Team:TPHS SanDiego

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Latest revision as of 04:03, 22 June 2013

Berkeley iGEM 2011

In an effort to expand the toolkit available to synthetic biologists, we've taken a system natively responsible for transcriptional activation and modified it to control transcriptional repression. The LasR system from Pseudomonas Aeruginosa requires the presence of a small molecule, C12-3-oxo-AHL, to induce activation of the Plas promoter. By modifying the -10 and -35 sites of the promoter, as well as shifting the location of the LasR binding sites, the new Plas promoter (Plas*) was changed from an inducible to a repressible promoter. Through adding this second functionality, the Plas* promoter could be used in conjunction with a wildtype Plas promoter to control two separate genes whose expression levels are always out of sync. Furthermore, if the bacteria are transfected with a plasmid encoding LasI, the bacteria will be able to turn off gene expression at a critical population density, instead of only being able to turn on gene expression.

Proteins discovered in Jellyfish that have the ability to emit fluorescence.

A clever way to normalize protein expression across cell populations.

A quorum sensing system from Pseudomonas aeruginosa.

Re-engineering the Plas promoter's response to LasR.

We have all been brought together by a mutual interest for synthetic biology. It fascinates us how easily organisms can be tweaked and altered to our liking in this expansive field, so we thought it’d be best get started early. Through reading, lectures, mentorship and hands on experience we opened our minds and fueled our thirst for control over these malleable organisms. We functioned well together and played each other’s strengths for maximum efficiency while simultaneously having the time of our lives. We have learned much and hope to keep putting our best efforts forward till the very end.

Attributions:

All experiments explained here were done by the TPHS students, any previously existing plasmids and materials were supplied by Spencer Scott and the Hasty Lab at UCSD. The plate reader data acquisition was done at a lab where the high school students could not enter, but they are versed on every step of the process. The website layout was adapted from Berkeley iGEM 2011's wiki with their permission. Almost all edits were done by members of the high school team, including the home page, project page, protocols page, daily journal, safety page, and much of the team page. Some things such as the team photos and the parts page were done by Spencer Scott as an instructive lesson on design and using Illustrator and Photoshop. Anything done by an advisor was done as a teaching lesson with close interaction with the students at all times.

The Torrey Pines High School iGEM team would like to thank New England Biolabs, the UCSD Biodynamics Labratory, and Mr. Brinn Belyea.

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-<p>Biosensors have widespread applications ranging from diagnostics to environmental monitoring. Vibrio cholerae's ToxR system can be used as a component in biological sensing devices. ToxS causes ToxR homodimerization, activating transcription of the ctx promoter. By replacing the periplasmic domain of ToxR with existing or engineered ligand-dependent homodimers, we hope to link ToxR dimerization (and gene expression) to the presence of specific ligands. Initially, ToxR constructs proved toxic to E. coli. We built a stress-regulated transcription system that drives relatively high expression of toxic proteins. This allowed us to further engineer ToxR chimeras. We fused an estrogen-dependent dimer with ToxR hoping to create an estrogen biosensor. We observed a range of constitutive phenotypes and plan more experiments to engineer a dose-dependent transcriptional response to estrogen. By fusing existing or engineered ligand dependent homodimers to ToxR, this modular system can be used to build new biosensors. </p>
+<p style="font-family:Georgia;color:black;font-size:14px;">In an effort to expand the toolkit available to synthetic biologists, we've taken a system natively responsible for transcriptional activation and modified it to control transcriptional repression. The LasR system from <i>Pseudomonas Aeruginosa</i> requires the presence of a small molecule, C12-3-oxo-AHL, to induce activation of the Plas promoter. By modifying the -10 and -35 sites of the promoter, as well as shifting the location of the LasR binding sites, the new Plas promoter (Plas*) was changed from an inducible to a repressible promoter. Through adding this second functionality, the Plas* promoter could be used in conjunction with a wildtype Plas promoter to control two separate genes whose expression levels are always out of sync. Furthermore, if the bacteria are transfected with a plasmid encoding LasI, the bacteria will be able to turn off gene expression at a critical population density, instead of only being able to turn on gene expression.</p>
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-  <p style="text-align:center; color:#CECECE;"> A protein with great potential as a general biosensor system.</p> </div>
+  <p style="font-family:Georgia;text-align:center; color:#CECECE;"> Proteins discovered in Jellyfish that have the
+ability to emit fluorescence.</p> </div>
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-<p style="text-align:center; color:#CECECE;"> Chimeric proteins that drive translation off of the Pctx promoter.</p></div>
+<p style="font-family:Georgia;text-align:center; color:#CECECE;"> A clever way to normalize protein expression across cell populations.</p></div>
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-<p style="text-align:center; color:#CECECE;"> Our method for expressing interesting (but toxic) proteins.</p> </div>
+<p style="font-family:Georgia;text-align:center; color:#CECECE;"> A quorum sensing system from <i>Pseudomonas aeruginosa</i>.</p> </div>
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-  <p style="text-align:center; color:#CECECE;"> Bacteria engineered to detect the presence of estrogen. <br></p></div>
+  <p style="font-family:Georgia;text-align:center; color:#CECECE;"> Re-engineering the Plas promoter's response to LasR. <br></p></div>
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 <img src="https://static.igem.org/mediawiki/2011/b/bd/Aboutus.jpg">
-<p>We are Team Berkeley, a cohesive unit of 7 undergraduates and 3 advisers. Earlier this year we planned a complex and risky project, given the short amount of time iGEM made available. We quickly learned each others strengths and weaknesses and developed standard systems of organizational management in order to synchronize our efforts for the many parallel tasks at hand. We created protocols, shared them with one another, and worked together on troubleshooting. Using google docs to keep up with the status of cloning projects, the results of assays, material logistics, or the final steps required to complete a project ensured that through the months of hard work, we fine-tuned our ability to work together. As a team, we have learned firsthand how the synthetic biology community relies on the goal-oriented cooperation of skilled individuals from very different backgrounds and with very different skill sets. Some of us have strong engineering backgrounds while others of us have strong biology backgrounds, but we all share a desire to build synthetic biological systems that solve human problems. We are proud of the project that we have created which we will present at the Jamboree together in October.</p>
+<p style="font-family:Georgia;color:black;font-size:14px;">
-</div>
+We have all been brought together by a mutual interest for synthetic biology. It fascinates us how easily organisms can be tweaked and altered to our liking in this expansive field, so we thought it’d be best get started early. Through reading, lectures, mentorship and hands on experience we opened our minds and fueled our thirst for control over these malleable organisms. We functioned well together and played each other’s strengths for maximum efficiency while simultaneously having the time of our lives. We have learned much and hope to keep putting our best efforts forward till the very end.
+</p></div>
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+<p style="font-family:Georgia;color:black;font-size:16px;"> <b>Attributions:</b></p>
+<p style="font-family:Georgia;color:black;font-size:12px;"> All experiments explained here were done by the TPHS students, any previously existing plasmids and materials were supplied by Spencer Scott and the Hasty Lab at UCSD. The plate reader data acquisition was done at a lab where the high school students could not enter, but they are versed on every step of the process. The website layout was adapted from Berkeley iGEM 2011's wiki with their permission. Almost all edits were done by members of the high school team, including the home page, project page, protocols page, daily journal, safety page, and much of the team page. Some things such as the team photos and the parts page were done by Spencer Scott as an instructive lesson on design and using Illustrator and Photoshop. Anything done by an advisor was done as a teaching lesson with close interaction with the students at all times.
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-<p style="text-align:left; color:#CECECE; font-size:13px; font-weight:bold; padding:3px"> The UC Berkeley iGEM team would like to thank Autodesk and Agilent for their financial support and Synberc, for their administrative support. </p>
+<p style="text-align:center; color:#CECECE; font-size:13px; font-weight:bold; padding:3px"> The Torrey Pines High School iGEM team would like to thank New England Biolabs, the UCSD Biodynamics Labratory, and Mr. Brinn Belyea. </p>
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