Team:MIT tconst

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<li><a href="#">Characterization</a></li>

                         <li><a href="#">Overview</a></li>

<li><a href="#">Standard and Design</a></li>

<li><a href="#">Experiments</a></li>

(<a href="http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html">Collins, 2000</a>). <br> <br>&nbsp;&nbsp;&nbsp;&nbsp;The 2010 MIT iGEM team saw that it was good, and decided to implement the Collins toggle in E.coli to create cells with bistable phenotypes.

&nbsp;&nbsp;&nbsp;&nbsp;We first wanted to make sure the parts we were working with were functional and reliable. As we wished to use the pTSMa plasmid as part of our circuit, we tested the original Collins toggle (pTSMa co-transformed with pCIRa). <div class="sidep"><a href="https://static.igem.org/mediawiki/2010/b/bf/Collins.jpg" class="thickbox" title="Our first patterned image created by exposing masked cells to UV light. The cells were made by co-transforming the Collins toggle plasmids pTSMa and pCIRa, in a recreation of the Collins toggle experiment."><img width=170px src="https://static.igem.org/mediawiki/2010/b/bf/Collins.jpg"></a><br>Our recreation of the Collins toggle experiment validated pTSMa as a functional bistable toggle plasmid.</div>According to the paper, the area exposed to UV light should turn bright fluorescent green, and the rest of the cells should remain non-fluorescent. We recreated this experiment and our results demonstrate that the Collins toggle was working as it is described in the paper.<br><div class="sidep" style="width: 350px; display: block; float: left;"><a href="https://static.igem.org/mediawiki/2010/b/ba/Collinstoggle.png" class="thickbox" title="A simplified Collins Toggle."><img width=350px src="https://static.igem.org/mediawiki/2010/b/ba/Collinstoggle.png"></a><br>The basic Collins Toggle plasmids pTSMa and pCIRa. We used pTSMa in our own experiments as a bistable toggle.</div><br><br><br><br><br><br><br><br><br><br><br>

&nbsp;&nbsp;&nbsp;&nbsp;With our newly verified (borrowed) plasmid, we got to work on building our own response to UV induction. Using the same promoter used in pCIRa (the PcI-OR promoter that is induced by AHL/LuxR complex and inhibited by cI), we created a biobrick that would respond to UV induction by producing mCherry fluorescent protein when co-transformed with pTSMa. Our part would also constitutively produce LuxR, the protein that binds AHL and becomes a transcription factor that encourages the activity of the PcI-OR promoter.<br><div class="sidep" style="width: 450px; float: left;"><a href="https://static.igem.org/mediawiki/igem.org/e/e3/K415006.png" class="thickbox" title="Our first working part with a readout was K415006."><img width=450px src="https://static.igem.org/mediawiki/igem.org/e/e3/K415006.png"></a><br>Our first working part with a readout was K415006.</div><div class="sidep"><a href="https://static.igem.org/mediawiki/2010/8/80/First.png" class="thickbox" title="The first patterned image created by exposing masked cells to UV light. The cells were made by co-transforming the Collins toggle plasmid pTSMa with our composite biobrick K415006."><img height=100px src="https://static.igem.org/mediawiki/2010/8/80/First.png"></a><br>The first picture of mCherry fluorescence induced by UV light.</div><br><br><br><br>

<br>&nbsp;&nbsp;&nbsp;&nbsp;After much fine-tuning of the power of the UV exposure, the concentrations of AHL and IPTG, and mask cutting, a pattern of fluorescence finally emerged -- the first image.<br>

&nbsp;&nbsp;&nbsp;&nbsp;As an intermediate in our project, we had created a bacterial camera with the ability to take and store images, reproduce the image on command (when AHL is added to the medium), and reset the entire plate to a blank slate whenever we pleased. All these abilities were a result of the bistable toggle we implemented in our design. In the context of past iGEM bacterial cameras, our toggle-based camera is a great improvement. UT-Austin created the first bacterial camera in 2005, exposing their cells to light for 12-15 hours to produce an image. Our toggle can snap and store an image in a matter of seconds.

&nbsp;&nbsp;&nbsp;&nbsp;We wanted our cells to be self-inducing and our signal to be self-propagating in addition to the circuit being sensitive and specific to user inputs. To do this, we added a part that would generate one of the necessary chemicals for transcription of the mCherry fluorescent protein. <br><br>

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R0065 is a hybrid promoter that the MIT 2010 iGEM team used in our composite parts to control transcription of mCherry fluorescence. In conjunction with our toggle, pLPTa, this promoter would only allow expression of fluorescence in the presence of AHL, LuxR, and UV exposure. While we found the promoter very useful, it is described many times on its <a href="http://partsregistry.org/Part:BBa_R0065">parts registry page</a> as being "leaky". After looking into the promoter, we realized this was due to two -10 promoter sequences, possibly due to an error when designing the hybrid promoter.