct.</p><br />
<h2>References</h2><br />
Topp, S. & Gallivan, J. P. 2007. Guiding bacteria with small molecules and RNA. J Am ChemSoc, 129, 6807-11<br />
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<p id="Top"> <br />
<h1><br />
Characterization of Parts</h1><br />
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<p>In order to characterize our final machines, we performed the following assays:</p><br />
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<a href="#Fluorometry">Fluorometry</a><br />
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<a href="#Microscopy1">Fluorescence microscopy: Liquid Culture</a><br />
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<a href="#Microscopy2">Fluorescence microscopy: Smeared Culture</a><br />
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<a href="#Motility">Motility</a><br />
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<a href="#Chemotaxis">Chemotaxis</a><br />
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<p id="Fluorometry"><br />
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<h2>Theophylline Riboswitches</h2><br />
<p>To characterise the theophylline riboswitches (1 and 2), we quantified their activation at different theophylline concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM and 2 mM) over a period of time using fluorometry. Competent <i>E. coli</i> (strain DH5a) cells were transformed with plasmid vectors containing the riboswitch and were cultured until the mid-log phase of growth. Thereafter, a different concentration of theophylline was added to each culture for induction. The activation of the riboswitch was detected as a fluorescent response as a result of increased translation of the fluorescent fusion protein CheZ-Venus, in the presence of the activator. A Jasco FP-6300 spectrofluorometer was used to excite the cultures at 514 nm and the intensity of the emission peak was detected at 528 nm. Twenty readings were taken every 15 minutes for each culture, for a total period of 135 minutes. An empty vector was used to correct for autofluorescence. Conversely, a construct that comprised of a Promoter-RBS-CheZ-Venus-double terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>) was used as a positive control. Fluorescent data for the 3D plots (Fig 1-4) were corrected for autofluorescence by subtracting the fluorescent values of the cultures transformed with the empty vector, from that of the riboswitches.<br />
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<td><p> <b>Figure 1:</b> The fluorescence of the CheZ-Venus fusion protein under the regulation of theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). <b>A)</b> The activation of the riboswitch over time at different concentrations of theophylline. The fluorescence increased over time, with an increasing concentration of theophylline, indicating the theophylline concentration-dependent expression of the CheZ-Venus fluorescent fusion protein. The fluorescence peaks 105 minutes after the addition of 1 mM theophylline. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 1 mM theophylline after 105 minutes incubation, and to that of baseline levels (0 minutes with 1 mM theophylline). 1 mM of theophylline induced the expression of CheZ-Venus to increase 4-fold after 105 minutes (fold change was calculated by dividing the fluorescence observed at 105 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values) (Click to enlarge)<br />
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<b>Figure 2:</b> The fluorescence of the CheZ-Venus fusion protein under the regulation of theophylline riboswitch 2 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>). <b>A)</b> The activation of the riboswitch over time at different concentrations of theophylline. The same trend of increased switch activation over time, with increasing theophylline concentrations was seen as with theophylline riboswitch 1. However, with the theophylline riboswitch 2, the fluorescence peaks 135 minutes after the addition of 1.5 mM theophylline. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 1.5 mM theophylline after 135 minutes incubation, and to that of baseline levels (0 minutes with 1.5 mM theophylline). 1.5 mM of theophylline induced a 37-fold increase in CheZ-Venus expression after 135 minutes (fold change was calculated by dividing the fluorescence observed at 135 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values). (Click to enlarge)</p> </td><br />
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<td><p> <b>Figure 3:</b> The fluorescence of the Venus reporter protein under the regulation of theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>). <b>A)</b> The fluorescent peak occurred prematurely after 75 minutes of incubation with 0.5 mM theophylline, compared to riboswitch 1, which contained the gene for the CheZ-Venus fusion protein. The maximum fluorescent intensity observed was also lower by a factor of 2 RFU. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 75 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 4.5-fold increase in protein expression was observed as the maximum (fold change was calculated by dividing the fluorescence observed at 75 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values). This fold change is comparable to that described in <b>Figure 1</b>. (Click to enlarge)<br />
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<td><p> <b>Figure 4:</b> The fluorescence of the Venus reporter protein under the regulation of the theophylline riboswitch 2 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>). <b>A)</b> The activation of the riboswitch was affected by the lack of the CheZ gene in the switch construct. The increase in fluorescence was minimal upon induction and incubation over time. Furthermore, the fluorescent peak occurred prematurely after 60 minutes of incubation with 0.5 mM theophylline, when compared to the theophylline riboswitch 2 that contained the gene for the CheZ-Venus fusion protein. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 60 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 2-fold increase in protein expression was observed as the maximum (fold change was calculated by dividing the fluorescence observed at 60 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values) . This is far less than the value described for the same switch in <b>Figure 2</b>.(Click to enlarge)<br />
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Both the theophylline riboswitches displayed activation in response to increasing theophylline concentrations and incubation times. Theophylline riboswitch 2 controlling CheZ-Venus expression (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>) clearly showed a superior level of activation compared to that theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). This was expected as it was selected by FACS for its optimised expression platform that allows for minimal leakiness at basal levels, and a strong activation in the presence of theophylline (Lynch and Gallivan, 2009). The activation shown by both the riboswitches regulating CheZ-Venus expression should be able to restore the motility of <i>E. coli</i> cells deficient in CheZ, in the presence of theophylline. More importantly, the theophylline concentration-dependent increase in CheZ-Venus expression alludes to the ability of directed cell movement, that is moving up concentration gradients of theophylline (Topp and Gallivan, 2007). Interestingly, the data suggests that the presence of the CheZ gene, as part of the CheZ-Venus fusion sequence, enhances the activation of the switches when compared to the constructs with venus alone. This may be due to some sort of structural stability conferred by the presence of the CheZ sequence.</P><br />
<h2> Atrazine Riboswitch</h2><br />
<p>Preliminary activation data was obtained for the atrazine riboswitch.Fluorometry was performed on <i>E. coli</i> (DH5a) cells transformed with a construct that contained a Promoter-atrazine riboswitch-mRFP-double terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a>). Similar to the theophylline riboswitches, different concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) of atrazine were added to the cultures for induction. The cultures were excited at 584 nm and detected at 607 nm using a Jasco FP-6300 spectrofluorometer. Readings were taken every 15 minutes for 120 minutes in total. The same empty vector was used as a negative control. The 3D plot data was normalised the same way as for the theophylline riboswitches.</p><br />
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<td><p> <b>Figure 5:</b> The fluorescence of the mRFP1 reporter protein under the regulation of the atrazine riboswitch(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a>). <b>A)</b> The activation of the atrazine riboswitch at different atrazine concentrations over time. There was an increase in fluorescence as the atrazine concentration and incubation time increased. The amount of fluorescence detected was very small, with a maximum of 0.1 RFU detected after 75 minutes of incubation with 2 mM atrazine. <b>B)</b> The maximum was compared to the negative control at 75 minutes, 2 mM atrazine, and to that of fluorescent levels detected at basal levels (0 minutes, 2 mM atrazine). A 1.7-fold increase in expression of mRFP1 was observed over the 75 minute period (values used for the calculation had the corresponding negative control value subtracted first).(Click to enlarge)<br />
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<h2>References</h2><br />
<p>Lynch, S. A. & Gallivan, J. P. 2009. A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Res, 37, 184-92.</p><br />
<p>Topp, S. & Gallivan, J. P. 2007. Guiding bacteria with small molecules and RNA. J Am Chem Soc, 129, 6807-11.</p><br />
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<h2>Epi-fluorescence Wide-Field Microscopy</h2><br />
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<p> Fluorescent imaging was performed for a qualitative assay of the engineered bacteria. <br />
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<p> Bacterial culture and Manipulation: <br />
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<p> 4 ml of LB broth with Ampicillin was inoculated from colonies off a master plate of positive clones and grown overnight at 37°C in a shaking incubator. 2 ml of each bacterial culture was then used to inoculate 25 ml of LB broth with Ampicillin and grown for ± 3 hours in a shaking incubator at 37°C until the cells reached an OD600 of 0.55 (mid-log phase). 2 x 5 ml of each sample culture was aliquoted into separate 15 ml falcon tubes. One tube was left untreated; 150 µl of 15 mM theophylline was added to the other (to make a final concentration of 1.5mM theophylline). These 5 ml aliquots were placed in a shaking incubator at 37°C for 30 min each to allow for activation. 200 µl of each treated and untreated sample was then used in a microscope plate for imaging. Imaging was done using Zeiss Axioscope (Andor EMCCD Camera, Till Photonics Polychrome V). <br />
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<p> Widefield fluorescence microscopy was used to assess the expression of the Venus protein. The bacterial samples were excited at 500 nm using the polychrome as an excitation source. The fluorescence was then observed by capturing light in the emission spectrum of Venus (528 nm). The figure below represents a schematic of the imaging setup. Corresponding bright-field images (using white light) were also captured for each experiment. <br />
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<p>Below are the images that were obtained for the parental strain (BW25113) of the <i>E. coli</i> CheZ-deletion mutant, which served as the negative, and the CheZ deletion mutants transformed with Promoter-theophylline riboswitch 1-venus (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) and Promoter-theophylline riboswitch 2-venus (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>).<br />
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<p>Please note: Bacteria which were transformed with constructs which contained the CheZ gene were not tested in this assay. This experiment served as a means of observing the activation of the theophylline riboswitches, hence only expression of the venus reporter protein was needed for detection of fluorescence. Activation of motility was not assessed in this case – it was examined in the motility assays on semi-solid agar plates. Differences in fluorescence intensities were not calculated from the images below since the fluorometry experiments served as the quantitative approach to measurement of riboswitch activation.<br />
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<b>Figure 6:</b> The parental strain (BW25113) of the <i>E. coli</i> CheZ deletion mutants were not transformed with any plasmid DNA. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In both the absence and presence of theophylline, the parental bacteria did not fluoresce – which was the expected result. (Click to enlarge)</p> </td><br />
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<b>Figure 7:</b> <i>E. coli</i> CheZ-deletion mutants which were transformed with the Promoter-theophylline riboswitch 1-venus (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) construct. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, almost no fluorescence occurred. Upon the addition of theophylline at a concentration of 1.5 mM, many of the cells emitted fluorescence showing activation of the theophylline riboswitch type 1.(Click to enlarge)</p> </td><br />
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<b>Figure 8:</b> <i>E.coli</i> CheZ-deletion mutants which were transformed with the Promoter-theophylline riboswitch 2-venus (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>) construct. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, some fluorescence was observed. This result showed the leakiness of the riboswitch. A substantial amount of venus translation is permitted because of the flexible conformation of this riboswitch. Upon the addition of theophylline at a concentration of 1.5 mM, much more fluorescence was detected. More venus was expressed in these cells due to the activation of the riboswitch via theophylline.(Click to enlarge)</p> </td><br />
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<p> Fluorescent imaging was performed again after the European Jamboree, using a different technique. The aim of this data is to depict the exact same brightfield and fluorescent static fields of view. <br />
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<p id="Motility"><br />
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<p> Semi-solid agar plates containing different concentrations of theophylline (0mM, 0.5mM, 1.0mM and 1.5mM) were made. Promoter-theophylline riboswitch 1-CheZ-Venus-double terminator (thRS1-Ch-V)(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>), Promoter-theophylline riboswitch 2-CheZ-Venus-double terminator (thRS2-Ch-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>), Promoter-theophylline riboswitch 1-Venus-double terminator (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) and Promoter-theophylline riboswitch 2-Venus-double terminator (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>) were transformed into CheZ deletion mutants and grown to mid-log phase (OD600 = 0.55). 1.5 µl of culture was plated into the centre of the plates containing the various concentrations of theophylline and incubated at 37°C for 24 hours. This experiment was performed in triplicate. The protocol for this experiment can be found here.<br />
</p><br />
<center><a href="https://static.igem.org/mediawiki/2011/a/a5/Motility_graph.jpg"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Characterization',title:'Figure 9',href:'https://static.igem.org/mediawiki/2011/a/a5/Motility_graph.jpg'"><br />
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<b>Figure 9:</b> The distance travelled by CheZ deletion mutants (mm) over a 24 hour period at 37°C on semi-solid agar plates containing different theophylline concentrations (mM)(Click to enlarge)</p> </td><br />
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The thRS1-V and thRS2-V transformants did not travel from the point of inoculation for all concentrations of theophylline. The CheZ deletion mutants containing thRS1-Ch-V travelled 0 mm at 0 mM theophylline and reached a maximum travelling distance of 5 mm at 1.5 mM theophylline. The thRS2-Ch-V containing CheZ deletion mutants travelled 2 mm in the absence of theophylline and at a concentration of 1.5 mM theophylline, were able to travel 22mm from the point of inoculation </p><br />
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<center><a href="https://static.igem.org/mediawiki/2011/2/2e/Motility_result_A.jpg"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Characterization',title:'Figure 10 A and B',href:'https://static.igem.org/mediawiki/2011/2/2e/Motility_result_A.jpg'"><br />
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<b>Figure 10:</b> 85 mm semi-solid agar plates containing 1.0 mM theophylline showing the point of inoculation and distance travelled by the bacteria. <b>A)</b> Inoculation of 1.5 µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS1-Ch-V(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). There is evidence of movement of bacteria from the point of inoculation. This can be observed as a faint halo surrounding the point of inoculation.<b>B)</b> Inoculation of 1.5 µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS1-V. Converse to <b>A</b>, there is no evidence of movement of the bacteria from the point of inoculation <b>C)</b> Inoculation of 1.5µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS2-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>). There is evidence of movement of the bacteria from the point of inoculation. This can be observed as a faint halo surrounding the point of inoculation. <b>D)</b> Inoculation of 1.5µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS2-V. Converse to <b>C</b>, there is no evidence of movement of bacteria from the point of inoculation (Click to enlarge)</p> </td><br />
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<h3> Discussion and conclusion</h3><br />
<p>The construct thRS1-Ch-V transformed into the CheZ deletion mutants are non-motile at a concentration of 0 mM theophylline, since in the absence of the activator, the riboswitch is sequestering the RBS and thus inhibiting the translation of CheZ (Topps and Gallivan, 2007). On the semi-solid agar plates containing theophylline, the motility of E.coli is evident, as the interaction of theophylline with the riboswitch enables the exposure of the RBS and the translation of the downstream CheZ, allowing for bacterial motility. The distance travelled by the bacteria transformed with the thRS1-Ch-V construct, from the start position (mm) in the presence of different concentrations of theophylline, is not significantly different (P=0.005). This indicates that the CheZ expression at 0.5 mM-1.5 mM is sufficient to allow the bacteria to move a distance of 5 mm. The construct thRS2-Ch-V transformed into the CheZ deletion mutants are slightly motile in the absence of theophylline, travelling 2mm over a 24 hour period indicating that this riboswitch is slightly leaky. Upon addition of 0.5 mM-1.5 mM theophylline, the bacteria were able to travel 20mm over a 24 hour period.</p><br />
<p>The distance travelled from the start position by the CheZ deletion mutants containing the thRS2-Ch-V is significantly higher than that those with the thRS1-Ch-V construct. This can be explained by the high fold-increase in CheZ protein expression by this riboswitch, as shown by fluorometry. Theophylline riboswitch 1 has a lower fold increase in CheZ expression from 0 mM to 1.5 mM theophylline, compared to the increase shown by the theophylline riboswitch 2. Therefore, in order to achieve maximum motility under the control of theophylline, at concentrations of between 0.5 mM and 1.5 mM, theophylline riboswitch 2 should be used.</p><br />
<h2>References</h3><br />
Topp, S. & Gallivan, J. P. 2007.Guiding bacteria with small molecules and RNA. J Am ChemSoc, 129, 6807-11 <br />
<p>Both the t1-V and t2-V constructs transformed into the CheZ deletion mutants are non-motile, each having travelled a distance of 0mm at all concentrations of theophylline (See A and B). This indicates that in the absence of the CheZ protein, the bacteria are non-motile (Topps and Gallivan, 2007).</p><br />
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<p id="Chemotaxis"><br />
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<p>In order to determine whether we had successfully engineered the <i>E. coli</i> CheZ deletion mutants to chemotactically migrate towards theophylline, we performed a capillary assay. Bacteria transformed with thRS1-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>), thRS2-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>), RBS-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>), thRS1-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>) and thRS2-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>) were grown to mid-log phase. Activation of chemotaxis was achieved by incubating the cultures with 1 mM theophylline prior to the assay. A 50 µl drop of each culture was then placed onto separate sterile surfaces and two capillary tubes were placed into each drop. The first capillary tube was a control, containing only PBS and 20 µM EDTA. The second tube contained PBS, 20 µM and 2mM theophylline. The two capillary tubes were suspended into each drop of culture for 30 minutes. A dilution series of the solutions in the tubes were plated and colonies counted after 12 hours. Further details of this protocol can be found here.<br />
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data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Characterization',title:'Figure 12: Riboswitch 2',href:'https://static.igem.org/mediawiki/2011/a/aa/Chemotaxis_figure_B.jpg'"><br />
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<h3>Discussion and conclusion</h3><br />
<p>Cells transformed with thRS1-V, thRS2-V and RBS-Ch-V all displayed an index of approximately 100, indicating the same number of bacteria moved towards the control (0mM theophylline) and the experiment (2mM theophylline) capillary tube. This proves that none of these constructs induce chemotactic behaviour. In terms of both thRS1-V and thRS2-V, the lack of CheZ results in the inability of CheY dephosphorylation and the swimming motion observed in chemotaxis, which is a key driver in directed movement towards an attractant (Topps and Gallivan, 2007). Although the RBS-Ch-V construct constitutively expresses CheZ, and bacterial swimming is achieved through CheY dephosphorylation, the expression levels remain constant regardless of the presence or absence of theophylline. Random movement will occur as a result.<p/><br />
<br />
<p>The thRS1-Ch-V and thRS2-Ch-V transformants, with chemotaxis indices of 180 and 227 respectively, both indicated a significantly higher number of bacteria that travelled to the 2mM theophylline solution in the capillary tube compared to that of the control. The chemotaxis index of the thRS2-Ch-V construct is significantly higher (p=0.005) than that of thRS1-Ch-V, indicating that cells transformed with thRS2-Ch-V is more attracted to theophylline than thRS1-Ch-V. Should maximum chemotaxis towards theophylline be desired, thRS2-Ch-V would be the more suitable construct.</p><br />
<h2>References</h2><br />
Topp, S. & Gallivan, J. P. 2007. Guiding bacteria with small molecules and RNA. J Am ChemSoc, 129, 6807-11<br />
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/ContactUs">Contact us</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
<div data-dojo-type="dijit.layout.ContentPane" data-dojo-props="region: 'top'" class="top"><br />
<a href="https://2011.igem.org/Team:WITS-CSIR_SA"><br />
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<div data-dojo-type="dijit.layout.ContentPane" data-dojo-props="region: 'center'"<br />
class="center"><br />
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<div style="width: 903px; margin: auto; height: 100%;"><br />
<div data-dojo-type="dijit.layout.BorderContainer" data-dojo-props="design: 'headline'"<br />
style="height: 100%;"><br />
<div data-dojo-type="dijit.layout.ContentPane" data-dojo-props="region: 'center'"><br />
<div style="height: 100%; overflow-y: auto;"><br />
<br />
<h1><br />
Parts Submitted</h1><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Table of parts submitted'"><br />
<p>Click on the part number to go to the associated registry page. Click on the part name for a brief description of the part.</p><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty" width="100%"><br />
<tr><br />
<th style="width: 80px;">Favourites</th><br />
<th style="width: 80px;"><br />
Part Number<br />
</th><br />
<th style="width: 160px;"><br />
Part Name<br />
</th><br />
<th><br />
Part Length<br />
</th><br />
<th>Sent</th><br />
</tr><br />
<tr><br />
<td><br />
<center><img src="https://static.igem.org/mediawiki/2011/1/15/Favourite_image.jpg" width="30px;"></center><br />
<a style="color:Green;" href="https://static.igem.org/mediawiki/2011/2/27/Datasheet1.pdf">Datasheet</a><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537001">BBa_K537001</a><br />
</td><br />
<td width ="200"><br />
<a href="#T1-Ch">Theophylline riboswitch 1 - CheZ</a><br />
</td><br />
<td><br />
698bp<br />
</td><br />
<br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
<center><img src="https://static.igem.org/mediawiki/2011/1/15/Favourite_image.jpg" width="30px;"></center><br />
<a style="color:Green;" href="http://partsregistry.org/wiki/images/f/f5/Datasheet2.pdf">Datasheet</a><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537002">BBa_K537002</a><br />
</td><br />
<td><br />
<a href="#T2-Ch">Theophylline riboswitch 2 - CheZ</a><br />
<br />
</td><br />
<td><br />
702bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537000">BBa_K537000</a><br />
</td><br />
<td><br />
<a href="#AtRS+CheZ">Atrazine riboswitch-CheZ</a><br />
<br />
</td><br />
<td><br />
730bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
<center><img src="https://static.igem.org/mediawiki/2011/1/15/Favourite_image.jpg" width="30px;"></center><br />
<a style="color:Green;" href="https://static.igem.org/mediawiki/2011/2/27/Datasheet1.pdf">Datasheet</a></td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537003">BBa_K537003</a><br />
</td><br />
<td><br />
<a href="#T1-V">Theophylline riboswitch 1- Venus</a><br />
<br />
</td><br />
<td><br />
780bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537004">BBa_K537004</a><br />
</td><br />
<td><br />
<a href="#T2-V">Theophylline riboswitch 2- Venus</a><br />
<br />
</td><br />
<td><br />
781bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537005">BBa_K537005</a><br />
</td><br />
<td><br />
<a href="#mRFP1 C-fusion">mRFP1 C-fusion</a><br />
<br />
</td><br />
<td><br />
679bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537006">BBa_K537006</a><br />
</td><br />
<td><br />
<a href="#Venus C-fusion">Venus C-fusion</a><br />
</td><br />
<td><br />
721bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537007">BBa_K537007</a><br />
</td><br />
<td><br />
<a href="#RBS CheZ N-fusion">RBS CheZ N-fusion</a><br />
<br />
</td><br />
<td><br />
658bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537008">BBa_K537008</a><br />
</td><br />
<td><br />
<a href="#Atrazine Riboswitch-mRFP">Atrazine Riboswitch-mRFP</a><br />
<br />
</td><br />
<td><br />
658bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a><br />
</td><br />
<td><br />
<a href="#Promoter-Theophylline riboswitch 1-Venus-Double terminator">Promoter-Theophylline riboswitch 1-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
960bp<br />
</td><br />
<td></center></td><br />
</tr><br />
<tr><br />
<td><br />
<br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a><br />
<br />
</td><br />
<td><br />
<a href="#Promoter-Theophylline riboswitch 2-Venus-Double terminator">Promoter-Theophylline riboswitch 2-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
961bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
<br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a><br />
</td><br />
<td><br />
<a href="#Promoter-Theophylline riboswitch 1-CheZ-Venus-Double terminator">Promoter-Theophylline riboswitch 1-CheZ-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
1583bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
<br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a><br />
</td><br />
<td><br />
<a href="#Promoter-Theophylline riboswitch 2-CheZ-Venus-Double terminator">Promoter-Theophylline riboswitch 2-CheZ-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
1587bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a><br />
</td><br />
<td><br />
<a href="#Promoter-RBS-CheZ-Venus-Double terminator">Promoter-RBS-CheZ-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
1543bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a><br />
</td><br />
<td><br />
<a href="#Promoter-Atrazine riboswitch-mCherry-Double terminator">Promoter-Atrazine riboswitch-mCherry-Double terminator</a><br />
<br />
</td><br />
<td><br />
946bp<br />
</td><br />
<td></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537015">BBa_K537015</a><br />
</td><br />
<td><br />
<a href="#Promoter-Atrazine riboswitch-CheZ-mCherry-Double terminator">Promoter-Atrazine riboswitch-CheZ-mCherry-Double terminator</a><br />
<br />
<br />
</td><br />
<td><br />
1570bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537016">BBa_K537016</a><br />
</td><br />
<td><br />
<a href="#Promoter-RBS-CheZ-mCherry-Double terminator">Promoter-RBS-CheZ-mCherry-Double terminator</a><br />
<br />
</td><br />
<td><br />
1501bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537019">BBa_K537019</a><br />
</td><br />
<td><br />
<a href="#lox66">lox66</a><br />
<br />
<br />
</td><br />
<td><br />
34bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537020">BBa_K537020</a><br />
</td><br />
<td><br />
<a href="#lox71">lox71</a><br />
<br />
</td><br />
<td><br />
34bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<br />
</table><br />
<br /><br />
</div><br />
<br /><br />
<p id ="T1-Ch"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Theophylline Riboswitch 1-CheZ'"><br />
<image src="https://static.igem.org/mediawiki/2011/f/fa/Wits_Parts_submitted_ThRS1-CheZ.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This DNA part will encode for an RNA riboswitch senstive to theophylline. The RBS is not exposed in the absence of theophylline. When theophylline is present, it binds to the riboswitch and causes a conformational change which leads to the exposure of the RBS and consequently translation of the adjoining gene. In this case, the gene which will be expressed is CheZ, which is a protein fundamental to bacterial movement.<br />
This theophylline riboswitch (type1) CheZ fusion BioBrick activates the expression of the CheZ gene in a theophylline-dependent fashion. It is composed of a theophylline-sensitive riboswitch clone 8.1 (Topp and Gallivan JACS 2007; BBa_K249026 and BBa_K411001) that is detached from its associated translation unit (coding region) and fused to a CheZ gene which has a N-fusion prefix and lacks a stop codon (although a TAG stop codon is provided by the RFC 25 suffix). The riboswitch regulated N-terminal part can be fused to a reporter or other CDS.</p><br />
<p>CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling) in the presence of a chemoattractant (in this case, theophylline).</p><br />
<br />
<br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="T2-Ch"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Theophylline Riboswitch 2-CheZ'"><br />
<image src="https://static.igem.org/mediawiki/2011/7/74/Wits_Parts_submitted_ThRS2-CheZ.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This DNA part will encode for an RNA riboswitch senstive to theophylline. When no theophylline is present, the RBS within the riboswitch sequence is not exposed to translation machinery. When theophylline is present, it binds to the riboswitch and causes a conformational change which results in the RBS being exposed. This allows for the translation of the adjoining gene. In this case, the gene which will be expressed is CheZ - a fundamental protein in the signalling cascade of bacterial chemotaxis.</p><br />
<p><br />
This theophylline riboswitch 2- CheZ fusion BioBrick activates the expression of the CheZ gene in a theophylline-dependent fashion. It consists of a newly improved theophylline riboswitch clone 12.1 (Lynch and Gallivan NAR 2009) that is detached from its associated translation unit (coding region) and fused to a CheZ gene which has a N-fusion prefix and lacks a stop codon (although a TAG stop codon is provided by the RFC 25 suffix). The riboswitch regulated N-terminal part can be fused to a reporter or other CDS.</p><br />
<p>CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling) in the presence of a chemoattractant (in this case, theophylline).<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<br />
<p id ="AtRS+CheZ"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Atrazine Riboswitch-CheZ fusion'"><br />
<image src="https://static.igem.org/mediawiki/2011/6/6c/Wits_Parts_submitted_AtRS-CheZ.png" align="left" style="padding: 10px;"/><br />
<br />
<p>When no atrazine is present, the RBS within the riboswitch sequence is not exposed to translation machinery. When atrazine is present, it binds to the riboswitch and causes a conformational change which results in the RBS being exposed. This allows for the translation of the adjoining gene. In this case, the gene which will be expressed is CheZ - a fundamental protein in the signalling cascade of bacterial chemotaxis.<br />
This riboswitch-CheZ fusion BioBrick regulates the expression of the CheZ gene in an atrazine-dependent fashion. It is composed of an atrazine-sensitive riboswitch, developed by Sinha et al (2010), which is detached from its associated translation unit (coding region) and fused to a CheZ gene which has the Freiburg N-fusion prefix and lacks a stop codon. While it is possible to fuse the riboswitch to the CheZ coding region by standard BioBrick assembly techniques, this approach was not used as it would increase the distance between the RBS and the ATG start codon and potentially decrease the efficiency of the riboswitch. This was shown in the work of the Taipei 2010 team who used this approach for a theophylline riboswitch. The riboswitch and the adjacent CheZ coding region are considered together and should be cloned together. CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the Chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling) in the presence of a chemoattractant (in this case atrazine).<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="T1-V"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Theophylline Riboswitch 1- Venus'"><br />
<image src="https://static.igem.org/mediawiki/2011/6/6d/Wits_Parts_submitted_ThRS1-Venus.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This part regulates the expression of venus in a theophylline-dependent fashion through an RNA aptamer specific to theophylline.<br />
This theophylline riboswitch 1 biobrick activates the expression of the Venus fluorescent protein in a theophylline-dependent fashion. It is composed of a theophylline sensitive riboswitch clone 8.1 (Topp and Gallivan JACS 2007; BBa_K249026 and BBa_K411001) fused upstream to an adjacent Venus coding region.<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="T2-V"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Theophylline Riboswitch 2- Venus'"><br />
<image src="https://static.igem.org/mediawiki/2011/f/fd/Wits_Parts_submitted_ThRS2-Venus.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This is a theophylline riboswitch clone 12.1 (Lynch and Gallivan NAR 2009) fusion with the Venus reporter BBa_K354002. This part regulates the expression of venus in a substrate-dependent fashion through an RNA aptamer specific to the antibiotic theophylline.<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="mRFP1 C-fusion"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'mCherry (mRFP1) C-Fusion'"><br />
<image src="https://static.igem.org/mediawiki/2011/d/da/Wits_Parts_submitted_mRFP1_C-fusion.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This BioBrick represents a C-terminal fusion part. The part contains a standard suffix and a “assembly standard 25” prefix (Freiburg Fusions/ RFC 25). This version of the mRFP reporter (BBa_E1010) has the standard BioBrick suffix and prefix. The standard Biobrick prefix, however, is followed by another restriction site – specific for NgoMIV (which forms the Freiburg fusion prefix). The part lacks an ATG start codon (provided by N-terminal part). It can therefore be fused to any gene in a modular fashion via the modular construction strategy of protein fusion developed by the Freiburg 2007 team.<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="Venus C-fusion"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Venus C-Fusion'"><br />
<image src="https://static.igem.org/mediawiki/2011/a/aa/Wits_Parts_submitted_Venus-C-fusion.png" align="left" style="padding: 10px;"/> <br />
<p>This BioBrick represents a C-terminal fusion part. The part contains a standard suffix and a “assembly standard 25” prefix (Freiburg Fusions/ RFC 25). This version of the Venus Fluorescent reporter (BBa_K354002) has the standard BioBrick suffix and prefix. The standard Biobrick prefix, however, is followed by another restriction site – specific for NgoMIV (which forms the Freiburg fusion prefix). The part lacks an ATG start codon (provided by N-terminal part). It can therefore be fused to any gene in a modular fashion via the modular construction strategy of protein fusion developed by the Freiburg 2007 team.<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="RBS CheZ N-fusion"> <br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'RBS CheZ N-Fusion'"><br />
<image src="https://static.igem.org/mediawiki/2011/8/8c/Wits_Parts_submitted_RBS-CheZ.png" align="left" style="padding: 10px;"/> <br />
<br />
<p>The RBS CheZ- Fusion BioBrick consists of a RBS fused to the E. coli CheZ motility factor that lacks a stop codon at the N-terminus (although a TAG stop codon is provided by the RFC 25 suffix). CheZ in this part contains the standard BioBrick prefix and the Freiburg (2007) fusion suffix. The N-terminal part can be fused to a reporter or other CDS. Using this BioBrick, CheZ is constitutively expressed.</p><br />
<p>CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling).</p><br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<br />
<p id ="Atrazine Riboswitch-mRFP"> <br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Atrazine Riboswitch-mRFP'"><br />
<image src="https://static.igem.org/mediawiki/2011/1/17/Wits_Parts_submitted_AtRS-mRFP1.png" align="left" style="padding: 10px;"/> <br />
<br />
<p>This part consists of an atrazine-sensitive riboswitch, developed by Sinha et al (2010), detached from its associated translation unit (coding region) and fused to mRFP1 fluorescent reporter (BBa_J61100). This part regulates the expression of mRFP in an atrazine-dependent fashion through an atrazine-specific RNA aptamer.</p><br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="Promoter-Theophylline riboswitch 1-Venus-Double terminator"> <br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-Theophylline riboswitch 1-Venus-Double terminator'"><br />
<image src="https://static.igem.org/mediawiki/2011/2/2f/Wits_Parts_submitted_Pr-thRS1-Venus-dt.png" align="left" style="padding: 10px;"/> <br />
<br />
<br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 1 (Topp and Gallivan JACS, 2007) which is fused to the Venus fluorescent reporter protein without a stop codon in between. The theophylline riboswitch1-venus fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).</p><br />
</p><br />
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<br/><br />
<p id ="Promoter-Theophylline riboswitch 2-Venus-Double terminator"> <br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-Theophylline riboswitch 2-Venus-Double terminator'"><br />
<image src="https://static.igem.org/mediawiki/2011/c/ca/Wits_Parts_submitted_Pr-ThRS2-Venus-dt.png" align="left" style="padding: 10px;"/> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 2 (Lynch and Gallivan NAR 2009) which is fused to the Venus fluorescent reporter protein without a stop codon in between. The theophylline riboswitch2-venus fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).</p><br />
<br />
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</p><br />
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<br/><br />
<p id ="Promoter-Theophylline riboswitch 1-CheZ-Venus-Double terminator"> <br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-Theophylline riboswitch 1-CheZ-Venus-Double terminator'"><br />
<center><image src="https://static.igem.org/mediawiki/2011/d/d9/Wits_Parts_submitted_Pr-thRS1-CheZ-Venus-dt.png"></center> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 1 (Topp and Gallivan JACS 2007) which is fused to CheZ and Venus fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The theophylline riboswitch1-CheZ fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).</p><br />
<br />
</p><br />
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<br/><br />
<p id ="Promoter-Theophylline riboswitch 2-CheZ-Venus-Double terminator"> <br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-Theophylline riboswitch 2-CheZ-Venus-Double terminator'"><br />
<center><image src="https://static.igem.org/mediawiki/2011/8/8e/Wits_Pr-thRS2-CheZ-Venus-dt.png"></center> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 2 (Lynch and Gallivan NAR 2009) which is fused to CheZ and Venus fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The theophylline riboswitch2-CheZ fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).</p><br />
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<br/><br />
<br />
<p id ="Promoter-RBS-CheZ-Venus-Double terminator"> <br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-RBS-CheZ-Venus-Double terminator'"><br />
<center><image src="https://static.igem.org/mediawiki/2011/8/85/Wits_Parts_submitted_Pr-RBS-CheZ-Venus-dt.png"></center> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an RBS which is fused to CheZ and Venus fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The RBS-CheZ fusion was constructed via PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015)<br />
</p><br />
</div><br />
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<br/><br />
<p id ="Promoter-Atrazine riboswitch-mCherry-Double terminator"> <br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-Atrazine riboswitch-mCherry-Double terminator'"><br />
<image src="https://static.igem.org/mediawiki/2011/4/43/Wits_Parts_submitted_Pr-AtRS-mRFP-dt.png" align="left" style="padding: 10px;"/> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an atrazine riboswitch riboswitch (Sinha et al, 2010) which is fused to the monomeric Red fluorescent reporter protein without a stop codon in between. The atrazine riboswitch-mRFP fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="Promoter-Atrazine riboswitch-CheZ-mCherry-Double terminator"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-Atrazine riboswitch-CheZ-mRFP-Double terminator'"><br />
<center><image src="https://static.igem.org/mediawiki/2011/c/c9/Wits_Parts_submitted_Pr-AtRS-CheZ-mRFP-dt.png"></center> <br />
<br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an atrazine riboswitch riboswitch (Sinha et al, 2010) which is fused to CheZ and monomeric Red fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The atrazine riboswitch-CheZ fusion was constructed via PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="Promoter-RBS-CheZ-mCherry-Double terminator"><br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-RBS-CheZ-mCherry-Double terminator'"><br />
<center><image src="https://static.igem.org/mediawiki/2011/5/52/Wits_Pr-RBS-CheZ-mRFP-dt.png"></center> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an RBS which is fused to CheZ and monomeric Red fluorescent reporter protein (mRFP) without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The RBS-CheZ fusion was constructed via PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).<br />
</p><br />
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<br/> <br />
<br />
<p id ="lox66"><br />
<br />
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<br />
<p>lox66 is a site specific recombination cassette. It belongs to the loxP family frequently used in genetics, particularily in mouse genetics.<br />
lox site recombination is catalysed by a Site specific recombinase, Cre.<br />
lox sequences are composed of an 8 bp Core sequence surrounded by two Arms.<br />
The particularity of lox66 is that it has an altered sequence at the end of it's left arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm. As a consequence, after a recombination between a lox66 and a lox71 (altered right arm sequence), one of the two resulting generated lox sites has very low recombination potential as it inherited both mutated arms. Use of lox66 & lox71 sites is potentially interresting when the recombination reaction must be "irreversible".<br />
</p><br />
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<br/> <br />
<br />
<p id ="lox71"><br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'lox71'"><br />
<br />
<p>Lox71 is a site specific recombination cassette. It belongs to the loxP family frequently used in genetics, particularly in mouse genetics.<br />
lox site recombination is catalysed by a Site specific recombinase, Cre.<br />
lox sequences are composed of an 8 bp Core sequence surrounded by two Arms.<br />
The particularity of lox66 is that it has an altered sequence at the end of it's left arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm. As a consequence, after a recombination between a lox71 and a lox66 (altered right arm sequence), one of the two resulting generated lox sites has very low recombination potential as it inherited both mutated arms. Use of lox71 & lox66 sites is potentially interesting when the recombination reaction must be "irreversible".<br />
</p><br />
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<br/> <br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Project/ProtocolsTeam:WITS-CSIR SA/Project/Protocols2011-10-22T20:28:57Z<p>Nkruger: </p>
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Project/AchievementsTeam:WITS-CSIR SA/Project/Achievements2011-10-22T20:28:02Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Concept">Overview</a></li><br />
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<br />
<p><b>1)</b> We have submitted 11 biobrick parts to the Registry of Standard Biological Parts. Three new biobrick parts representing our “favourite” biobricks, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537001">BBa_K537001</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537002">BBa_K537002</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537003">BBa_K537003</a>, have been fully characterized.</p><br />
<br />
<p><b>2)</b> We are the first team to characterize the function of two different types of theophylline-responsive riboswitches, and this was achieved for two separate coding sequences. These riboswitch designs are improvements over previously-developed iGEM riboswitches.</p><br />
<br />
<p><b>3)</b> Theophylline-response riboswitch 2 (thRS2-CheZ, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537002">BBa_K537002</a>) was capable of significantly higher activation than Theophylline-response riboswitch 1 (thRS1, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537001">BBa_K537001</a>) when expressing motility factor CheZ (37-fold vs 4-fold). Moreover, thRS2 showed 6-fold improved motility function over thRS1 and 1-fold increased chemotactic sensitivity to theophylline.</p><br />
<br />
<p><b>4)</b> Theophylline-responsive riboswitch 2-CheZ (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537002">BBa_K537002</a>) represents an ideal biobrick for N-terminal fusion to proteins of interest for theophylline-activated messenger activity. This part could have numerous advantages in several other synthetic biology applications. </p><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Project/AchievementsTeam:WITS-CSIR SA/Project/Achievements2011-10-22T20:27:17Z<p>Nkruger: </p>
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<p><b>1)</b> We have submitted 11 biobrick parts to the Registry of Standard Biological Parts. Three new biobrick parts representing our “favourite” biobricks, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537001">BBa_K537001</a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537002">BBa_K537002</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537003">BBa_K537003</a>, have been fully characterized.</p><br />
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<p><b>2)</b> We are the first team to characterize the function of two different types of theophylline-responsive riboswitches, and this was achieved for two separate coding sequences. These riboswitch designs are improvements over previously-developed iGEM riboswitches.</p><br />
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<p><b>3)</b> Theophylline-response riboswitch 2 (thRS2-CheZ, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537002">BBa_K537002</a>) was capable of significantly higher activation than Theophylline-response riboswitch 1 (thRS1, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537001">BBa_K537001</a>) when expressing motility factor CheZ (37-fold vs 4-fold). Moreover, thRS2 showed 6-fold improved motility function over thRS1 and 1-fold increased chemotactic sensitivity to theophylline.</p><br />
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<p><b>4)</b> Theophylline-responsive riboswitch 2-CheZ (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537002">BBa_K537002</a>) represents an ideal biobrick for N-terminal fusion to proteins of interest for theophylline-activated messenger activity. This part could have numerous advantages in several other synthetic biology applications. </p><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Project/ApplicationsTeam:WITS-CSIR SA/Project/Applications2011-10-22T20:26:23Z<p>Nkruger: </p>
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<h1><br />
Potential Applications</h1><br />
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<p>Our completed machine serves as the first of a chain of events which could be expanded for multiple applications. The ‘Biotweet’ machine enables the movement of bacteria to an attractant (A). Once the source of the attractant is found (IPTG), the bacteria return to the starting position through an attraction towards B, the attraction towards A is switched off at this point. The bacteria that complete this circular journey are the messenger bacteria. Upon returning to the start position, they recruit other bacteria to send them to the site of the attractant to perform a particular function, such as degradation. Important to note is that since the bacteria will return to the start, this means that none will remain in the environment in which they were employed to work. This provides assured safety whilst working with our machine (See our safety page).</p><br />
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<p>Our machine utilizes two riboswitches, one sensitive to theophylline (at A) and the other sensitive to atrazine. The beauty of the use of riboswitches in our project is that they can be substituted with riboswitches sensitive to other substances. This ensures that our machine can be used as a template, where attraction towards virtually any substances can be engineered.</p><br />
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<p> The above template can be employed in the industrial sector to identify and degrade contaminants or alternately to collect substances of economic importance. </p><br />
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<p>Example: Messenger bacteria attracted to a contaminant such as Mercury. Upon finding the Mercury, they will return to the start to recruit a number of bacteria which have the ability to absorb it. Once the Mercury has been absorbed, the transcription of the riboswitch inducing the attraction of the bacteria to the starting position will be activated. The bacteria will then return to the start position (home), leaving the environment free of genetically modified organisms. </p><br />
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<p>There is much room for the improvement of diagnostic technology, where some diagnoses can take months to perform, such as is the case for TB. A riboswitch specific to a disease biomarker can be engineered to allow the bacteria to search a blood sample, or biomarkers at low concentrations present in the saliva or urine. Upon contact with the biomarker, chemotaxis can then be toggled where the bacteria will return to the start position and report on their findings. </p><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Project/NotebookTeam:WITS-CSIR SA/Project/Notebook2011-10-22T20:25:41Z<p>Nkruger: </p>
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<h1><br />
Lab Notebook</h1><br />
<p>Note: Lab work was carried out from June onwards</p><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Collaboration/IndexTeam:WITS-CSIR SA/Collaboration/Index2011-10-22T20:24:18Z<p>Nkruger: </p>
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Collaboration</h1><br />
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<p>The Wits CSIR iGEM team collaborated with Imperial College London, both for the modelling and the potential suitability of motility experiments in the testing of our machines. The biologists in our team and those at Imperial College London had a number of skype conferences to discuss the progress of our lab work as well as to share insight into both qualitative and quantitative assay for motility. </p><br />
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<p>We provided Imperial College London with some ideas regarding motility assays using TTC in stab agar. Their team gave us some tips about the preparation of cells for motility experiments and recommended spinning down the bacteria and resuspending them in a low volume of broth to increase the density of cells. They also recommended the use of capillary motility assays. There was an exchange of protocols both ways to aid in the set-up of our experiments. A week later we discussed the progress we had made in the lab. We gave each other tips and suggested more assays to indicate the ability of our bacteria to move towards a stimulus. Imperial College London shared with us a type of capillary assay they were performing at the time and we, too, shared a protocol with them which is detailed in Gullivan and Topps (2006) paper, Guiding bacteria with small molecules and RNA.</p><br />
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<p>Collaboration was also performed between the engineers to improve the modeling for both the teams. Several Skype conferences were held between the team from Imperial College London and the Wits CSIR team to discuss the design and theory behind our own chemotaxis models. The collaborative effort resulted in the Imperial College London kindly providing us with a model for our theophylline riboswitches. Their modelling results can be found on our href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Modelling">modelling</a> page.<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Project/ModellingTeam:WITS-CSIR SA/Project/Modelling2011-10-22T20:23:05Z<p>Nkruger: </p>
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<h1><br />
Modelling</h1><br />
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In order to model the chemotactic motion of the engineered bacteria, a simulation program was designed and produced. The simulation models the motion of a population of bacteria in a small environment composed of two chemoattractants. This page provides an overview of the simulator followed by an in-depth discussion of the mathematical model used [1,2]. The inputs to the model and the possible outputs are highlighted. An analysis of the assumptions made in development, and resulting shortcomings of the tool is also conducted.<br />
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<p><br />
The simulation tool, as shown in the alongside figure, models the chemotactic motion of a population of bacteria in a small environment (a petri dish). As in the project setup, two chemoattractants (shown in red and green) are present in the environment and influence the motile action of the bacteria. The representative bacteria take the colour of the chemical towards which they are attracted, and move about the environment according to a movement algorithm. Various options are available to the user of the tool to specify the experiment setup (see later) and the panel on the left allows for this.<br />
</p><br />
<p>The tool may be viewed <b><a href="http://www.bioinf.wits.ac.za/igem/WebPlayer.html">at this link</a></b>. It is necessary to install the <b><a href="http://unity3d.com/webplayer/">Unity Web Player</a></b> to run the application.</p><br />
<br />
<h2>Development Tools Used</h2><br />
<p><br />
The simulator was developed in the C# language and utilises the Unity3D graphics engine. This engine was chosen as a development tool above other platforms such as MATLAB or Java for a variety of reasons. Firstly, the engine is free to use and supports different development environments including easy web deployment. Secondly, the ease of implementation allowed the developer to prototype and deploy iteratively for the team. Functionality such as graphical user interface integration was provided and allowed more time to be dedicated to the modelling of the bacterial motility.<br />
</p><br />
<h2>The Movement Algorithm</h2><br />
<p><br />
Chemotaxis in E. coli bacteria is achieved by the altering of a signalling pathway to the bacterial flagellar motors [1]. The flagella of the bacterium are capable of both clockwise and counter-clockwise motion. The former leads to a reorientation of the bacterium: a tumble [1,2]. The latter leads to a fairly straight propulsion of the bacterium through the medium: a run [1,2]. By altering the frequency of tumbles and runs, the bacterium effectively undergoes selective locomotion towards more favourable regions. This so-called biased random walk [2] is simulated in the program with a two-state model.<br />
</p><br />
<p><br />
Effectively, each bacterium is associated with the particular chemoattractant that dictates its motion (specified by the functioning of the riboswitch). The diffusion of this chemical is simulated by the inverse square law; the point concentration decreases with time, whilst the spread (or radius) of the chemical increases. The first state of the model occurs when a bacterium is outside this chemical radius. In this instance, a bacterium’s movement is dominated by tumbles. The second instance is when the bacterium lies within the chemical concentration gradient. In this case, the frequency of runs is higher and dictated by the distance between the bacterium and the chemical point-source. Again, an inverse square relationship was used.<br />
</p><br />
<h2>Inputs and Outputs</h2><br />
<p><br />
The simulator allows users to specify the experimental setup. Alterable variables include:<br />
<ul><br />
<li>The number of bacteria to simulate</li><br />
<li>The bacterial speed</li><br />
<li>The bacterial sensitivity to the chemoattractants</li><br />
<li>The initial concentration of attractants</li><br />
<li>The diffusion rate constant of the attractants</li><br />
<li>The distribution grid resolution and size (see below)</li><br />
</ul><br />
<p><br />
The simulation program models the bacterial motion and creates useful measurements of the scenario. These include:<br />
</p><br />
<ul><br />
<li>A discreet probability density function of bacterial location (as in the figure below)</li><br />
<li>The maximum and average times of flight it took a bacterium to reach each attractant</li><br />
</ul><br />
<br />
<h2>Verification</h2><br />
<br />
<p>In order to verify the model, it was necessary to determine the extent to which the directed chemotaxis varied the bacteria’s motion from that of random movement. To this end, the number of bacteria in the toggled state (after coming into contact with the chemoattractant) over time, as a function of the initial chemical concentration was determined. A number of runs of the simulation tool under the same initial chemical concentration were performed. The simulator output the number of toggled bacteria sampled every 2 seconds. These data were then averaged to obtain a statistical mean of the particular experiment. This process was repeated over a range of concentration values, and plotted as below:</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/4/4a/ToggledBacteria.png" width = "550" height = "400" alt = ""/><br />
<br />
<p>As may be seen from the above figure, the initial chemical concentration does indeed have an effect on the rate at which bacteria successfully navigate to the attractant. The chosen arbitrary reference concentration sees an almost 10 fold increase of the number of bacteria in the toggled state over the chosen 2 minutes of simulation time. Even at half of the reference concentration, a six fold increase over pure random motion was obtained. Concentration increases above 10 times the reference concentration had increasingly minimal impact on the chemotaxis of the bacteria. This is due to the overall underlying stochastic nature of the movement and physical distance between where the bacteria are initially located and the chemoattractant.</p><br />
<br />
<h2>Collaboration</h2><br />
<p><br />
The modelling software was discussed with the Imperial College team. Specifically, the use of a non steady-state model was discussed and ultimately decided upon due to the team's implementation of the environment. A non steady-state model is used since the chemoattractant is not replenished with time, and so will diffuse uniformly over the entire environment.<br />
</p><br />
<br />
<br />
<h2>Shortcomings</h2><br />
<p><br />
The two-state model used to simulate the bacterial motion neglects the half-life and phosphorylation times of the signalling pathways. The three-state model of [2] could be used to provide a more accurate model. However, it is assumed that these times are negligible when compared to the time of flight of the bacteria. Larger chemical networks consisting of three or four attractants (or the inclusion of repellents) are also not catered for. As of yet, the variables in the simulator are also unitless. More detailed investigation into the underlying model is needed to fully characterise them.<br />
</p></div><br />
<br/><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'References'"><br />
<br />
<p>[1] P. Spiro, J. Parkinson, H., Othmer, A model of excitation and adaptation in bacterial chemotaxis, Proceedings of the Nation Academy of Sciences, United States of America, Biochemistry, Vol 94, pp. 7263 – 7268, July 1997.</p><br />
<p>[2] C. Rao, J. Kirby, A. Arkin, Design and Diversity in Bacterial Chemotaxis: A Comparative Study in Escherichia coli and Bacillus subtilis, PloS Biology, Issue 2, Vol 2, pp. 239 – 252, February 2004.</p><br />
</div><br />
<br/><br />
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<p>Collaboration was also performed between the engineers to improve the modeling for both the teams. Several Skype conferences were held between the team from Imperial College London and the Wits CSIR team to discuss the design and theory behind our own chemotaxis models. The collaborative effort resulted in the Imperial College London kindly providing us with a model for our theophylline riboswitches. Their modelling results are as follows:</p><br />
<p>The Wits CSIR iGEM team intend to use a riboswitch to reprogram the chemotactic behavior of <i>E.coli</i>. The project includes engineering the attraction of the bacteria to theophylline[1]. CheZ is an important protein controlling the chemotaxis of bacteria. They used a theophylline riboswitch to control the expression of CheZ in CheZ deletion mutants in order to engineer the bacteria's movement towards theophylline[1]. We're using a riboswitch sensitive to theophylline to control the expression of CheZ. In the absence of theophylline, the start codon is covered so the translation of the strand cannot occur. In the presence of theophylline, the conformation of the riboswitch changes and the ribosome binding site is exposed[1]. Thus, the higher the concentration of the theophylline, the more will enter the cell resulting in the up regulation of CheZ expression. This will increase the frequency of directed movement[1].</p> <br />
<p>The theophylline riboswitch can be modeled in three differential equations [2].</p><br />
<p><center>M ?= a?(T)-?M</center></p><br />
<p><center>(CheZ) ?= ßMT(P)-?CheZ</center></p><br />
<p><center>T ?= ?(P_ext )CheZ-dT</center></p><br />
<p>M, CheZ and T respectively stands for the concentration of CheZ mRNA, the concentration of protein CheZ and the concentration of theophylline. The constants a,ß,?,? and d are all positive, and respectively denote the CheZ-promoter transcription rate, the CheZ-mRNA translation rate and the mRNA, CheZ and theophylline degradation-plus-dilution-rates. ?(Text) is the theophylline transport rate per unit CheZ concentration. It is a function of number of theophylline receptors and external theophylline concentration. The function ?(T) and T(T) denote the theophylline-governed regulation ay the transcriptional and translation levels respectively (equation below [2]). KF is the equilibrium constant at transcriptional level and KT is the equilibrium constant at translation level.</p><br />
<p><center>F(T)= K_F/(K_F+T)</center></p><br />
<p><center>T(T)= K_T/(K_T+T)</center></p><br />
<p>Varying the parameter ?(Text) of above model could help us to understand how the number of receptors and external theophylline concentration effect the intracellular concentration of theothyline and hence the expression level of CheZ. The results are shown in Fig 1 and Fig 2 below. In addition, the response curve of CheZ against theophylline concentration with different theophylline transport rate was illustrated in Fig 3.</p><br />
<center><a href="https://static.igem.org/mediawiki/2011/c/c6/Wits_collaboration_Figure_1.png"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Collaboration',title:'Figure 1',href:'https://static.igem.org/mediawiki/2011/c/c6/Wits_collaboration_Figure_1.png'"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c6/Wits_collaboration_Figure_1.png"<br />
alt="Collaboration fig 1" height="470" /></a></center> <br />
<center><a href="https://static.igem.org/mediawiki/2011/6/6a/Wits_collaboration_Figure_2.png"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Collaboration',title:'Figure 2',href:'https://static.igem.org/mediawiki/2011/6/6a/Wits_collaboration_Figure_2.png'"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6a/Wits_collaboration_Figure_2.png"<br />
alt="Collaboration fig 2" height="470" /></a></center> <br />
<center><a href="https://static.igem.org/mediawiki/2011/6/6f/Wits_COllaboration_Figure_3.png"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Collaboration',title:'Figure 3',href:'https://static.igem.org/mediawiki/2011/6/6f/Wits_COllaboration_Figure_3.png'"><br />
<img src="https://static.igem.org/mediawiki/2011/6/6f/Wits_COllaboration_Figure_3.png"<br />
alt="Collaboration fig 3" height="470" /></a></center> <br />
<p>Both the expression of CheZ and intracellular concentration increases with increase of transport rate. The bacteria will produce more CheZ and therefore higher level of directed movement. The CheZ response curve shows that the transport rate can be used to tune the CheZ response, as the threshold intracellular concentration of theophylline required to trigger the CheZ response increases with transport rate.</p><br />
<p><center>Parameters:</center></p><br />
<center><img src="https://static.igem.org/mediawiki/2011/6/61/Wits_collaboration_Collab_table.jpg"></center> <br />
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<p>[2] C. Rao, J. Kirby, A. Arkin, Design and Diversity in Bacterial Chemotaxis: A Comparative Study in Escherichia coli and Bacillus subtilis, PloS Biology, Issue 2, Vol 2, pp. 239 – 252, February 2004.</p><br />
<p>[1] P. Spiro, J. Parkinson, H., Othmer, A model of excitation and adaptation in bacterial chemotaxis, Proceedings of the Nation Academy of Sciences, United States of America, Biochemistry, Vol 94, pp. 7263 – 7268, July 1997.</p><br />
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<h1><br />
Characterization of Parts</h1><br />
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<p>In order to characterize our final machines, we performed the following assays:</p><br />
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<a href="#Fluorometry">Fluorometry</a><br />
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<a href="#Microscopy1">Fluorescence microscopy: Liquid Culture</a><br />
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<a href="#Microscopy2">Fluorescence microscopy: Smeared Culture</a><br />
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<a href="#Motility">Motility</a><br />
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<a href="#Chemotaxis">Chemotaxis</a><br />
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<p id="Fluorometry"><br />
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<h2>Theophylline Riboswitches</h2><br />
<p>To characterise the theophylline riboswitches (1 and 2), we quantified their activation at different theophylline concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM and 2 mM) over a period of time using fluorometry. Competent <i>E. coli</i> (strain DH5a) cells were transformed with plasmid vectors containing the riboswitch and were cultured until the mid-log phase of growth. Thereafter, a different concentration of theophylline was added to each culture for induction. The activation of the riboswitch was detected as a fluorescent response as a result of increased translation of the fluorescent fusion protein CheZ-Venus, in the presence of the activator. A Jasco FP-6300 spectrofluorometer was used to excite the cultures at 514 nm and the intensity of the emission peak was detected at 528 nm. Twenty readings were taken every 15 minutes for each culture, for a total period of 135 minutes. An empty vector was used to correct for autofluorescence. Conversely, a construct that comprised of a Promoter-RBS-CheZ-Venus-double terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>) was used as a positive control. Fluorescent data for the 3D plots (Fig 1-4) were corrected for autofluorescence by subtracting the fluorescent values of the cultures transformed with the empty vector, from that of the riboswitches.<br />
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<td><p> <b>Figure 1:</b> The fluorescence of the CheZ-Venus fusion protein under the regulation of theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). <b>A)</b> The activation of the riboswitch over time at different concentrations of theophylline. The fluorescence increased over time, with an increasing concentration of theophylline, indicating the theophylline concentration-dependent expression of the CheZ-Venus fluorescent fusion protein. The fluorescence peaks 105 minutes after the addition of 1 mM theophylline. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 1 mM theophylline after 105 minutes incubation, and to that of baseline levels (0 minutes with 1 mM theophylline). 1 mM of theophylline induced the expression of CheZ-Venus to increase 4-fold after 105 minutes (fold change was calculated by dividing the fluorescence observed at 105 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values) (Click to enlarge)<br />
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<b>Figure 2:</b> The fluorescence of the CheZ-Venus fusion protein under the regulation of theophylline riboswitch 2 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>). <b>A)</b> The activation of the riboswitch over time at different concentrations of theophylline. The same trend of increased switch activation over time, with increasing theophylline concentrations was seen as with theophylline riboswitch 1. However, with the theophylline riboswitch 2, the fluorescence peaks 135 minutes after the addition of 1.5 mM theophylline. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 1.5 mM theophylline after 135 minutes incubation, and to that of baseline levels (0 minutes with 1.5 mM theophylline). 1.5 mM of theophylline induced a 37-fold increase in CheZ-Venus expression after 135 minutes (fold change was calculated by dividing the fluorescence observed at 135 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values). (Click to enlarge)</p> </td><br />
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data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Characterization',title:'Figure 3B',href:'https://static.igem.org/mediawiki/2011/d/d2/Flourometry_RS1_V_B.jpg'"><br />
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<td><p> <b>Figure 3:</b> The fluorescence of the Venus reporter protein under the regulation of theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>). <b>A)</b> The fluorescent peak occurred prematurely after 75 minutes of incubation with 0.5 mM theophylline, compared to riboswitch 1, which contained the gene for the CheZ-Venus fusion protein. The maximum fluorescent intensity observed was also lower by a factor of 2 RFU. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 75 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 4.5-fold increase in protein expression was observed as the maximum (fold change was calculated by dividing the fluorescence observed at 75 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values). This fold change is comparable to that described in <b>Figure 1</b>. (Click to enlarge)<br />
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<td><p> <b>Figure 4:</b> The fluorescence of the Venus reporter protein under the regulation of the theophylline riboswitch 2 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>). <b>A)</b> The activation of the riboswitch was affected by the lack of the CheZ gene in the switch construct. The increase in fluorescence was minimal upon induction and incubation over time. Furthermore, the fluorescent peak occurred prematurely after 60 minutes of incubation with 0.5 mM theophylline, when compared to the theophylline riboswitch 2 that contained the gene for the CheZ-Venus fusion protein. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 60 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 2-fold increase in protein expression was observed as the maximum (fold change was calculated by dividing the fluorescence observed at 60 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values) . This is far less than the value described for the same switch in <b>Figure 2</b>.(Click to enlarge)<br />
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Both the theophylline riboswitches displayed activation in response to increasing theophylline concentrations and incubation times. Theophylline riboswitch 2 controlling CheZ-Venus expression (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>) clearly showed a superior level of activation compared to that theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). This was expected as it was selected by FACS for its optimised expression platform that allows for minimal leakiness at basal levels, and a strong activation in the presence of theophylline (Lynch and Gallivan, 2009). The activation shown by both the riboswitches regulating CheZ-Venus expression should be able to restore the motility of <i>E. coli</i> cells deficient in CheZ, in the presence of theophylline. More importantly, the theophylline concentration-dependent increase in CheZ-Venus expression alludes to the ability of directed cell movement, that is moving up concentration gradients of theophylline (Topp and Gallivan, 2007). Interestingly, the data suggests that the presence of the CheZ gene, as part of the CheZ-Venus fusion sequence, enhances the activation of the switches when compared to the constructs with venus alone. This may be due to some sort of structural stability conferred by the presence of the CheZ sequence.</P><br />
<h2> Atrazine Riboswitch</h2><br />
<p>Preliminary activation data was obtained for the atrazine riboswitch.Fluorometry was performed on <i>E. coli</i> (DH5a) cells transformed with a construct that contained a Promoter-atrazine riboswitch-mRFP-double terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a>). Similar to the theophylline riboswitches, different concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) of atrazine were added to the cultures for induction. The cultures were excited at 584 nm and detected at 607 nm using a Jasco FP-6300 spectrofluorometer. Readings were taken every 15 minutes for 120 minutes in total. The same empty vector was used as a negative control. The 3D plot data was normalised the same way as for the theophylline riboswitches.</p><br />
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data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Characterization',title:'Figure 5B',href:'https://static.igem.org/mediawiki/2011/e/ec/Flourometry_atRS_R_B.jpg'"><br />
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<td><p> <b>Figure 5:</b> The fluorescence of the mRFP1 reporter protein under the regulation of the atrazine riboswitch(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a>). <b>A)</b> The activation of the atrazine riboswitch at different atrazine concentrations over time. There was an increase in fluorescence as the atrazine concentration and incubation time increased. The amount of fluorescence detected was very small, with a maximum of 0.1 RFU detected after 75 minutes of incubation with 2 mM atrazine. <b>B)</b> The maximum was compared to the negative control at 75 minutes, 2 mM atrazine, and to that of fluorescent levels detected at basal levels (0 minutes, 2 mM atrazine). A 1.7-fold increase in expression of mRFP1 was observed over the 75 minute period (values used for the calculation had the corresponding negative control value subtracted first).(Click to enlarge)<br />
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<h2>References</h2><br />
<p>Lynch, S. A. & Gallivan, J. P. 2009. A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Res, 37, 184-92.</p><br />
<p>Topp, S. & Gallivan, J. P. 2007. Guiding bacteria with small molecules and RNA. J Am Chem Soc, 129, 6807-11.</p><br />
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<p id="Microscopy1"><br />
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<h2>Epi-fluorescence Wide-Field Microscopy</h2><br />
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<p> Fluorescent imaging was performed for a qualitative assay of the engineered bacteria. <br />
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<p> Bacterial culture and Manipulation: <br />
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<p> 4 ml of LB broth with Ampicillin was inoculated from colonies off a master plate of positive clones and grown overnight at 37°C in a shaking incubator. 2 ml of each bacterial culture was then used to inoculate 25 ml of LB broth with Ampicillin and grown for ± 3 hours in a shaking incubator at 37°C until the cells reached an OD600 of 0.55 (mid-log phase). 2 x 5 ml of each sample culture was aliquoted into separate 15 ml falcon tubes. One tube was left untreated; 150 µl of 15 mM theophylline was added to the other (to make a final concentration of 1.5mM theophylline). These 5 ml aliquots were placed in a shaking incubator at 37°C for 30 min each to allow for activation. 200 µl of each treated and untreated sample was then used in a microscope plate for imaging. Imaging was done using Zeiss Axioscope (Andor EMCCD Camera, Till Photonics Polychrome V). <br />
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<p> Widefield fluorescence microscopy was used to assess the expression of the Venus protein. The bacterial samples were excited at 500 nm using the polychrome as an excitation source. The fluorescence was then observed by capturing light in the emission spectrum of Venus (528 nm). The figure below represents a schematic of the imaging setup. Corresponding bright-field images (using white light) were also captured for each experiment. <br />
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<p>Below are the images that were obtained for the parental strain (BW25113) of the <i>E. coli</i> CheZ-deletion mutant, which served as the negative, and the CheZ deletion mutants transformed with Promoter-theophylline riboswitch 1-venus (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) and Promoter-theophylline riboswitch 2-venus (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>).<br />
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<p>Please note: Bacteria which were transformed with constructs which contained the CheZ gene were not tested in this assay. This experiment served as a means of observing the activation of the theophylline riboswitches, hence only expression of the venus reporter protein was needed for detection of fluorescence. Activation of motility was not assessed in this case – it was examined in the motility assays on semi-solid agar plates. Differences in fluorescence intensities were not calculated from the images below since the fluorometry experiments served as the quantitative approach to measurement of riboswitch activation.<br />
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<b>Figure 6:</b> The parental strain (BW25113) of the <i>E. coli</i> CheZ deletion mutants were not transformed with any plasmid DNA. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In both the absence and presence of theophylline, the parental bacteria did not fluoresce – which was the expected result. (Click to enlarge)</p> </td><br />
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<b>Figure 7:</b> <i>E. coli</i> CheZ-deletion mutants which were transformed with the Promoter-theophylline riboswitch 1-venus (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) construct. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, almost no fluorescence occurred. Upon the addition of theophylline at a concentration of 1.5 mM, many of the cells emitted fluorescence showing activation of the theophylline riboswitch type 1.(Click to enlarge)</p> </td><br />
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<b>Figure 8:</b> <i>E.coli</i> CheZ-deletion mutants which were transformed with the Promoter-theophylline riboswitch 2-venus (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>) construct. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, some fluorescence was observed. This result showed the leakiness of the riboswitch. A substantial amount of venus translation is permitted because of the flexible conformation of this riboswitch. Upon the addition of theophylline at a concentration of 1.5 mM, much more fluorescence was detected. More venus was expressed in these cells due to the activation of the riboswitch via theophylline.(Click to enlarge)</p> </td><br />
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<p> Fluorescent imaging was performed again after the European Jamboree, using a different technique. The aim of this data is to depict the exact same brightfield and fluorescent static fields of view. <br />
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<p> Semi-solid agar plates containing different concentrations of theophylline (0mM, 0.5mM, 1.0mM and 1.5mM) were made. Promoter-theophylline riboswitch 1-CheZ-Venus-double terminator (thRS1-Ch-V)(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>), Promoter-theophylline riboswitch 2-CheZ-Venus-double terminator (thRS2-Ch-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>), Promoter-theophylline riboswitch 1-Venus-double terminator (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) and Promoter-theophylline riboswitch 2-Venus-double terminator (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>) were transformed into CheZ deletion mutants and grown to mid-log phase (OD600 = 0.55). 1.5 µl of culture was plated into the centre of the plates containing the various concentrations of theophylline and incubated at 37°C for 24 hours. This experiment was performed in triplicate. The protocol for this experiment can be found here.<br />
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<b>Figure 9:</b> The distance travelled by CheZ deletion mutants (mm) over a 24 hour period at 37°C on semi-solid agar plates containing different theophylline concentrations (mM)(Click to enlarge)</p> </td><br />
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The thRS1-V and thRS2-V transformants did not travel from the point of inoculation for all concentrations of theophylline. The CheZ deletion mutants containing thRS1-Ch-V travelled 0 mm at 0 mM theophylline and reached a maximum travelling distance of 5 mm at 1.5 mM theophylline. The thRS2-Ch-V containing CheZ deletion mutants travelled 2 mm in the absence of theophylline and at a concentration of 1.5 mM theophylline, were able to travel 22mm from the point of inoculation </p><br />
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<b>Figure 10:</b> 85 mm semi-solid agar plates containing 1.0 mM theophylline showing the point of inoculation and distance travelled by the bacteria. <b>A)</b> Inoculation of 1.5 µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS1-Ch-V(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). There is evidence of movement of bacteria from the point of inoculation. This can be observed as a faint halo surrounding the point of inoculation.<b>B)</b> Inoculation of 1.5 µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS1-V. Converse to <b>A</b>, there is no evidence of movement of the bacteria from the point of inoculation <b>C)</b> Inoculation of 1.5µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS2-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>). There is evidence of movement of the bacteria from the point of inoculation. This can be observed as a faint halo surrounding the point of inoculation. <b>D)</b> Inoculation of 1.5µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS2-V. Converse to <b>C</b>, there is no evidence of movement of bacteria from the point of inoculation (Click to enlarge)</p> </td><br />
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<h3> Discussion and conclusion</h3><br />
<p>The construct thRS1-Ch-V transformed into the CheZ deletion mutants are non-motile at a concentration of 0 mM theophylline, since in the absence of the activator, the riboswitch is sequestering the RBS and thus inhibiting the translation of CheZ (Topps and Gallivan, 2007). On the semi-solid agar plates containing theophylline, the motility of E.coli is evident, as the interaction of theophylline with the riboswitch enables the exposure of the RBS and the translation of the downstream CheZ, allowing for bacterial motility. The distance travelled by the bacteria transformed with the thRS1-Ch-V construct, from the start position (mm) in the presence of different concentrations of theophylline, is not significantly different (P=0.005). This indicates that the CheZ expression at 0.5 mM-1.5 mM is sufficient to allow the bacteria to move a distance of 5 mm. The construct thRS2-Ch-V transformed into the CheZ deletion mutants are slightly motile in the absence of theophylline, travelling 2mm over a 24 hour period indicating that this riboswitch is slightly leaky. Upon addition of 0.5 mM-1.5 mM theophylline, the bacteria were able to travel 20mm over a 24 hour period.</p><br />
<p>The distance travelled from the start position by the CheZ deletion mutants containing the thRS2-Ch-V is significantly higher than that those with the thRS1-Ch-V construct. This can be explained by the high fold-increase in CheZ protein expression by this riboswitch, as shown by fluorometry. Theophylline riboswitch 1 has a lower fold increase in CheZ expression from 0 mM to 1.5 mM theophylline, compared to the increase shown by the theophylline riboswitch 2. Therefore, in order to achieve maximum motility under the control of theophylline, at concentrations of between 0.5 mM and 1.5 mM, theophylline riboswitch 2 should be used.</p><br />
<h2>References</h3><br />
Topp, S. & Gallivan, J. P. 2007.Guiding bacteria with small molecules and RNA. J Am ChemSoc, 129, 6807-11 <br />
<p>Both the t1-V and t2-V constructs transformed into the CheZ deletion mutants are non-motile, each having travelled a distance of 0mm at all concentrations of theophylline (See A and B). This indicates that in the absence of the CheZ protein, the bacteria are non-motile (Topps and Gallivan, 2007).</p><br />
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<p id="Chemotaxis"><br />
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<p>In order to determine whether we had successfully engineered the <i>E. coli</i> CheZ deletion mutants to chemotactically migrate towards theophylline, we performed a capillary assay. Bacteria transformed with thRS1-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>), thRS2-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>), RBS-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>), thRS1-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>) and thRS2-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>) were grown to mid-log phase. Activation of chemotaxis was achieved by incubating the cultures with 1 mM theophylline prior to the assay. A 50 µl drop of each culture was then placed onto separate sterile surfaces and two capillary tubes were placed into each drop. The first capillary tube was a control, containing only PBS and 20 µM EDTA. The second tube contained PBS, 20 µM and 2mM theophylline. The two capillary tubes were suspended into each drop of culture for 30 minutes. A dilution series of the solutions in the tubes were plated and colonies counted after 12 hours. Further details of this protocol can be found here.<br />
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<h3>Discussion and conclusion</h3><br />
<p>Cells transformed with thRS1-V, thRS2-V and RBS-Ch-V all displayed an index of approximately 100, indicating the same number of bacteria moved towards the control (0mM theophylline) and the experiment (2mM theophylline) capillary tube. This proves that none of these constructs induce chemotactic behaviour. In terms of both thRS1-V and thRS2-V, the lack of CheZ results in the inability of CheY dephosphorylation and the swimming motion observed in chemotaxis, which is a key driver in directed movement towards an attractant (Topps and Gallivan, 2007). Although the RBS-Ch-V construct constitutively expresses CheZ, and bacterial swimming is achieved through CheY dephosphorylation, the expression levels remain constant regardless of the presence or absence of theophylline. Random movement will occur as a result.<p/><br />
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<p>The thRS1-Ch-V and thRS2-Ch-V transformants, with chemotaxis indices of 180 and 227 respectively, both indicated a significantly higher number of bacteria that travelled to the 2mM theophylline solution in the capillary tube compared to that of the control. The chemotaxis index of the thRS2-Ch-V construct is significantly higher (p=0.005) than that of thRS1-Ch-V, indicating that cells transformed with thRS2-Ch-V is more attracted to theophylline than thRS1-Ch-V. Should maximum chemotaxis towards theophylline be desired, thRS2-Ch-V would be the more suitable construct.</p><br />
<h2>References</h2><br />
Topp, S. & Gallivan, J. P. 2007. Guiding bacteria with small molecules and RNA. J Am ChemSoc, 129, 6807-11<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Project/ConceptTeam:WITS-CSIR SA/Project/Concept2011-10-22T19:53:34Z<p>Nkruger: </p>
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<h1>Overview</h1><br />
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<p>The WITS-CSIR_SA iGEM 2011 team decided to create a microscopic biological communication network, in which there will be the transfer of data between bacterial populations as chemical signals. This type of system has been dubbed “Biotweet”. The generic framework of “Biotweet” may serve as the basis for the functioning of complex biological communication networks and may be useful in various applications, including those of the medical and industrial sectors.</p><br />
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<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Biotweet: A framework for bacterial networks using programmed bacterial motility '"><br />
<p>Any network requires the establishment of connections between nodes of the network, allowing for the transport of node-to-node data packets. With “Biotweet”, we have attempted to construct such a network within a biological network: a challenging task, considering that bacterial cells cannot be linked by wires. Analogous to the transfer of data as electronic packets in a computer network, the concept of engineering bacteria to transport packets of chemical signals between bacterial populations arose. In this way, the bacteria themselves can form the network connections. In order to do this, we decided to reprogram the motility of bacteria, such that each bacterium travels in a stimulus-directed fashion (eg. chemotactic response) to specific points of the network (the nodes), where they can send and receive signals. Such a stimulus should be unique to the bacterium and not be part of the natural environment. With these specifications in mind, we needed a way of manipulating the chemotactic behaviour of bacteria that would allow for us to easily adapt the bacterial chemotactic response to the stimuli associated with any application. Specifically, the chemotactic response in E.coli lends itself to easy manipulation. The activation of CheZ allows for counter-clockwise (CCW) flagellar activity resulting in a “run and tumble” movement. By linking CheZ expression to post-transcriptional activation by a specific anayte (using for example a analyte-specific riboswitch), bacterial motility can be guided and controlled (Fig.1). For more on bacterial chemotaxis,<a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Motility">see here</a>. </p><br />
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<p><b>Figure 1.</b>The reprogramming of bacterial chemotactic response by using post-transcriptional activation of CheZ using a analyte-responsive riboswitch. CheZ expression results in counter-clockwise flagellar rotation and a “run and tumble” movement. (Click to enlarge)<br />
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<p>Firstly, we have chosen to use artificial riboswitches to control the expression of the flagella rotation regulator protein CheZ. To do this, we borrowed heavily from studies which have described riboswitch-controlled bacterial chemotaxis(Topps and Gallivan. 2007).Riboswitches are ligand-inducible RNA protein expression regulators that are comprised of an aptamer domain and an expression platform (Gallivan, 2007). The aptamer is a sequence of nucleotides that is designed to specifically bind to ligands, while the expression platform consists of a ribosome binding site (RBS) and a downstream coding region (CDS). When the specific ligand or analyte binds to the aptamer domain, the riboswitch undergoes a structural change. This results in the exposure of the RBS and the expression of the downstream CDS (Fig.2). Using this riboswitch mechanism, the expression of CheZ can be regulated in a ligand concentration-dependent manner, outside the control of the natural chemotaxis pathway (Fig2, bottom panel and right). Topps and Gallivan (2007) demonstrated that these riboswitches can be used to reprogram the chemotactic response of bacteria, so that they move up a concentration gradient, towards the source of the stimulus that activates the riboswitch (a process known aspseudotaxis). Bacteria can theoretically be reprogrammed to respond to any stimulus, if the appropriate riboswitch is made. This provides the versatility needed for biological network connections to be established in any application. Furthermore, this eliminates the need to engineer novel chemoreceptors.</p><br />
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<p><b>Figure 2. </b>Using ligand-responsive riboswitch activation of CheZ expression. CheZ can be fused to a reporter and direct bacterial motility in a ligand-dependent manner up a concentration gradient.(Click to enlarge) </p><br />
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<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Designing a “send” and “receive” network using riboswitch-controlled bacterial motility'"><br />
<p>The aim was to reprogram E. coli (which lack natural CheZ functionality) with riboswitch-activated CheZ. This enables bacteria to search, localise to the riboswitch activator, and return to a set location in order to report. This, in principle, will show that bacteria can be programmed to move in a directed manner towards a new stimulus and establish a connection between two points in space. The generic design for this is depicted below. The “send” module is essentially a riboswitch-activated CheZresponsive to analyte A, and the “receive” module is a riboswitch-activated CheZ responsive to analyte B. We have decided to include two fluorescent reporters as fusion proteins since both N- and C-terminal CheZ fusions are well-tolerated (Gallivan, 2007). To ensure that the “send” and “receive” modules act independently, Analyte/repressor B can be in a biphasic toggle switch with repressor A (analyte A). Therefore A, which represses Cre-recombinase, flips the toggle at a critical concentration. Cre recombination allows for irreversible switching of constitutive promoter activity (by lox site recombination) and convert motility from “send” to “receive” such that bacterial motility is now dictated by a concentration gradient of analyte B.</p><br />
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<center><a href="https://static.igem.org/mediawiki/2011/b/bc/Send_and_recieve_module.jpg"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Overview',title:'Figure 3',href:'https://static.igem.org/mediawiki/2011/b/bc/Send_and_recieve_module.jpg'"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bc/Send_and_recieve_module.jpg"<br />
alt="Flowchart" width="400" /></a></center><br />
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<p><b>Figure 3.</b>“send” and “receive” riboswitch-controlled motility module. Featured is also an Analyte B-responsive toggle switch which activates Crerecombinase expression. Cre, in turn, causes “receive” motility activationby flipping constitutively active promoter, and allowing motility to be controlled by Analyte A. (Click to enlarge)</p><br />
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<p>Unfortunately, other than Tetracycline (Dox) riboswitches for use in yeast, at present, there isn’t an engineeredriboswitch which is sensitive to a known transcriptional repressor. Therefore, we designed system which allows the “receive” function (Crerecombinase) to be activated by IPTG (lacI). Also, we focused this design on the use of two known synthetic riboswitches – against theophylline and atrazine (see riboswitch design considerations below).</p><br />
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<center><a href="https://static.igem.org/mediawiki/2011/2/2b/Wits_Overview_Page_Large_Flowchart_Figure_4.png"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Overview',title:'Figure 4',href:'https://static.igem.org/mediawiki/2011/2/2b/Wits_Overview_Page_Large_Flowchart_Figure_4.png'"><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Wits_Overview_Page_Large_Flowchart_Figure_4.png"<br />
alt="Flowchart" width="400" /></a></center><br />
<br />
<p><b>Figure 4.</b> “send” and “receive” riboswitch-controlled motility module. In this design, Cre-recombinase is activated by IPTG which is present only at the theophylline position (not present in the theophylline gradient - green). As with Fig.3, Cre, in turn, causes “receive” motility activation by flipping constitutively active promoter, and allowing motility to be controlled by an Atrazine gradient (red). (Click to enlarge)</p><br />
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<p>Our initial aim was to develop a theophylline-sensitive riboswitch which is capable of regulating CheZ (motility factor) translation. Several theophylline-sensitive riboswitches have been developed, most notably by the Gallivan lab (Emory, USA).</p><br />
<p><br />
To summarize, using a high-throughput screen(Lynch et al., 2007) and a high-throughput selection (Topp and Gallivan, 2008) in E.coli, the Gallivan lab has been able to develop a powerful theophylline-dependent riboswitch. It is worth noting that the motility-based screen(Topp and Gallivan, 2008) made use of a theophylline riboswitch library activating the expression of CheZ, the motility factor in E.coli which allows for running (as opposed to tumbling) movement. Therefore, activation of CheZ allows bacteria to move along the theophylline gradient. It is worth mentioning that the best riboswitch identified in these earlier screens was the theophylline riboswitch clone 8.1 (see figure bellow), which displays 36-fold expression activation ratio (Fig.5).</p><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/4/4d/Wits_Overview_Riboswitch_Considerations_1.jpg"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Overview',title:'Figure 4',href:'https://static.igem.org/mediawiki/2011/4/4d/Wits_Overview_Riboswitch_Considerations_1.jpg'"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4d/Wits_Overview_Riboswitch_Considerations_1.jpg"<br />
alt="riboswitch" width="400" /></a></center><br />
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<p><b>Figure 5. </b>Theophylline responsive riboswitch (clone 8.1). We have referred to this riboswitch as theophylline riboswitch type 1 (thRS1). (Click to enlarge)</p><br />
<p>In order to improve on their earlier riboswitches, Topp and Gallivan developed an in vivo-based selection strategy aimed at selecting bacteria based on fluorescence activated cell sorting (FACS) (Wieland et al., 2008; Lynch et al., 2009) (Fig.6). By randomizing the sequence encoding the RBS (+ Shine Delgarno/SD), and by sorting a N12 library, clone 12.1 was identified, which displays a 96-fold expression activation in the presence of theophylline (1 mM) (see figure below). The authors of this study suggest this is the largest activation ratio to date, for synthetic or natural riboswitches. It is worth noting that this riboswitch contains a strong (and longer) UAAGG SD which is found in a tight (inaccessible) duplex in the “OFF” (ie no theophylline) state. Moreover, the UAAGG sequence is spaced optimally, with 6 nt separating the 5’ A of the anti-SD sequence and the start codon of the translation (AUG) (Fig.6).</p><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/e/eb/Wits_Overview_Riboswitch_Considerations_2.jpg"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Overview',title:'Figure 4',href:'https://static.igem.org/mediawiki/2011/e/eb/Wits_Overview_Riboswitch_Considerations_2.jpg'"><br />
<img src="https://static.igem.org/mediawiki/2011/e/eb/Wits_Overview_Riboswitch_Considerations_2.jpg"<br />
alt="riboswitch" width="400" /></a></center><br />
<br />
<p><b>Figure 6.</b>Theophylline responsive riboswitch (clone 12.1). We have referred to this riboswitch as theophylline riboswitch type 2 (thRS2). (Click to enlarge)</p><br />
<p>The atrazine riboswitch was developed recently by Sinha et al (2010). In order to make this riboswitch compatible with biobrick assembly, Pst1 site was removed by creating a G:U wobble base pair in lower stem region (Fig 7). </p><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/f/f7/Wits_Overview_Riboswitch_Considerations_3.jpg"<br />
data-dojo-type="dojox.image.Lightbox" data-dojo-props="group:'Overview',title:'Figure 4',href:'https://static.igem.org/mediawiki/2011/f/f7/Wits_Overview_Riboswitch_Considerations_3.jpg'"><br />
<img src="https://static.igem.org/mediawiki/2011/f/f7/Wits_Overview_Riboswitch_Considerations_3.jpg"<br />
alt="riboswitch" width="330" /></a></center><br />
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<p><b>Figure 7. </b>Atrazine responsive riboswitch. We have referred to this riboswitch as atrazine riboswitch (atRS) (Sinha et al., 2010). (Click to enlarge)</p><br />
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<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Problems associated with riboswitches that were designed by past iGEM teams'"><br />
<p>We have identified that several theophylline riboswitches have been previously described by other iGEM teams (For reference, see Lethbridge 2007, Lethbridge 2009 and NYMU Taipei 2010). One of our initial concerns was whether it was possible to isolate the riboswitch from the associated translational unit (coding region). Being able to do so would allow the riboswitch to be defined independently as a BioBrick, with the useful ability to act in a modular fashion for fusion to an adjacent translational unit. All previous teams have made this assumption. We will show that the riboswitch and its adjacent coding region should be considered together and should be cloned as one biobrick, or an alternative "acanonical" assembly standard should be used.</p><br />
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<p>Team NYMU Taipei assumed that a created theophylline riboswitch (Gallivan lab clone 8.1) biobrick could be fused to a GFP coding region by standard biobrick assembly techniques. While it is possible to do this, we feel that the resulting 6 nt scar sequence insertion increases the distance between the RBS and the ATG start codon, a result which is likely to cause a decrease in riboswitch efficiency (see team Taipei switch below, riboswitch highlighted in yellow, inserted biobrick “scar” region highlighted in violet, ATG start underlined). In fact, Taipei 2010 did show data which showed only a 2.5 fold increase in gene activation at approx. 4 mM theophylline. Compared to the 36-fold activation at 1mM theophylline by Gallivan lab, this seems too low! We therefore feel that this riboswitch is not behaving efficiently.</p><br />
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<br />
<img src="https://static.igem.org/mediawiki/2011/6/63/Wits_Overview_Sequence_Yellow.jpg" width="500"><br />
<p><b>Figure 8.</b> <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K411001">BBa_K411001</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K411003">BBa_K411003</a> - Team NYMU-Taipei iGEM2010</p><br />
<p>Team Lethbridge 2009, used the same theophylline riboswitch (clone 8.1) to generate a riboswitch-CheZ construct. However, this team created a 11 nt insert after the ATG start codon. This result created a TAG stop codon (in the 3rd amino acid position) as well as a frameshift mutation. Therefore it is unlikely that a functional CheZ could have been produced from this system. (see team Lethbridge 2009 below, riboswitch highlighted in yellow, inserted biobrick “scar” region highlighted in violet, ATG start underlined) </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/6/6c/Wits_Overview_Sequence_Yellow_2.jpg" width="500"><br />
<p>Fig 9.<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K249028">BBa_K249028</a>, TaemLethbridge iGEM2009. Theophylline riboswitch - scar - CheZ -dT</p><br />
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<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'References'"><br />
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<p>Gallivan JP. Toward reprogramming bacteria with small molecules and RNA.CurrOpinChemBiol 2007;11:612-9</p><br />
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<p>Topp S, Gallivan JP. Guiding bacteria with small molecules and RNA. J Am ChemSoc 2007;129:6807-11.</p><br />
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<p>Lynch S.A. Desai S.K.,Sajja,H.K., and Gallivan J.P.A high-trhoughput screen for synthetic riboswitches reveals mechanistic insight into their function. 2007, ChemBiol 14:173-184</p><br />
<p>Topp S. and Gallivan J.P. Random walks to synthetic riboswitches – a high throughput selection based on cell motility. 2008, ChemBiochem 9:210-213</p><br />
<p>Wieland M., and Hartig, J.S. Improved aptazyme design and in vivo screening enable riboswitches in bacteria. 2008, Agnew.Chem.Iny.Ed.Engl 47:2604-2607</p><br />
<p>Lynch S.A. and Gallivan J.P., A flow cytometry based screen for synthetic riboswitches. 2009, Nucleic Acids Res. 37(1)184-192</p><br />
<p>Sinha J, Reyes SJ, Gallivan JP.Reprogramming bacteria to seek and destroy an herbicide.Nat Chem Biol. 2010 Jun;6(6):464-70. Epub 2010 May 9.</p><br />
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class="center"><br />
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<div style="width: 903px; margin: auto; height: 100%;"><br />
<div data-dojo-type="dijit.layout.BorderContainer" data-dojo-props="design: 'headline'"<br />
style="height: 100%;"><br />
<div data-dojo-type="dijit.layout.ContentPane" data-dojo-props="region: 'center'"><br />
<div style="height: 100%; overflow-y: auto;"><br />
<br />
<h1><br />
Parts Submitted</h1><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Table of parts submitted'"><br />
<p>Click on the part number to go to the associated registry page. Click on the part name for a brief description of the part.</p><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty" width="100%"><br />
<tr><br />
<th style="width: 80px;">Favourites</th><br />
<th style="width: 80px;"><br />
Part Number<br />
</th><br />
<th style="width: 160px;"><br />
Part Name<br />
</th><br />
<th><br />
Part Length<br />
</th><br />
<th>Sent</th><br />
</tr><br />
<tr><br />
<td><br />
<center><img src="https://static.igem.org/mediawiki/2011/1/15/Favourite_image.jpg" width="30px;"></center><br />
<a style="color:Green;" href="https://static.igem.org/mediawiki/2011/2/27/Datasheet1.pdf">Datasheet</a><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537001">BBa_K537001</a><br />
</td><br />
<td width ="200"><br />
<a href="#T1-Ch">Theophylline riboswitch 1 - CheZ</a><br />
</td><br />
<td><br />
698bp<br />
</td><br />
<br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
<center><img src="https://static.igem.org/mediawiki/2011/1/15/Favourite_image.jpg" width="30px;"></center><br />
<a style="color:Green;" href="http://partsregistry.org/wiki/images/f/f5/Datasheet2.pdf">Datasheet</a><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537002">BBa_K537002</a><br />
</td><br />
<td><br />
<a href="#T2-Ch">Theophylline riboswitch 2 - CheZ</a><br />
<br />
</td><br />
<td><br />
702bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537000">BBa_K537000</a><br />
</td><br />
<td><br />
<a href="#AtRS+CheZ">Atrazine riboswitch-CheZ</a><br />
<br />
</td><br />
<td><br />
730bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
<center><img src="https://static.igem.org/mediawiki/2011/1/15/Favourite_image.jpg" width="30px;"></center><br />
<a style="color:Green;" href="https://static.igem.org/mediawiki/2011/2/27/Datasheet1.pdf">Datasheet</a></td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537003">BBa_K537003</a><br />
</td><br />
<td><br />
<a href="#T1-V">Theophylline riboswitch 1- Venus</a><br />
<br />
</td><br />
<td><br />
780bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537004">BBa_K537004</a><br />
</td><br />
<td><br />
<a href="#T2-V">Theophylline riboswitch 2- Venus</a><br />
<br />
</td><br />
<td><br />
781bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537005">BBa_K537005</a><br />
</td><br />
<td><br />
<a href="#mRFP1 C-fusion">mRFP1 C-fusion</a><br />
<br />
</td><br />
<td><br />
679bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537006">BBa_K537006</a><br />
</td><br />
<td><br />
<a href="#Venus C-fusion">Venus C-fusion</a><br />
</td><br />
<td><br />
721bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537007">BBa_K537007</a><br />
</td><br />
<td><br />
<a href="#RBS CheZ N-fusion">RBS CheZ N-fusion</a><br />
<br />
</td><br />
<td><br />
658bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537008">BBa_K537008</a><br />
</td><br />
<td><br />
<a href="#Atrazine Riboswitch-mRFP">Atrazine Riboswitch-mRFP</a><br />
<br />
</td><br />
<td><br />
658bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a><br />
</td><br />
<td><br />
<a href="#Promoter-Theophylline riboswitch 1-Venus-Double terminator">Promoter-Theophylline riboswitch 1-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
960bp<br />
</td><br />
<td></center></td><br />
</tr><br />
<tr><br />
<td><br />
<br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a><br />
<br />
</td><br />
<td><br />
<a href="#Promoter-Theophylline riboswitch 2-Venus-Double terminator">Promoter-Theophylline riboswitch 2-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
961bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
<br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a><br />
</td><br />
<td><br />
<a href="#Promoter-Theophylline riboswitch 1-CheZ-Venus-Double terminator">Promoter-Theophylline riboswitch 1-CheZ-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
1583bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
<br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a><br />
</td><br />
<td><br />
<a href="#Promoter-Theophylline riboswitch 2-CheZ-Venus-Double terminator">Promoter-Theophylline riboswitch 2-CheZ-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
1587bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a><br />
</td><br />
<td><br />
<a href="#Promoter-RBS-CheZ-Venus-Double terminator">Promoter-RBS-CheZ-Venus-Double terminator</a><br />
<br />
</td><br />
<td><br />
1543bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a><br />
</td><br />
<td><br />
<a href="#Promoter-Atrazine riboswitch-mCherry-Double terminator">Promoter-Atrazine riboswitch-mCherry-Double terminator</a><br />
<br />
</td><br />
<td><br />
946bp<br />
</td><br />
<td></center></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537015">BBa_K537015</a><br />
</td><br />
<td><br />
<a href="#Promoter-Atrazine riboswitch-CheZ-mCherry-Double terminator">Promoter-Atrazine riboswitch-CheZ-mCherry-Double terminator</a><br />
<br />
<br />
</td><br />
<td><br />
1570bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537016">BBa_K537016</a><br />
</td><br />
<td><br />
<a href="#Promoter-RBS-CheZ-mCherry-Double terminator">Promoter-RBS-CheZ-mCherry-Double terminator</a><br />
<br />
</td><br />
<td><br />
1501bp<br />
</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537019">BBa_K537019</a><br />
</td><br />
<td><br />
<a href="#lox66">lox66</a><br />
<br />
<br />
</td><br />
<td><br />
34bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<br />
<tr><br />
<td><br />
</td><br />
<td><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537020">BBa_K537020</a><br />
</td><br />
<td><br />
<a href="#lox71">lox71</a><br />
<br />
</td><br />
<td><br />
34bp<br />
</td><br />
<td><center><img src="https://static.igem.org/mediawiki/2011/c/c0/Parts_tick.png" width="30px;"></center></td><br />
</tr><br />
<br />
</table><br />
<br /><br />
</div><br />
<br /><br />
<p id ="T1-Ch"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Theophylline Riboswitch 1-CheZ'"><br />
<image src="https://static.igem.org/mediawiki/2011/f/fa/Wits_Parts_submitted_ThRS1-CheZ.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This DNA part will encode for an RNA riboswitch senstive to theophylline. The RBS is not exposed in the absence of theophylline. When theophylline is present, it binds to the riboswitch and causes a conformational change which leads to the exposure of the RBS and consequently translation of the adjoining gene. In this case, the gene which will be expressed is CheZ, which is a protein fundamental to bacterial movement.<br />
This theophylline riboswitch (type1) CheZ fusion BioBrick activates the expression of the CheZ gene in a theophylline-dependent fashion. It is composed of a theophylline-sensitive riboswitch clone 8.1 (Topp and Gallivan JACS 2007; BBa_K249026 and BBa_K411001) that is detached from its associated translation unit (coding region) and fused to a CheZ gene which has a N-fusion prefix and lacks a stop codon (although a TAG stop codon is provided by the RFC 25 suffix). The riboswitch regulated N-terminal part can be fused to a reporter or other CDS.</p><br />
<p>CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling) in the presence of a chemoattractant (in this case, theophylline).</p><br />
<br />
<br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="T2-Ch"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Theophylline Riboswitch 2-CheZ'"><br />
<image src="https://static.igem.org/mediawiki/2011/7/74/Wits_Parts_submitted_ThRS2-CheZ.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This DNA part will encode for an RNA riboswitch senstive to theophylline. When no theophylline is present, the RBS within the riboswitch sequence is not exposed to translation machinery. When theophylline is present, it binds to the riboswitch and causes a conformational change which results in the RBS being exposed. This allows for the translation of the adjoining gene. In this case, the gene which will be expressed is CheZ - a fundamental protein in the signalling cascade of bacterial chemotaxis.</p><br />
<p><br />
This theophylline riboswitch 2- CheZ fusion BioBrick activates the expression of the CheZ gene in a theophylline-dependent fashion. It consists of a newly improved theophylline riboswitch clone 12.1 (Lynch and Gallivan NAR 2009) that is detached from its associated translation unit (coding region) and fused to a CheZ gene which has a N-fusion prefix and lacks a stop codon (although a TAG stop codon is provided by the RFC 25 suffix). The riboswitch regulated N-terminal part can be fused to a reporter or other CDS.</p><br />
<p>CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling) in the presence of a chemoattractant (in this case, theophylline).<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<br />
<p id ="AtRS+CheZ"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Atrazine Riboswitch-CheZ fusion'"><br />
<image src="https://static.igem.org/mediawiki/2011/6/6c/Wits_Parts_submitted_AtRS-CheZ.png" align="left" style="padding: 10px;"/><br />
<br />
<p>When no atrazine is present, the RBS within the riboswitch sequence is not exposed to translation machinery. When atrazine is present, it binds to the riboswitch and causes a conformational change which results in the RBS being exposed. This allows for the translation of the adjoining gene. In this case, the gene which will be expressed is CheZ - a fundamental protein in the signalling cascade of bacterial chemotaxis.<br />
This riboswitch-CheZ fusion BioBrick regulates the expression of the CheZ gene in an atrazine-dependent fashion. It is composed of an atrazine-sensitive riboswitch, developed by Sinha et al (2010), which is detached from its associated translation unit (coding region) and fused to a CheZ gene which has the Freiburg N-fusion prefix and lacks a stop codon. While it is possible to fuse the riboswitch to the CheZ coding region by standard BioBrick assembly techniques, this approach was not used as it would increase the distance between the RBS and the ATG start codon and potentially decrease the efficiency of the riboswitch. This was shown in the work of the Taipei 2010 team who used this approach for a theophylline riboswitch. The riboswitch and the adjacent CheZ coding region are considered together and should be cloned together. CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the Chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling) in the presence of a chemoattractant (in this case atrazine).<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="T1-V"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Theophylline Riboswitch 1- Venus'"><br />
<image src="https://static.igem.org/mediawiki/2011/6/6d/Wits_Parts_submitted_ThRS1-Venus.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This part regulates the expression of venus in a theophylline-dependent fashion through an RNA aptamer specific to theophylline.<br />
This theophylline riboswitch 1 biobrick activates the expression of the Venus fluorescent protein in a theophylline-dependent fashion. It is composed of a theophylline sensitive riboswitch clone 8.1 (Topp and Gallivan JACS 2007; BBa_K249026 and BBa_K411001) fused upstream to an adjacent Venus coding region.<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="T2-V"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Theophylline Riboswitch 2- Venus'"><br />
<image src="https://static.igem.org/mediawiki/2011/f/fd/Wits_Parts_submitted_ThRS2-Venus.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This is a theophylline riboswitch clone 12.1 (Lynch and Gallivan NAR 2009) fusion with the Venus reporter BBa_K354002. This part regulates the expression of venus in a substrate-dependent fashion through an RNA aptamer specific to the antibiotic theophylline.<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="mRFP1 C-fusion"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'mCherry (mRFP1) C-Fusion'"><br />
<image src="https://static.igem.org/mediawiki/2011/d/da/Wits_Parts_submitted_mRFP1_C-fusion.png" align="left" style="padding: 10px;"/><br />
<br />
<p>This BioBrick represents a C-terminal fusion part. The part contains a standard suffix and a “assembly standard 25” prefix (Freiburg Fusions/ RFC 25). This version of the mRFP reporter (BBa_E1010) has the standard BioBrick suffix and prefix. The standard Biobrick prefix, however, is followed by another restriction site – specific for NgoMIV (which forms the Freiburg fusion prefix). The part lacks an ATG start codon (provided by N-terminal part). It can therefore be fused to any gene in a modular fashion via the modular construction strategy of protein fusion developed by the Freiburg 2007 team.<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="Venus C-fusion"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Venus C-Fusion'"><br />
<image src="https://static.igem.org/mediawiki/2011/a/aa/Wits_Parts_submitted_Venus-C-fusion.png" align="left" style="padding: 10px;"/> <br />
<p>This BioBrick represents a C-terminal fusion part. The part contains a standard suffix and a “assembly standard 25” prefix (Freiburg Fusions/ RFC 25). This version of the Venus Fluorescent reporter (BBa_K354002) has the standard BioBrick suffix and prefix. The standard Biobrick prefix, however, is followed by another restriction site – specific for NgoMIV (which forms the Freiburg fusion prefix). The part lacks an ATG start codon (provided by N-terminal part). It can therefore be fused to any gene in a modular fashion via the modular construction strategy of protein fusion developed by the Freiburg 2007 team.<br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="RBS CheZ N-fusion"> <br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'RBS CheZ N-Fusion'"><br />
<image src="https://static.igem.org/mediawiki/2011/8/8c/Wits_Parts_submitted_RBS-CheZ.png" align="left" style="padding: 10px;"/> <br />
<br />
<p>The RBS CheZ- Fusion BioBrick consists of a RBS fused to the E. coli CheZ motility factor that lacks a stop codon at the N-terminus (although a TAG stop codon is provided by the RFC 25 suffix). CheZ in this part contains the standard BioBrick prefix and the Freiburg (2007) fusion suffix. The N-terminal part can be fused to a reporter or other CDS. Using this BioBrick, CheZ is constitutively expressed.</p><br />
<p>CheZ is the chief regulator of the molecular events that lead the counter clockwise rotation of the flagella motor during the chemotaxis signal transduction pathway of E.coli. This counter clockwise flagella motor rotation results in bacterial swimming (instead of tumbling).</p><br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<br />
<p id ="Atrazine Riboswitch-mRFP"> <br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Atrazine Riboswitch-mRFP'"><br />
<image src="https://static.igem.org/mediawiki/2011/1/17/Wits_Parts_submitted_AtRS-mRFP1.png" align="left" style="padding: 10px;"/> <br />
<br />
<p>This part consists of an atrazine-sensitive riboswitch, developed by Sinha et al (2010), detached from its associated translation unit (coding region) and fused to mRFP1 fluorescent reporter (BBa_J61100). This part regulates the expression of mRFP in an atrazine-dependent fashion through an atrazine-specific RNA aptamer.</p><br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="Promoter-Theophylline riboswitch 1-Venus-Double terminator"> <br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-Theophylline riboswitch 1-Venus-Double terminator'"><br />
<image src="https://static.igem.org/mediawiki/2011/2/2f/Wits_Parts_submitted_Pr-thRS1-Venus-dt.png" align="left" style="padding: 10px;"/> <br />
<br />
<br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 1 (Topp and Gallivan JACS, 2007) which is fused to the Venus fluorescent reporter protein without a stop codon in between. The theophylline riboswitch1-venus fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).</p><br />
</p><br />
</div><br />
<a href="#Top">Back to Top</a><br />
<br/><br />
<p id ="Promoter-Theophylline riboswitch 2-Venus-Double terminator"> <br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Promoter-Theophylline riboswitch 2-Venus-Double terminator'"><br />
<image src="https://static.igem.org/mediawiki/2011/c/ca/Wits_Parts_submitted_Pr-ThRS2-Venus-dt.png" align="left" style="padding: 10px;"/> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 2 (Lynch and Gallivan NAR 2009) which is fused to the Venus fluorescent reporter protein without a stop codon in between. The theophylline riboswitch2-venus fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).</p><br />
<br />
<br />
</p><br />
</div><br />
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<br/><br />
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<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 1 (Topp and Gallivan JACS 2007) which is fused to CheZ and Venus fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The theophylline riboswitch1-CheZ fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).</p><br />
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<p id ="Promoter-Theophylline riboswitch 2-CheZ-Venus-Double terminator"> <br />
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<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by a theophylline riboswitch 2 (Lynch and Gallivan NAR 2009) which is fused to CheZ and Venus fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The theophylline riboswitch2-CheZ fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).</p><br />
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<center><image src="https://static.igem.org/mediawiki/2011/8/85/Wits_Parts_submitted_Pr-RBS-CheZ-Venus-dt.png"></center> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an RBS which is fused to CheZ and Venus fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The RBS-CheZ fusion was constructed via PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015)<br />
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<p id ="Promoter-Atrazine riboswitch-mCherry-Double terminator"> <br />
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<image src="https://static.igem.org/mediawiki/2011/4/43/Wits_Parts_submitted_Pr-AtRS-mRFP-dt.png" align="left" style="padding: 10px;"/> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an atrazine riboswitch riboswitch (Sinha et al, 2010) which is fused to the monomeric Red fluorescent reporter protein without a stop codon in between. The atrazine riboswitch-mRFP fusion was constructed via 2 rounds of PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).<br />
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<p id ="Promoter-Atrazine riboswitch-CheZ-mCherry-Double terminator"><br />
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<center><image src="https://static.igem.org/mediawiki/2011/c/c9/Wits_Parts_submitted_Pr-AtRS-CheZ-mRFP-dt.png"></center> <br />
<br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an atrazine riboswitch riboswitch (Sinha et al, 2010) which is fused to CheZ and monomeric Red fluorescent reporter protein without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The atrazine riboswitch-CheZ fusion was constructed via PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).<br />
</p><br />
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<p id ="Promoter-RBS-CheZ-mCherry-Double terminator"><br />
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<center><image src="https://static.igem.org/mediawiki/2011/5/52/Wits_Pr-RBS-CheZ-mRFP-dt.png"></center> <br />
<p>This composite BioBrick begins with a strong, constitutively active promoter of This composite BioBrick begins with a strong, constitutively active promoter of E.coli (BBa_J23119) followed by an RBS which is fused to CheZ and monomeric Red fluorescent reporter protein (mRFP) without stop codons in between. The Freiberg iGEM 2007 BioBrick 3.0 fusion protein assembly was used to construct this BioBrick. The RBS-CheZ fusion was constructed via PCR. This part ends with a standard double terminator transcriptional terminator for E.coli (BBa_B0015).<br />
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<p>lox66 is a site specific recombination cassette. It belongs to the loxP family frequently used in genetics, particularily in mouse genetics.<br />
lox site recombination is catalysed by a Site specific recombinase, Cre.<br />
lox sequences are composed of an 8 bp Core sequence surrounded by two Arms.<br />
The particularity of lox66 is that it has an altered sequence at the end of it's left arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm. As a consequence, after a recombination between a lox66 and a lox71 (altered right arm sequence), one of the two resulting generated lox sites has very low recombination potential as it inherited both mutated arms. Use of lox66 & lox71 sites is potentially interresting when the recombination reaction must be "irreversible".<br />
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<br />
<p>Lox71 is a site specific recombination cassette. It belongs to the loxP family frequently used in genetics, particularly in mouse genetics.<br />
lox site recombination is catalysed by a Site specific recombinase, Cre.<br />
lox sequences are composed of an 8 bp Core sequence surrounded by two Arms.<br />
The particularity of lox66 is that it has an altered sequence at the end of it's left arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm. As a consequence, after a recombination between a lox71 and a lox66 (altered right arm sequence), one of the two resulting generated lox sites has very low recombination potential as it inherited both mutated arms. Use of lox71 & lox66 sites is potentially interesting when the recombination reaction must be "irreversible".<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SATeam:WITS-CSIR SA2011-10-22T18:05:23Z<p>Nkruger: </p>
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<td> <p>A defining characteristic of humanity is the constantly evolving sophistication with which human beings communicate and store information. Biologically, data communication networks exist too. The transfer of data occurs via signalling molecules incorporated into highly regulated and integrated networks.Our goal is to construct a bacterial communication network that will allow a group of engineered bacteria to transport messages in a directed manner, with the final aim of completing location-based functional tasks.We have focused on one aspect of this network: the directed transport of the message-carrying “communication modules” within our biological network. These communication modules will take the form of bacteria, which can move over physical distances carrying information. We have achieved this through engineering the exogenously controlled chemotactic behaviour of bacteria using synthetic riboswitches to regulate the expression of the CheZ protein required for bacterial motility.<br />
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<h1> Acknowledgements </h1><br />
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<p>The Wits-CSIR 2011 iGEM team was proudly sponsored by:</p><br />
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<p>The Wits-CSIR 2011 iGEM team would like to thank all the above sponsors for their generous financial contributions, which lightened the team’s financial burden thus allowing them to focus their attention on the project. Your generosity has inspired the team to help others and give back to the community, through human advances. </p> <br />
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<p>The Wits_CSIR 2011 iGEM team would like to thank the following advisors and supervisors for their moral and scientific support, especially; the supervisors for leading the team effectively and efficiently in this competitive, fast-paced and ever-changing world of synthetic biology; and Michelle Robinson for her tenacious effort to keep the team on track by assisting the team with implementing time management and organisational skills of the highest calibre. </p><br />
<h2>Supervisors </h2><br />
<p>Karl Rumbold: </p><br />
<p>Marco Weinberg: </p> <br />
<p>Musa Mhlanga: Continuous academic and financial assistance from the Synthetic Biology unit at the CSIR </p><br />
<p>Raymond Sparrow: </p><br />
<br />
<br />
<h2>Advisors </h2><br />
<p>Michelle Robinson </p><br />
<p>Laura Millroy </p><br />
<p>Youtaro Shibayama</p> <br />
<p>Scott Hazelhurst </p><br />
<p>Robyn Brackin </p><br />
<br />
<h2>We would also like to extend our gratitude to the following people:</h2><br />
<p>Prof Kramer from the Wits faculty of Health Science and Prof Crouch from the the Wits faculty of Science for their continued support of the iGEM team.</p><br />
<p>Prof Patrick Arbuthnot for access to of the Anti-viral Gene Therapy Research Unit (AGTRU) laboratory facilities.</p> <br />
<p>Prof Theresa Coetzer for the use of Malaria Research Unit (MRU) laboratory space.</p><br />
<p>Dr Sylvia Fanucchi and Prof Heini Dirr for the use of the Jasco Spectrofluorometer.</p><br />
<p>David Balchin for his assistance with the Jasco Spectrofluorometer.<br />
<p>Robyn Brackin for all her assistance with the Epi-Fluorescence Microscopy imaging.</p><br />
<p>Dr Clement Penny for all his help and advice with the Fluorescent microscopy imaging.</p><br />
<p>The Synthetic Biology Research Unit at the CSIR, for their academic assistance and use of equipment in their lab facilities.</p><br />
<p>Dr Sonja Lauterbach and Kubendran Naidoo for all their help, advice and care in the lab.</p><br />
<p> Dr Toru Nakayashiki and Prof Hirotada Mori from the Nara Institute of Science and Technology for their kind donation of the <i>E. coli</i> CheZ deletion mutant and parental strain.</p><br />
<p>Dr Joy Sinha and Prof Justin Gallivan from Emory University for their time and the invaluable advice on riboswitches and bacterial chemotaxis.</p> <br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/AcknowledgementsTeam:WITS-CSIR SA/AboutUs/Acknowledgements2011-10-22T17:49:43Z<p>Nkruger: </p>
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<h1> Acknowledgements </h1><br />
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<p>The Wits-CSIR 2011 iGEM team was proudly sponsored by:</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a1/Wits_Home_Page_Sponsors.jpg"></center><br />
<p>The Wits-CSIR 2011 iGEM team would like to thank all the above sponsors for their generous financial contributions, which lightened the team’s financial burden thus allowing them to focus their attention on the project. Your generosity has inspired the team to help others and give back to the community, through human advances. </p> <br />
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<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Acknowledgements'"><br />
<p>The Wits_CSIR 2011 iGEM team would like to thank the following advisors and supervisors for their moral and scientific support, especially; the supervisors for leading the team effectively and efficiently in this competitive, fast-paced and ever-changing world of synthetic biology; and Michelle Robinson for her tenacious effort to keep the team on track by assisting the team with implementing time management and organisational skills of the highest calibre. </p><br />
<h2>Supervisors </h2><br />
<p>Karl Rumbold: </p><br />
<p>Marco Weinberg: </p> <br />
<p>Musa Mhlanga: Continuous academic and financial assistance from the Synthetic Biology unit at the CSIR </p><br />
<p>Raymond Sparrow: </p><br />
<br />
<br />
<h2>Advisors </h2><br />
<p>Michelle Robinson </p><br />
<p>Laura Millroy </p><br />
<p>Youtaro Shibayama</p> <br />
<p>Scott Hazelhurst </p><br />
<p>Robyn Brackin </p><br />
<br />
<h2>We would also like to extend our gratitude to the following people:</h2><br />
<p>Prof Kramer from the Wits faculty of Health Science and Prof Crouch from the the Wits faculty of Science for their continued support of the iGEM team.</p><br />
<p>Prof Patrick Arbuthnot for access to of the Anti-viral Gene Therapy Research Unit (AGTRU) laboratory facilities.</p> <br />
<p>Prof Theresa Coetzer for the use of Malaria Research Unit (MRU) laboratory space.</p><br />
<p>Dr Sylvia Fanucchi and Prof Heini Dirr for the use of the Jasco Spectrofluorometer.</p><br />
<p>David Balchin for his assistance with the Jasco Spectrofluorometer.<br />
<p>Robyn Brackin for all her assistance with the Epi-Fluorescence Microscopy imaging.</p><br />
<p>Dr Clement Penny for all his help and advice with the Fluorescent microscopy imaging.</p><br />
<p>The Synthetic Biology Research Unit at the CSIR, for their academic assistance and use of equipment in their lab facilities.</p><br />
<p>Dr Sonja Lauterbach and Kubendran Naidoo for all their help, advice and care in the lab.</p><br />
<p> Dr Toru Nakayashiki and Prof Hirotada Mori from the Nara Institute of Science and Technology for their kind donation of the <i>E. coli</i> CheZ deletion mutant and parental strain.</p><br />
<p>Dr Joy Sinha and Prof Justin Gallivan from Emory University for their time and the invaluable advice on riboswitches and bacterial chemotaxis.</p> <br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SATeam:WITS-CSIR SA2011-10-22T17:41:40Z<p>Nkruger: </p>
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<td> <p>A defining characteristic of humanity is the constantly evolving sophistication with which human beings communicate and store information. Biologically, data communication networks exist too. The transfer of data occurs via signalling molecules incorporated into highly regulated and integrated networks.Our goal is to construct a bacterial communication network that will allow a group of engineered bacteria to transport messages in a directed manner, with the final aim of completing location-based functional tasks.We have focused on one aspect of this network: the directed transport of the message-carrying “communication modules” within our biological network. These communication modules will take the form of bacteria, which can move over physical distances carrying information. We have achieved this through engineering the exogenously controlled chemotactic behaviour of bacteria using synthetic riboswitches to regulate the expression of the CheZ protein required for bacterial motility.<br />
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Collaboration</h1><br />
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<p>The Wits CSIR iGEM team collaborated with Imperial College London, both for the modelling and the potential suitability of motility experiments in the testing of our machines. The biologists in our team and those at Imperial College London had a number of skype conferences to discuss the progress of our lab work as well as to share insight into both qualitative and quantitative assay for motility. </p><br />
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<p>We provided Imperial College London with some ideas regarding motility assays using TTC in stab agar. Their team gave us some tips about the preparation of cells for motility experiments and recommended spinning down the bacteria and resuspending them in a low volume of broth to increase the density of cells. They also recommended the use of capillary motility assays. There was an exchange of protocols both ways to aid in the set-up of our experiments. A week later we discussed the progress we had made in the lab. We gave each other tips and suggested more assays to indicate the ability of our bacteria to move towards a stimulus. Imperial College London shared with us a type of capillary assay they were performing at the time and we, too, shared a protocol with them which is detailed in Gullivan and Topps (2006) paper, Guiding bacteria with small molecules and RNA.</p><br />
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<p>Collaboration was also performed between the engineers to improve the modeling for both the teams. Several Skype conferences were held between the team from Imperial College London and the Wits CSIR team to discuss the design and theory behind our own chemotaxis models. The collaborative effort resulted in the Imperial College London kindly providing us with a model for our theophylline riboswitches. Their modelling results can be found on our href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Modelling">modelling</a> page.<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/CharacterizationTeam:WITS-CSIR SA/Characterization2011-10-22T17:35:04Z<p>Nkruger: </p>
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<h1><br />
Characterization of Parts</h1><br />
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<p>In order to characterize our final machines, we performed the following assays:</p><br />
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<a href="#Fluorometry">Fluorometry</a><br />
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<a href="#Microscopy">Fluorescence microscopy</a><br />
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<a href="#Motility">Motility</a><br />
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<a href="#Chemotaxis">Chemotaxis</a><br />
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<h2>Theophylline Riboswitches</h2><br />
<p>To characterise the theophylline riboswitches (1 and 2), we quantified their activation at different theophylline concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM and 2 mM) over a period of time using fluorometry. Competent <i>E. coli</i> (strain DH5a) cells were transformed with plasmid vectors containing the riboswitch and were cultured until the mid-log phase of growth. Thereafter, a different concentration of theophylline was added to each culture for induction. The activation of the riboswitch was detected as a fluorescent response as a result of increased translation of the fluorescent fusion protein CheZ-Venus, in the presence of the activator. A Jasco FP-6300 spectrofluorometer was used to excite the cultures at 514 nm and the intensity of the emission peak was detected at 528 nm. Twenty readings were taken every 15 minutes for each culture, for a total period of 135 minutes. An empty vector was used to correct for autofluorescence. Conversely, a construct that comprised of a Promoter-RBS-CheZ-Venus-double terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>) was used as a positive control. Fluorescent data for the 3D plots (Fig 1-4) were corrected for autofluorescence by subtracting the fluorescent values of the cultures transformed with the empty vector, from that of the riboswitches.<br />
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<td><p> <b>Figure 1:</b> The fluorescence of the CheZ-Venus fusion protein under the regulation of theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). <b>A)</b> The activation of the riboswitch over time at different concentrations of theophylline. The fluorescence increased over time, with an increasing concentration of theophylline, indicating the theophylline concentration-dependent expression of the CheZ-Venus fluorescent fusion protein. The fluorescence peaks 105 minutes after the addition of 1 mM theophylline. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 1 mM theophylline after 105 minutes incubation, and to that of baseline levels (0 minutes with 1 mM theophylline). 1 mM of theophylline induced the expression of CheZ-Venus to increase 4-fold after 105 minutes (fold change was calculated by dividing the fluorescence observed at 105 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values) (Click to enlarge)<br />
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<b>Figure 2:</b> The fluorescence of the CheZ-Venus fusion protein under the regulation of theophylline riboswitch 2 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>). <b>A)</b> The activation of the riboswitch over time at different concentrations of theophylline. The same trend of increased switch activation over time, with increasing theophylline concentrations was seen as with theophylline riboswitch 1. However, with the theophylline riboswitch 2, the fluorescence peaks 135 minutes after the addition of 1.5 mM theophylline. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 1.5 mM theophylline after 135 minutes incubation, and to that of baseline levels (0 minutes with 1.5 mM theophylline). 1.5 mM of theophylline induced a 37-fold increase in CheZ-Venus expression after 135 minutes (fold change was calculated by dividing the fluorescence observed at 135 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values). (Click to enlarge)</p> </td><br />
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<td><p> <b>Figure 3:</b> The fluorescence of the Venus reporter protein under the regulation of theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>). <b>A)</b> The fluorescent peak occurred prematurely after 75 minutes of incubation with 0.5 mM theophylline, compared to riboswitch 1, which contained the gene for the CheZ-Venus fusion protein. The maximum fluorescent intensity observed was also lower by a factor of 2 RFU. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 75 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 4.5-fold increase in protein expression was observed as the maximum (fold change was calculated by dividing the fluorescence observed at 75 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values). This fold change is comparable to that described in <b>Figure 1</b>. (Click to enlarge)<br />
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<td><p> <b>Figure 4:</b> The fluorescence of the Venus reporter protein under the regulation of the theophylline riboswitch 2 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>). <b>A)</b> The activation of the riboswitch was affected by the lack of the CheZ gene in the switch construct. The increase in fluorescence was minimal upon induction and incubation over time. Furthermore, the fluorescent peak occurred prematurely after 60 minutes of incubation with 0.5 mM theophylline, when compared to the theophylline riboswitch 2 that contained the gene for the CheZ-Venus fusion protein. <b>B)</b> The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 60 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 2-fold increase in protein expression was observed as the maximum (fold change was calculated by dividing the fluorescence observed at 60 minutes by the fluorescence at 0 minutes, after subtracting the corresponding autofluorescence values) . This is far less than the value described for the same switch in <b>Figure 2</b>.(Click to enlarge)<br />
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Both the theophylline riboswitches displayed activation in response to increasing theophylline concentrations and incubation times. Theophylline riboswitch 2 controlling CheZ-Venus expression (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>) clearly showed a superior level of activation compared to that theophylline riboswitch 1 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). This was expected as it was selected by FACS for its optimised expression platform that allows for minimal leakiness at basal levels, and a strong activation in the presence of theophylline (Lynch and Gallivan, 2009). The activation shown by both the riboswitches regulating CheZ-Venus expression should be able to restore the motility of <i>E. coli</i> cells deficient in CheZ, in the presence of theophylline. More importantly, the theophylline concentration-dependent increase in CheZ-Venus expression alludes to the ability of directed cell movement, that is moving up concentration gradients of theophylline (Topp and Gallivan, 2007). Interestingly, the data suggests that the presence of the CheZ gene, as part of the CheZ-Venus fusion sequence, enhances the activation of the switches when compared to the constructs with venus alone. This may be due to some sort of structural stability conferred by the presence of the CheZ sequence.</P><br />
<h2> Atrazine Riboswitch</h2><br />
<p>Preliminary activation data was obtained for the atrazine riboswitch.Fluorometry was performed on <i>E. coli</i> (DH5a) cells transformed with a construct that contained a Promoter-atrazine riboswitch-mRFP-double terminator (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a>). Similar to the theophylline riboswitches, different concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) of atrazine were added to the cultures for induction. The cultures were excited at 584 nm and detected at 607 nm using a Jasco FP-6300 spectrofluorometer. Readings were taken every 15 minutes for 120 minutes in total. The same empty vector was used as a negative control. The 3D plot data was normalised the same way as for the theophylline riboswitches.</p><br />
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<td><p> <b>Figure 5:</b> The fluorescence of the mRFP1 reporter protein under the regulation of the atrazine riboswitch(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537014">BBa_K537014</a>). <b>A)</b> The activation of the atrazine riboswitch at different atrazine concentrations over time. There was an increase in fluorescence as the atrazine concentration and incubation time increased. The amount of fluorescence detected was very small, with a maximum of 0.1 RFU detected after 75 minutes of incubation with 2 mM atrazine. <b>B)</b> The maximum was compared to the negative control at 75 minutes, 2 mM atrazine, and to that of fluorescent levels detected at basal levels (0 minutes, 2 mM atrazine). A 1.7-fold increase in expression of mRFP1 was observed over the 75 minute period (values used for the calculation had the corresponding negative control value subtracted first).(Click to enlarge)<br />
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<h2>References</h2><br />
<p>Lynch, S. A. & Gallivan, J. P. 2009. A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Res, 37, 184-92.</p><br />
<p>Topp, S. & Gallivan, J. P. 2007. Guiding bacteria with small molecules and RNA. J Am Chem Soc, 129, 6807-11.</p><br />
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<p> Fluorescent imaging was performed for a qualitative assay of the engineered bacteria. <br />
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<p> Bacterial culture and Manipulation: <br />
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<p> 4 ml of LB broth with Ampicillin was inoculated from colonies off a master plate of positive clones and grown overnight at 37°C in a shaking incubator. 2 ml of each bacterial culture was then used to inoculate 25 ml of LB broth with Ampicillin and grown for ± 3 hours in a shaking incubator at 37°C until the cells reached an OD600 of 0.55 (mid-log phase). 2 x 5 ml of each sample culture was aliquoted into separate 15 ml falcon tubes. One tube was left untreated; 150 µl of 15 mM theophylline was added to the other (to make a final concentration of 1.5mM theophylline). These 5 ml aliquots were placed in a shaking incubator at 37°C for 30 min each to allow for activation. 200 µl of each treated and untreated sample was then used in a microscope plate for imaging. Imaging was done using Zeiss Axioscope (Andor EMCCD Camera, Till Photonics Polychrome V). <br />
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<p> Widefield fluorescence microscopy was used to assess the expression of the Venus protein. The bacterial samples were excited at 500 nm using the polychrome as an excitation source. The fluorescence was then observed by capturing light in the emission spectrum of Venus (528 nm). The figure below represents a schematic of the imaging setup. Corresponding bright-field images (using white light) were also captured for each experiment. <br />
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<p>Below are the images that were obtained for the parental strain (BW25113) of the <i>E. coli</i> CheZ-deletion mutant, which served as the negative, and the CheZ deletion mutants transformed with Promoter-theophylline riboswitch 1-venus (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) and Promoter-theophylline riboswitch 2-venus (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>).<br />
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<p>Please note: Bacteria which were transformed with constructs which contained the CheZ gene were not tested in this assay. This experiment served as a means of observing the activation of the theophylline riboswitches, hence only expression of the venus reporter protein was needed for detection of fluorescence. Activation of motility was not assessed in this case – it was examined in the motility assays on semi-solid agar plates. Differences in fluorescence intensities were not calculated from the images below since the fluorometry experiments served as the quantitative approach to measurement of riboswitch activation.<br />
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<b>Figure 6:</b> The parental strain (BW25113) of the <i>E. coli</i> CheZ deletion mutants were not transformed with any plasmid DNA. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In both the absence and presence of theophylline, the parental bacteria did not fluoresce – which was the expected result. (Click to enlarge)</p> </td><br />
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<b>Figure 7:</b> <i>E. coli</i> CheZ-deletion mutants which were transformed with the Promoter-theophylline riboswitch 1-venus (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) construct. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, almost no fluorescence occurred. Upon the addition of theophylline at a concentration of 1.5 mM, many of the cells emitted fluorescence showing activation of the theophylline riboswitch type 1.(Click to enlarge)</p> </td><br />
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<b>Figure 8:</b> <i>E.coli</i> CheZ-deletion mutants which were transformed with the Promoter-theophylline riboswitch 2-venus (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>) construct. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, some fluorescence was observed. This result showed the leakiness of the riboswitch. A substantial amount of venus translation is permitted because of the flexible conformation of this riboswitch. Upon the addition of theophylline at a concentration of 1.5 mM, much more fluorescence was detected. More venus was expressed in these cells due to the activation of the riboswitch via theophylline.(Click to enlarge)</p> </td><br />
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<p> Semi-solid agar plates containing different concentrations of theophylline (0mM, 0.5mM, 1.0mM and 1.5mM) were made. Promoter-theophylline riboswitch 1-CheZ-Venus-double terminator (thRS1-Ch-V)(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>), Promoter-theophylline riboswitch 2-CheZ-Venus-double terminator (thRS2-Ch-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>), Promoter-theophylline riboswitch 1-Venus-double terminator (thRS1-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>) and Promoter-theophylline riboswitch 2-Venus-double terminator (thRS2-V) (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>) were transformed into CheZ deletion mutants and grown to mid-log phase (OD600 = 0.55). 1.5 µl of culture was plated into the centre of the plates containing the various concentrations of theophylline and incubated at 37°C for 24 hours. This experiment was performed in triplicate. The protocol for this experiment can be found here.<br />
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<b>Figure 9:</b> The distance travelled by CheZ deletion mutants (mm) over a 24 hour period at 37°C on semi-solid agar plates containing different theophylline concentrations (mM)(Click to enlarge)</p> </td><br />
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The thRS1-V and thRS2-V transformants did not travel from the point of inoculation for all concentrations of theophylline. The CheZ deletion mutants containing thRS1-Ch-V travelled 0 mm at 0 mM theophylline and reached a maximum travelling distance of 5 mm at 1.5 mM theophylline. The thRS2-Ch-V containing CheZ deletion mutants travelled 2 mm in the absence of theophylline and at a concentration of 1.5 mM theophylline, were able to travel 22mm from the point of inoculation </p><br />
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<b>Figure 10:</b> 85 mm semi-solid agar plates containing 1.0 mM theophylline showing the point of inoculation and distance travelled by the bacteria. <b>A)</b> Inoculation of 1.5 µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS1-Ch-V(<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>). There is evidence of movement of bacteria from the point of inoculation. This can be observed as a faint halo surrounding the point of inoculation.<b>B)</b> Inoculation of 1.5 µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS1-V. Converse to <b>A</b>, there is no evidence of movement of the bacteria from the point of inoculation <b>C)</b> Inoculation of 1.5µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS2-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>). There is evidence of movement of the bacteria from the point of inoculation. This can be observed as a faint halo surrounding the point of inoculation. <b>D)</b> Inoculation of 1.5µl of mid-log phase <i>E. coli</i> CheZ deletion mutants transformed with thRS2-V. Converse to <b>C</b>, there is no evidence of movement of bacteria from the point of inoculation (Click to enlarge)</p> </td><br />
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<h3> Discussion and conclusion</h3><br />
<p>The construct thRS1-Ch-V transformed into the CheZ deletion mutants are non-motile at a concentration of 0 mM theophylline, since in the absence of the activator, the riboswitch is sequestering the RBS and thus inhibiting the translation of CheZ (Topps and Gallivan, 2007). On the semi-solid agar plates containing theophylline, the motility of E.coli is evident, as the interaction of theophylline with the riboswitch enables the exposure of the RBS and the translation of the downstream CheZ, allowing for bacterial motility. The distance travelled by the bacteria transformed with the thRS1-Ch-V construct, from the start position (mm) in the presence of different concentrations of theophylline, is not significantly different (P=0.005). This indicates that the CheZ expression at 0.5 mM-1.5 mM is sufficient to allow the bacteria to move a distance of 5 mm. The construct thRS2-Ch-V transformed into the CheZ deletion mutants are slightly motile in the absence of theophylline, travelling 2mm over a 24 hour period indicating that this riboswitch is slightly leaky. Upon addition of 0.5 mM-1.5 mM theophylline, the bacteria were able to travel 20mm over a 24 hour period.</p><br />
<p>The distance travelled from the start position by the CheZ deletion mutants containing the thRS2-Ch-V is significantly higher than that those with the thRS1-Ch-V construct. This can be explained by the high fold-increase in CheZ protein expression by this riboswitch, as shown by fluorometry. Theophylline riboswitch 1 has a lower fold increase in CheZ expression from 0 mM to 1.5 mM theophylline, compared to the increase shown by the theophylline riboswitch 2. Therefore, in order to achieve maximum motility under the control of theophylline, at concentrations of between 0.5 mM and 1.5 mM, theophylline riboswitch 2 should be used.</p><br />
<h2>References</h3><br />
Topp, S. & Gallivan, J. P. 2007.Guiding bacteria with small molecules and RNA. J Am ChemSoc, 129, 6807-11 <br />
<p>Both the t1-V and t2-V constructs transformed into the CheZ deletion mutants are non-motile, each having travelled a distance of 0mm at all concentrations of theophylline (See A and B). This indicates that in the absence of the CheZ protein, the bacteria are non-motile (Topps and Gallivan, 2007).</p><br />
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<p id="Chemotaxis"><br />
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<p>In order to determine whether we had successfully engineered the <i>E. coli</i> CheZ deletion mutants to chemotactically migrate towards theophylline, we performed a capillary assay. Bacteria transformed with thRS1-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537009">BBa_K537009</a>), thRS2-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537010">BBa_K537010</a>), RBS-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537013">BBa_K537013</a>), thRS1-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537011">BBa_K537011</a>) and thRS2-Ch-V (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K537012">BBa_K537012</a>) were grown to mid-log phase. Activation of chemotaxis was achieved by incubating the cultures with 1 mM theophylline prior to the assay. A 50 µl drop of each culture was then placed onto separate sterile surfaces and two capillary tubes were placed into each drop. The first capillary tube was a control, containing only PBS and 20 µM EDTA. The second tube contained PBS, 20 µM and 2mM theophylline. The two capillary tubes were suspended into each drop of culture for 30 minutes. A dilution series of the solutions in the tubes were plated and colonies counted after 12 hours. Further details of this protocol can be found here.<br />
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<h3>Discussion and conclusion</h3><br />
<p>Cells transformed with thRS1-V, thRS2-V and RBS-Ch-V all displayed an index of approximately 100, indicating the same number of bacteria moved towards the control (0mM theophylline) and the experiment (2mM theophylline) capillary tube. This proves that none of these constructs induce chemotactic behaviour. In terms of both thRS1-V and thRS2-V, the lack of CheZ results in the inability of CheY dephosphorylation and the swimming motion observed in chemotaxis, which is a key driver in directed movement towards an attractant (Topps and Gallivan, 2007). Although the RBS-Ch-V construct constitutively expresses CheZ, and bacterial swimming is achieved through CheY dephosphorylation, the expression levels remain constant regardless of the presence or absence of theophylline. Random movement will occur as a result.<p/><br />
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<p>The thRS1-Ch-V and thRS2-Ch-V transformants, with chemotaxis indices of 180 and 227 respectively, both indicated a significantly higher number of bacteria that travelled to the 2mM theophylline solution in the capillary tube compared to that of the control. The chemotaxis index of the thRS2-Ch-V construct is significantly higher (p=0.005) than that of thRS1-Ch-V, indicating that cells transformed with thRS2-Ch-V is more attracted to theophylline than thRS1-Ch-V. Should maximum chemotaxis towards theophylline be desired, thRS2-Ch-V would be the more suitable construct.</p><br />
<h2>References</h2><br />
Topp, S. & Gallivan, J. P. 2007. Guiding bacteria with small molecules and RNA. J Am ChemSoc, 129, 6807-11<br />
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<h1> Acknowledgements </h1><br />
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<p>The Wits-CSIR 2011 iGEM team was proudly sponsored by:</p><br />
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<p>The Wits_CSIR 2011 iGEM team would like to thank the following advisors and supervisors for their moral and scientific support, especially; the supervisors for leading the team effectively and efficiently in this competitive, fast-paced and ever-changing world of synthetic biology; and Michelle Robinson for her tenacious effort to keep the team on track by assisting the team with implementing time management and organisational skills of the highest calibre. </p><br />
<h2>Supervisors </h2><br />
<p>Karl Rumbold: </p><br />
<p>Marco Weinberg: </p> <br />
<p>Musa Mhlanga: Continuous academic and financial assistance from the Synthetic Biology unit at the CSIR </p><br />
<p>Raymond Sparrow: </p><br />
<br />
<br />
<h2>Advisors </h2><br />
<p>Michelle Robinson </p><br />
<p>Laura Millroy </p><br />
<p>Youtaro Shibayama</p> <br />
<p>Scott Hazelhurst </p><br />
<p>Robyn Brackin </p><br />
<br />
<h2>We would also like to extend our gratitude to the following people:</h2><br />
<p>Prof Kramer from the Wits faculty of Health Science and Prof Crouch from the the Wits faculty of Science for their continued support of the iGEM team.</p><br />
<p>Prof Patrick Arbuthnot for access to of the Anti-viral Gene Therapy Research Unit (AGTRU) laboratory facilities.</p> <br />
<p>Prof Theresa Coetzer for the use of Malaria Research Unit (MRU) laboratory space.</p><br />
<p>Dr Sylvia Fanucchi and Prof Heini Dirr for the use of the Jasco Spectrofluorometer.</p><br />
<p>David Balchin for his assistance with the Jasco Spectrofluorometer.<br />
<p>Robyn Brackin for all her assistance with the Epi-Fluorescence Microscopy imaging.</p><br />
<p>Dr Clement Penny for all his help and advice with the Fluorescent microscopy imaging.</p><br />
<p>The Synthetic Biology Research Unit at the CSIR, for their academic assistance and use of equipment in their lab facilities.</p><br />
<p>Dr Sonja Lauterbach and Kubendran Naidoo for all their help, advice and care in the lab.</p><br />
<p> Dr Toru Nakayashiki and Prof Hirotada Mori from the Nara Institute of Science and Technology for their kind donation of the <i>E. coli</i> CheZ deletion mutant and parental strain.</p><br />
<p>Dr Joy Sinha and Prof Justin Gallivan from Emory University for their time and the invaluable advice on riboswitches and bacterial chemotaxis.</p> <br />
<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/AcknowledgementsTeam:WITS-CSIR SA/AboutUs/Acknowledgements2011-10-22T17:20:53Z<p>Nkruger: </p>
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<h1> Acknowledgements </h1><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Sponsors'"><br />
<p>The Wits-CSIR 2011 iGEM team was proudly sponsored by:</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a1/Wits_Home_Page_Sponsors.jpg"></center><br />
<p>The Wits_CSIR 2011 iGEM team would like to thank all the above sponsors for their generous financial contributions, which lightened the team’s financial burden thus allowing them to focus their attention on the project. Your generosity has inspired the team to help others and give back to the community, through human advances. </p> <br />
</div><br />
<br> <br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Acknowledgements'"><br />
<p>The Wits_CSIR 2011 iGEM team would like to thank the following advisors and supervisors for their moral and scientific support, especially; the supervisors for leading the team effectively and efficiently in this competitive, fast-paced and ever-changing world of synthetic biology; and Michelle Robinson for her tenacious effort to keep the team on track by assisting the team with implementing time management and organisational skills of the highest calibre. </p><br />
<h2>Supervisors </h2><br />
<p>Karl Rumbold: </p><br />
<p>Marco Weinberg: </p> <br />
<p>Musa Mhlanga: Continuous academic and financial assistance from the Synthetic Biology unit at the CSIR </p><br />
<p>Raymond Sparrow: </p><br />
<br />
<br />
<h2>Advisors </h2><br />
<p>Michelle Robinson </p><br />
<p>Laura Millroy </p><br />
<p>Youtaro Shibayama</p> <br />
<p>Scott Hazelhurst </p><br />
<p>Robyn Brackin </p><br />
<br />
<h2>We would also like to extend our gratitude to the following people:</h2><br />
<p>Prof Kramer from the Wits faculty of Health Science and Prof Crouch from the the Wits faculty of Science for their continued support of the iGEM team.</p><br />
<p>Prof Patrick Arbuthnot for access to of the Anti-viral Gene Therapy Research Unit (AGTRU) laboratory facilities.</p> <br />
<p>Prof Theresa Coetzer for the use of Malaria Research Unit (MRU) laboratory space.</p><br />
<p>Dr Sylvia Fanucchi and Prof Heini Dirr for the use of the Jasco Spectrofluorometer.</p><br />
<p>David Balchin for his assistance with the Jasco Spectrofluorometer.<br />
<p>Robyn Brackin for all her assistance with the Epi-Fluorescence Microscopy imaging.</p><br />
<p>Dr Clement Penny for all his help and advice with the Fluorescent microscopy imaging.</p><br />
<p>The Synthetic Biology Research Unit at the CSIR, for their extensive academic assistance and use of equipment in their lab facilities.</p><br />
<p>Dr Sonja Lauterbach and Kubendran Naidoo for all their help, advice and care in the lab.</p><br />
<p> Dr Toru Nakayashiki and Prof Hirotada Mori from the Nara Institute of Science and Technology for their kind donation of the <i>E. coli</i> CheZ deletion mutant and parental strain.</p><br />
<p>Dr Joy Sinha and Prof Justin Gallivan from Emory University for their time and the invaluable advice on riboswitches and bacterial chemotaxis.</p> <br />
<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/AcknowledgementsTeam:WITS-CSIR SA/AboutUs/Acknowledgements2011-10-22T17:19:36Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
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<h1> Acknowledgements </h1><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Sponsors'"><br />
<p>The Wits-CSIR 2011 iGEM team was proudly sponsored by:</p><br />
<center><img src="https://static.igem.org/mediawiki/2011/a/a1/Wits_Home_Page_Sponsors.jpg"></center><br />
<p>The Wits_CSIR 2011 iGEM team would like to thank all the above sponsors for their generous financial contributions, which lightened the team’s financial burden thus allowing them to focus their attention on the project. Your generosity has inspired the team to help others and give back to the community, through human advances. </p> <br />
<br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Acknowledgements'"><br />
<p>The Wits_CSIR 2011 iGEM team would like to thank the following advisors and supervisors for their moral and scientific support, especially; the supervisors for leading the team effectively and efficiently in this competitive, fast-paced and ever-changing world of synthetic biology; and Michelle Robinson for her tenacious effort to keep the team on track by assisting the team with implementing time management and organisational skills of the highest calibre. </p><br />
<h2>Supervisors </h2><br />
<p>Karl Rumbold: </p><br />
<p>Marco Weinberg: </p> <br />
<p>Musa Mhlanga: Continuous academic and financial assistance from the Synthetic Biology unit at the CSIR </p><br />
<p>Raymond Sparrow: </p><br />
<br />
<br />
<h2>Advisors </h2><br />
<p>Michelle Robinson </p><br />
<p>Laura Millroy </p><br />
<p>Youtaro Shibayama</p> <br />
<p>Scott Hazelhurst </p><br />
<p>Robyn Brackin </p><br />
<br />
<h2>We would also like to extend our gratitude to the following people:</h2><br />
<p>Prof Kramer from the Wits faculty of Health Science and Prof Crouch from the the Wits faculty of Science for their continued support of the iGEM team.</p><br />
<p>Prof Patrick Arbuthnot for access to of the Anti-viral Gene Therapy Research Unit (AGTRU) laboratory facilities.</p> <br />
<p>Prof Theresa Coetzer for the use of Malaria Research Unit (MRU) laboratory space.</p><br />
<p>Dr Sylvia Fanucchi and Prof Heini Dirr for the use of the Jasco Spectrofluorometer.</p><br />
<p>David Balchin for his assistance with the Jasco Spectrofluorometer.<br />
<p>Robyn Brackin for all her assistance with the Epi-Fluorescence Microscopy imaging.</p><br />
<p>Dr Clement Penny for all his help and advice with the Fluorescent microscopy imaging.</p><br />
<p>The Synthetic Biology Research Unit at the CSIR, for their extensive academic assistance and use of equipment in their lab facilities.</p><br />
<p>Dr Sonja Lauterbach and Kubendran Naidoo for all their help, advice and care in the lab.</p><br />
<p> Dr Toru Nakayashiki and Prof Hirotada Mori from the Nara Institute of Science and Technology for their kind donation of the <i>E. coli</i> CheZ deletion mutant and parental strain.</p><br />
<p>Dr Joy Sinha and Prof Justin Gallivan from Emory University for their time and the invaluable advice on riboswitches and bacterial chemotaxis.</p> <br />
<br />
<br />
</div><br />
<br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</div><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/ContactUsTeam:WITS-CSIR SA/AboutUs/ContactUs2011-10-22T17:11:20Z<p>Nkruger: </p>
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Cnr Yale and Empire Road,<br /><br />
Johannesburg,<br /><br />
South Africa<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugsTeam:WITS-CSIR SA/AboutUs/MeetOurBugs2011-10-22T17:09:32Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Parts">Parts submitted</a></li><br />
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
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<p>Some crazy scientists tried to PCR out my CheZ gene. Don't they know that i'm non-motile. It's a pity i couldn't swim away!</p> <br />
<h2>XL1 Blue</h2><br />
<p>Nervous for tomorrow's PCR</p><br />
<h2>XL1 Blue</h2><br />
<p>Robbed of my CheZ gene. Geez, that's Jo'burg for you</p><br />
<h2>CheZ deletion mutant</h2><br />
<p>Leaving for South Africa tomorrow. Bye bye Japan!</p><br />
<h2>CheZ deletion mutant</h2><br />
<p>Made it safely through customs!!!</p><br />
<h2>CheZ deletion mutant</h2><br />
<p>Geez! It's been 7 days! When will i arrive? My neighbours won't stop multiplying and i'm SO hungry!</p><br />
<h2>DH5 alpha</h2><br />
<p>Doing Kiegel exercises to keep my cell wall tight!</p><br />
<h2>DH5 alpha</h2><br />
<p>My favourite daughter cell was left behind when our colony was picked. Noooo! :'( </p><br />
<h2>CheZ deletion mutant</h2><br />
<p>Finally! A PLATE of food!</p><br />
<h2>XL1 Blue</h2><br />
<p>John Lennon, YOU ROCK</p><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/AcknowledgementsTeam:WITS-CSIR SA/AboutUs/Acknowledgements2011-10-22T17:07:08Z<p>Nkruger: </p>
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<p>The Wits_CSIR 2011 iGEM team would like to thank the following advisors and supervisors for their moral and scientific support, especially; the supervisors for leading the team effectively and efficiently in this competitive, fast-paced and ever-changing world of synthetic biology; and Michelle Robinson for her tenacious effort to keep the team on track by assisting the team with implementing time management and organisational skills of the highest calibre. </p><br />
<h2>Supervisors </h2><br />
<p>Karl Rumbold: </p><br />
<p>Marco Weinberg: </p> <br />
<p>Musa Mhlanga: Continuous academic and financial assistance from the Synthetic Biology unit at the CSIR </p><br />
<p>Raymond Sparrow: </p><br />
<br />
<br />
<h2>Advisors </h2><br />
<p>Michelle Robinson </p><br />
<p>Laura Millroy </p><br />
<p>Youtaro Shibayama</p> <br />
<p>Scott Hazelhurst </p><br />
<p>Robyn Brackin </p><br />
<br />
<h2>We would also like to extend our gratitude to the following people:</h2><br />
<p>Prof Kramer from the Wits faculty of Health Science and Prof Crouch from the the Wits faculty of Science for their continued support of the iGEM team.</p><br />
<p>Prof Patrick Arbuthnot for access to of the Anti-viral Gene Therapy Research Unit (AGTRU) laboratory facilities.</p> <br />
<p>Prof Theresa Coetzer for the use of Malaria Research Unit (MRU) laboratory space.</p><br />
<p>Dr Sylvia Fanucchi and Prof Heini Dirr for the use of the Jasco Spectrofluorometer.</p><br />
<p>David Balchin for his assistance with the Jasco Spectrofluorometer.<br />
<p>Robyn Brackin for all her assistance with the Epi-Fluorescence Microscopy imaging.</p><br />
<p>Dr Clement Penny for all his help and advice with the Fluorescent microscopy imaging.</p><br />
<p>The Synthetic Biology Research Unit at the CSIR, for their extensive academic assistance and use of equipment in their lab facilities.</p><br />
<p>Dr Sonja Lauterbach and Kubendran Naidoo for all their help, advice and care in the lab.</p><br />
<p> Dr Toru Nakayashiki and Prof Hirotada Mori from the Nara Institute of Science and Technology for their kind donation of the <i>E. coli</i> CheZ deletion mutant and parental strain.</p><br />
<p>Dr Joy Sinha and Prof Justin Gallivan from Emory University for their time and the invaluable advice on riboswitches and bacterial chemotaxis.</p> <br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SATeam:WITS-CSIR SA2011-10-22T17:05:42Z<p>Nkruger: </p>
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<td> <p>A defining characteristic of humanity is the constantly evolving sophistication with which human beings communicate and store information. Biologically, data communication networks exist too. The transfer of data occurs via signalling molecules incorporated into highly regulated and integrated networks.Our goal is to construct a bacterial communication network that will allow a group of engineered bacteria to transport messages in a directed manner, with the final aim of completing location-based functional tasks.We have focused on one aspect of this network: the directed transport of the message-carrying “communication modules” within our biological network. These communication modules will take the form of bacteria, which can move over physical distances carrying information. We have achieved this through engineering the exogenously controlled chemotactic behaviour of bacteria using synthetic riboswitches to regulate the expression of the CheZ protein required for bacterial motility.<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/AttributionsTeam:WITS-CSIR SA/AboutUs/Attributions2011-10-22T17:04:41Z<p>Nkruger: </p>
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<h2> Lab work</h2> <br />
<p> All of the lab work presented on this wiki is the work of the team members. </p><br />
<p>- Cloning was done by all team members. </p><br />
<p>- Fluorometry experiments were conducted by Ezio and Gloria. </p><br />
<p>- Fluorescence microscopy was conducted by Sasha, with the help of Robyn Brackin and Dr. Clement Penny. </p><br />
<p>- Motility and Chemotaxis Assays were performed by Natasia, with some help from all the other team members. </p><br />
<br />
<h2> Modelling</h2> <p><br />
All modelling was completed by Brad, our information engineer.</p><br />
<h2> Logo</h2> <p>Our logo was also designed by Brad</p><br />
<h2> Animation</h2> <br />
<p>This task was performed by a professional animator, Jess Scholtz.</p><br />
<h2> Website</h2> <p><br />
Our site was built by a contracted web developer, Shaun Farrell.</p><br />
<p>All team members were involved in uploading, editing and updating the wiki content. </p><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/AttributionsTeam:WITS-CSIR SA/AboutUs/Attributions2011-10-22T17:04:09Z<p>Nkruger: </p>
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelationsTeam:WITS-CSIR SA/Outreach/PublicRelations2011-10-22T17:02:28Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Characterization</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Modelling">Modelling</a></li><br />
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<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Forum">Synthetic biology forum</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Survey">Survey</a></li><br />
</ul><br />
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<br />
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<li><a href="./" class="dir">About us</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeam">Meet the team</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelations">Media attention</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
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<img src="https://static.igem.org/mediawiki/2011/6/63/Wits_Logo_Biotweet_Rearrange.gif" alt="Biotweet" height="60px"<br />
style="float: left; margin-bottom: 5px; border: none;" /><br />
</a><a href="https://2011.igem.org/Main_Page" target="_blank"><br />
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style="height: 100%;"><br />
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<div style="height: 100%; overflow-y: auto;"><br />
<br />
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<p>The South African press was very supportive of our entry into this competition. We had interviews on Cape Talk and Talk Radio 702. We also had an article featured in <i>The New Age</i> newspaper. Wits university published an article about Biotweet on their website home page. </p><br />
<img src="https://static.igem.org/mediawiki/2011/9/9a/Wits_PR_Newspaper.JPG" align="right" style="padding: 5px;" /><br />
<p>For the material, click the links below:</p><br />
<br />
<a href="https://static.igem.org/mediawiki/2011/e/e9/Wits_PR702_inteview_Part_1.mp3">702 interview with Sasha Part 1</a><br />
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<a href="https://static.igem.org/mediawiki/2011/7/7b/Wits_702_interview_Part2_final_final.mp3">702 interview with Sasha Part 2</a><br />
<br/><br />
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<a href="https://static.igem.org/mediawiki/2011/2/2b/Wits_Cape_talk_interview.mp3">Cape talk interview with Ezio</a><br />
<br/><br />
<br/><br />
<a href="http://www.wits.ac.za/newsroom/newsitems/201109/13841/news_item_13841.html">Wits News article</a><br />
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<a href="http://thenewage.co.za/29458-1008-53-Wits_students_put_social_networking_to_use">New Age news article</a><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA.test1Team:WITS-CSIR SA.test12011-10-22T16:59:18Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA">Home</a></li><br />
<li><a href="#" class="dir">Project</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Concept">Overview</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Characterization</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Modelling">Modelling</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Collaboration/Index">Collaboration</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Notebook">Lab notebook</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Applications">Potential applications</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Achievements">Achievements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Parts">Parts submitted</a></li><br />
<li><a href="./" class="dir">Outreach</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Forum">Synthetic biology forum</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Survey">Survey</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Safety">Safety</a></li><br />
<br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Diary/Gallery">Gallery</a></li><br />
<li><a href="./" class="dir">About us</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeam">Meet the team</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelations">Media attention</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
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<img src="https://static.igem.org/mediawiki/2011/6/63/Wits_Logo_Biotweet_Rearrange.gif" alt="Biotweet" height="60px"<br />
style="float: left; margin-bottom: 5px; border: none;" /><br />
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class="center"><br />
<div style="width: 903px; margin: auto;"><br />
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Biotweet: A collaboration between the University of the Witwatersrand and the CSIR'"><br />
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<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'What is Biotweet?'"><br />
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<td> <p>A defining characteristic of humanity is the constantly evolving sophistication with which human beings communicate and store information. Biologically, data communication networks exist too. The transfer of data occurs via signalling molecules incorporated into highly regulated and integrated networks.Our goal is to construct a bacterial communication network that will allow a group of engineered bacteria to transport messages in a directed manner, with the final aim of completing location-based functional tasks.We have focused on one aspect of this network: the directed transport of the message-carrying “communication modules” within our biological network. These communication modules will take the form of bacteria, which can move over physical distances carrying information. We have achieved this through engineering the exogenously controlled chemotactic behaviour of bacteria using synthetic riboswitches to regulate the expression of the CheZ protein required for bacterial motility.<br />
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<td> <p>A defining characteristic of humanity is the constantly evolving sophistication with which human beings communicate and store information. Biologically, data communication networks exist too. The transfer of data occurs via signalling molecules incorporated into highly regulated and integrated networks.Our goal is to construct a bacterial communication network that will allow a group of engineered bacteria to transport messages in a directed manner, with the final aim of completing location-based functional tasks.We have focused on one aspect of this network: the directed transport of the message-carrying “communication modules” within our biological network. These communication modules will take the form of bacteria, which can move over physical distances carrying information. We have achieved this through engineering the exogenously controlled chemotactic behaviour of bacteria using synthetic riboswitches to regulate the expression of the CheZ protein required for bacterial motility.<br />
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<td> <p>A defining characteristic of humanity is the constantly evolving sophistication with which human beings communicate and store information. Biologically, data communication networks exist too. The transfer of data occurs via signalling molecules incorporated into highly regulated and integrated networks.Our goal is to construct a bacterial communication network that will allow a group of engineered bacteria to transport messages in a directed manner, with the final aim of completing location-based functional tasks.We have focused on one aspect of this network: the directed transport of the message-carrying “communication modules” within our biological network. These communication modules will take the form of bacteria, which can move over physical distances carrying information. We have achieved this through engineering the exogenously controlled chemotactic behaviour of bacteria using synthetic riboswitches to regulate the expression of the CheZ protein required for bacterial motility.<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA.test1Team:WITS-CSIR SA.test12011-10-22T16:53:30Z<p>Nkruger: </p>
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<td> <p>A defining characteristic of humanity is the constantly evolving sophistication with which human beings communicate and store information. Biologically, data communication networks exist too. The transfer of data occurs via signalling molecules incorporated into highly regulated and integrated networks.Our goal is to construct a bacterial communication network that will allow a group of engineered bacteria to transport messages in a directed manner, with the final aim of completing location-based functional tasks.We have focused on one aspect of this network: the directed transport of the message-carrying “communication modules” within our biological network. These communication modules will take the form of bacteria, which can move over physical distances carrying information. We have achieved this through engineering the exogenously controlled chemotactic behaviour of bacteria using synthetic riboswitches to regulate the expression of the CheZ protein required for bacterial motility.<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA.test1Team:WITS-CSIR SA.test12011-10-22T16:51:09Z<p>Nkruger: </p>
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<td> <p>A defining characteristic of humanity is the constantly evolving sophistication with which human beings communicate and store information. Biologically, data communication networks exist too. The transfer of data occurs via signalling molecules incorporated into highly regulated and integrated networks.Our goal is to construct a bacterial communication network that will allow a group of engineered bacteria to transport messages in a directed manner, with the final aim of completing location-based functional tasks.We have focused on one aspect of this network: the directed transport of the message-carrying “communication modules” within our biological network. These communication modules will take the form of bacteria, which can move over physical distances carrying information. We have achieved this through engineering the exogenously controlled chemotactic behaviour of bacteria using synthetic riboswitches to regulate the expression of the CheZ protein required for bacterial motility.<br />
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<td> <p>A defining characteristic of humanity is the constantly evolving sophistication with which human beings communicate and store information. Biologically, data communication networks exist too. The transfer of data occurs via signalling molecules incorporated into highly regulated and integrated networks.Our goal is to construct a bacterial communication network that will allow a group of engineered bacteria to transport messages in a directed manner, with the final aim of completing location-based functional tasks.We have focused on one aspect of this network: the directed transport of the message-carrying “communication modules” within our biological network. These communication modules will take the form of bacteria, which can move over physical distances carrying information. We have achieved this through engineering the exogenously controlled chemotactic behaviour of bacteria using synthetic riboswitches to regulate the expression of the CheZ protein required for bacterial motility.<br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeamTeam:WITS-CSIR SA/AboutUs/MeetTheTeam2011-10-22T16:35:01Z<p>Nkruger: </p>
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<h1><br />
Meet the team</h1><br />
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Name:<br />
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Sasha Reznichenko<br />
</td><br />
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<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/c/c5/WITS_SASH_final_.JPG" class="thumbnail" align="right"/><br />
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Field of Study:<br />
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Molecular Medicine<br />
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About Sasha:<br />
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<p>Sasha is the “MOM” of the team – making sure everyone eats on time and has a substantial meal. She is always the one that is most stressed out. She is very dedicated to her work – only arriving 10-15min late, like a true lady. While waiting for a PCR reaction, Sasha can be found in the common room painting her nails, talking and laughing (too loud for anyone else in the department to concentrate). Her primary interest is the field human genetics and her passion lies in the medical successes that can be achieved through developments such as synthetic biology and gene therapy. She has thoroughly enjoyed doing a project with a team of colleagues since she always has someone to talk to and there is never a dull moment with all the crazy conversations that arise in the lab. </p><br />
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Name:<br />
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<td width=45%><br />
Ezio Fok<br />
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Field of Study:<br />
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Molecular Medicine<br />
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<p>Ezio has a keen interest in all things that are fun. He enjoys day dreaming and jamming. His drink of preference is Schweppes Dry Lemon. He claims that it is wildly delicious and provides him with a regular, prophylactic dose of the malaria drug quinine (30 mg/l). He is commonly known for his wisdom, which is demonstrated by his mane of grey hair. His hobbies involve sleeping, eating food and learning how to swim. He also enjoys things like iGEM and science.</p><br />
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Name:<br />
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Gloria Hlongwane<br />
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Field of Study:<br />
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Microbiology and biotechnology <br />
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About Gloria:<br />
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<td colspan="2"><br />
<p>Gloria is a super crazy stickler for perfection with a broad sense of humour. She likes organizing all her thoughts and ideas that occur throughout the busy day on paper towels, particularly serviettes. She talks non-stop, that is,every time, all day and every day. She believes that, she is probably the most affable and aloof person she has ever met. If not doing work, she is somewhere doing one of three things; (1)laughing, (2) thinking of what to eat or (3) eating like a horse. </p><br />
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Name:<br />
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Natasia Kruger<br />
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<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/a/a5/WITS_TASH.JPG" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
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Field of Study:<br />
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<td><br />
Microbiology and biotechnology <br />
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About Natasia:<br />
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<p>Natasia is a workaholic, which means if you can't find her at any time of the day or night, you probably haven't checked the lab. Her passion lies with biotechnology and iGEM has narrowed this interest to the specific field of synthetic biology. She is the “listener” of the team and helps team members calm down when they are STRESSED! She has many late night lab activities including trips to the vending machine and inventing new games to pass time, such as the ability to play tetris manually o_O. She thoroughly enjoyed the iGEM process and would recommend it to engineering or biology undergrads! </p><br />
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Bradley Marques<br />
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<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/9/93/BRAD_-_WITS_cropped_Me_copy.png" class="thumbnail" /><br />
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Field of Study:<br />
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<td><br />
Information engineering </td><br />
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<th><br />
About Bradley:<br />
</th><br />
<td colspan="2"><br />
<p>As a biomedical engineer, Bradley likes to think of blood flow as an electrical current, and is occasionally known to model people as spheres. He can usually be found whittling the early hours of the morning away banging out an emergent behaviour simulation. His information engineering degree has piqued his interest in pervasive computing, the human-computer interface as well as an active interest in digital arts. When not contemplating his lack of existence after Ray Kurzweils’ singularity, he may found trolling game design forums with the judicious use of smileys.</p><br />
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Michelle Robinson<br />
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<th><br />
Field of Study:<br />
</th><br />
<td><br />
Master's in Molecular Medicine<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Michelle:<br />
</th><br />
<td colspan="2"><br />
<p>After experiencing firsthand the fun and insanity of iGEM as a member of the first-ever Wits iGEM team, Michelle Robinson elected to become a student advisor to the 2011 team. In between working towards her Masters of Science in Medicine Michelle practices her nagging skills and makes sure that the team knows that ‘we only have 62 days, 13 hours, 42 minutes and 4 seconds until the Wiki Freeze!’ The iGEM competition inspired her to stay within synthetic biology and Michelle is currently working on a gene therapy-based project with a synthetic biology twist. When she isn’t slaving in the lab or slave-driving the iGEM team, Michelle pursues some of her other interests such as activism and advocacy - she is involved in various projects that advocate for public and rural health; as well as scientific literacy and the interface between science and the media.</p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Laura Millroy<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/2/23/Laura_image.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
PhD Molecular Medicine<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Laura:<br />
</th><br />
<td colspan="2"><br />
<p>Laura has a keen interest in synthetic biology and genetic engineering, especially in relation to advances in human health. Her PhD project involves the development of aptamer siRNA chimeras for the treatment of HIV. Beyond lab work and research, Laura spends her time baking, painting and editing iGEM documents! Laura is a student advisor to the team for a second year. She enjoys helping the team to realise their goals while understanding the importance of having fun.</p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
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<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Youtaro Shibayama<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/0/0c/CIMG6093.JPG" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
Genetics<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Youtaro:<br />
</th><br />
<td colspan="2"><br />
<p>During his postgraduate studies, Youtaro specialized in the genetics of<br />
bacteriophages of Gram positive pathogens and also in the structures of<br />
endogenous plasmids occurring in these bacteria. He is currently pursuing his<br />
interests in the nuclear architecture of both eukaryotic and prokaryotic cells as a<br />
postdoc in Synthetic Biology of the CSIR.</p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Robyn Brackin<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/2/25/WITS_ROBYN.JPG" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field:<br />
</th><br />
<td><br />
Gene Expression and Biophysics<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Robyn:<br />
</th><br />
<td colspan="2"><br />
<p>Robyn has always had a thrist for acquiring scientific knowledge, from her early years when<br />
<br />
she dissected stamens from the flowers in her mothers garden to later on in life when she<br />
<br />
acquired degrees in both Molecular Biology and Electrical Engineering. Now working as<br />
<br />
a Biomedical Engineer she gets to play not only with pipettes, eppies and solutions but<br />
<br />
also with soft polymers, resistors, wago controllers and really cool microscopes. Robyn<br />
<br />
is an advisor to team for a second year and enjoys helping the team with imaging on the<br />
<br />
microscope. Robyn and the team really enjoy seeing flourescently labelled bacteria! </p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Dr Karl Rumbold<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/5/52/Karl_image.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
Industrial <br> Biotechnology </br> </td><br />
</tr><br />
<tr><br />
<th><br />
About Karl:<br />
</th><br />
<td colspan="2"><br />
<p>Dr Rumbold is a microbiologist by training; he graduated from the University of Graz, Austria, and started an international academic career at the University of Stellenbosch, South Africa, where he received his PhD. He then held research positions at University College London, Ghent University and the Dutch research organization TNO. He returned to South Africa after accepting a faculty position at the University of the Witwatersrand, Johannesburg in 2009. He thinks iGEM is an awesome experience and that the team is so fortunate to be in it for the second time. </p> </td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Prof Marco Weinberg<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/1/1a/MarcoAdvisorProfileFinal4.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
RNA Biology and <br> Gene Therapy </br> </td><br />
</tr><br />
<tr><br />
<th><br />
About Marco:<br />
</th><br />
<td colspan="2"><br />
<p> Prof Marco Weinberg loves science…and good coffee…but mostly science. An associate professor of the University of the Witwatersrand, Marco works within the Antiviral Gene Therapy Research Unit and became entangled with the iGEM competition quite by accident. He has been one of the team faculty advisors for the past two years, working closely with the biology students in particular. He believes that it is important to push the team members to approach their project with scientific rigour and a strong work ethic, but also to remind them of the importance of being imaginative, and to inspire them to actively enjoy the experience of working in such a stimulating and creative environment. </p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Dr Raymond Sparrow<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/3/35/Raymond_image.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field:<br />
</th><br />
<td><br />
CSIR: Biosciences<br />
</td><br />
</tr><br />
<tr><br />
<th><br />
About Raymond:<br />
</th><br />
<td colspan="2"><br />
<p> Dr Sparrow is from the UK and came over to South Africa in 2005 to the National Laser Centre then transferred to Biosciences in 2007. He is currently the Human Capital Development Manager for the Synthetic Biology ERA.<br />
<br />
In the UK he conducted research into ultra-fast photosynthetic light harvesting and energy transfer reactions.<br />
<br />
He was a Post-Doctoral Fellow at the University of Central Lancashire and conducted research at the Lasers For Science Facility at the Rutherford Appleton Laboratory and Daresbury Synchrotron Radiation Source. Then a Higher Scientific Officer at Daresbury on a Vacuum Ultra Violet Beamline.<br />
<br />
From 1992 - 2005 he was a lecturer in Life Sciences at Bromley College of F&HE.</p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
<table border="0" cellpadding="2" cellspacing="2" class="pretty"><br />
<tr><br />
<th width=20%><br />
Name:<br />
</th><br />
<td width=45%><br />
Dr Musa Mhlanga<br />
</td><br />
<td rowspan="2" valign="top"><br />
<img alt="Team Member" src="https://static.igem.org/mediawiki/2011/5/59/Musa_image2.jpg" class="thumbnail" /><br />
<div class="ui-helper-clearfix"><br />
</div><br />
</td><br />
</tr><br />
<tr><br />
<th><br />
Field of Study:<br />
</th><br />
<td><br />
Cell Biology and Biophysics </td><br />
</tr><br />
<tr><br />
<th><br />
About Musa:<br />
</th><br />
<td colspan="2"><br />
<p>Dr Musa Mhlanga is the research leader of the Synthetic Biology ERA of the CSIR’s gene expression and biophysics research group. He studied at the New York University Medical School and the Rockefeller University before heading off to France for postdoctoral research. He has research interests in gene expression, imaging and microscopy, nuclear architecture, single molecule biology and high-throughput screening, and has presented a number of papers internationally in these fields. Musa has been instrumental in setting-up synthetic biology in South Africa and has played a significant role in helping both the 2010 and 2011 iGEM teams. </p><br />
</td><br />
</tr><br />
<tr><br />
<br />
</tr><br />
</table><br />
</p><br />
</div><br />
<br />
<br /><br />
<br />
<br />
<br />
</div><br />
<br />
<br />
<br />
</div><br />
</div><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Diary/GalleryTeam:WITS-CSIR SA/Diary/Gallery2011-10-22T16:32:00Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Diary/GalleryTeam:WITS-CSIR SA/Diary/Gallery2011-10-22T16:27:16Z<p>Nkruger: </p>
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Forum">Synthetic biology forum</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Survey">Survey</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Safety">Safety</a></li><br />
<br />
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<li><a href="./" class="dir">About us</a><br />
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<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeam">Meet the team</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelations">Media attention</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
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</html></div>Nkrugerhttp://2011.igem.org/Team:WITS-CSIR_SA/Diary/GalleryTeam:WITS-CSIR SA/Diary/Gallery2011-10-22T16:25:08Z<p>Nkruger: </p>
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<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Concept">Overview</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Characterization">Characterization</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Modelling">Modelling</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Collaboration/Index">Collaboration</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Notebook">Lab notebook</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Applications">Potential applications</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Achievements">Achievements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Parts">Parts submitted</a></li><br />
<li><a href="./" class="dir">Outreach</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/SciBono">Scibono experience</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Forum">Synthetic biology forum</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/Survey">Survey</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Safety">Safety</a></li><br />
<br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Diary/Gallery">Gallery</a></li><br />
<li><a href="./" class="dir">About us</a><br />
<ul><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetTheTeam">Meet the team</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/Outreach/PublicRelations">Media attention</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Attributions">Attributions</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/Acknowledgements">Acknowledgements</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/MeetOurBugs">Meet our bugs!</a></li><br />
<li><a href="https://2011.igem.org/Team:WITS-CSIR_SA/AboutUs/ContactUs">Contact us</a></li><br />
</ul><br />
</li><br />
</ul><br />
</div><br />
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