Team:MIT toggle

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  <li class="sel"><a href="#">Toggle Overview</a><br></li>

    <ul><li><a href="#">Toggle Construction</a><br></li>

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

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

    <ul><li><a href="#">Phage Construction</a></li>

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

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

    <ul><li><a href="#">Protocol & Design</a></li>

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

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<div class="bodybaby" style="font-size: 12px;">toggle by parts</div><a href="https://static.igem.org/mediawiki/2010/c/cc/UV_Circuit1.jpg" class="thickbox" title="Bacterial UV Toggle for controlling phage polymerization"><img src="https://static.igem.org/mediawiki/2010/c/cc/UV_Circuit1.jpg" width=215px></a>

<div class="bodybaby" style="font-size: 12px;">toggle abstraction</div><a href="https://static.igem.org/mediawiki/2010/4/43/Abstractcircuit.png" class="thickbox" title="Abstracted bacterial toggle circuit to logic gates."><img src="https://static.igem.org/mediawiki/2010/4/43/Abstractcircuit.png" width=215px></a><br><br><br>

<div class="bodybaby" style="font-size: 12px;">toggle black-boxed</div><a href="https://static.igem.org/mediawiki/2010/1/18/Circuit.png" class="thickbox" title="Black-boxed bacterial toggle circuit. Also shown are the phage linker constructs."><img src="https://static.igem.org/mediawiki/2010/1/18/Circuit.png" width=215px></a>

<table width=70%><tr><td><div class="bodybaby">bacterial biobrick construction</div></td>

<tr><td><br>The Bacterial team focused on implementing and improving the Collins toggle. The ultimate goal is to connect our toggle to our phage module, so that exposing UV light in a pattern on a lawn of bacteria will result in phage production and polymerization in the exposed area. As an intermediate in our project, we have managed to create what is essentially an improved bacterial camera capable of instantaneous photography.<br><br>We implemented part of the <a href="http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html">Collins toggle</a> in our bacteria, improving upon previous iGEM bacterial cameras by shortening the exposure time from hours <a href="http://parts.mit.edu/wiki/index.php/UT_Austin_2005">(UT-Austin 2005)</a> to seconds. During our experiments, noticeable cell death in UV-exposed regions prompted development of an additional feature. Our bacterial circuit improves upon the Collins toggle in that the power of UV light required to switch the toggle is reduced, resulting in significantly more cells surviving the image capture process.<br><br></td>

<tr><td><a href="https://static.igem.org/mediawiki/2010/d/dc/Uvoverview.png" class="thickbox"><img src="https://static.igem.org/mediawiki/2010/d/dc/Uvoverview.png"></a></td>

(<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.

<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>

We planned for the toggle to create patterned fluorescence and phage polymerization in response to exposing the cells to UV light. <br>&nbsp;&nbsp;&nbsp;&nbsp;Our first construction resulted in a composite biobrick K415006 that, once co-transformed with pTSMa from the Collins toggle, induced mCherry fluorescence in cells that were exposed to UV light. 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>

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&nbsp;&nbsp;&nbsp;&nbsp;Then we decided to make the signal amplify itself. We added a gene onto our composite biobrick that would produce additional AHL if induced by UV light. The new composite biobrick was dubbed K415022. In order to view the propagation, we recorded a movie beginning from the moment the cells were induced with UV. In addition to viewing the progression of the fluorescence, we were able to record how long it took for a true pattern of fluorescence to emerge in our cell lawn.<br><object width="300" height="180" style="display: inline; margin: 0px 16%;"><param name="movie" value="http://www.youtube.com/v/6KHabnSzCJ8?fs=1&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/6KHabnSzCJ8?fs=1&amp;hl=en_US" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="400" height="240"></embed></object>

&nbsp;&nbsp;&nbsp;&nbsp;After further inspection of the movie, we realized it might not be an accurate representation of the fluorescent propagation because we noticed a circle of cell death where the UV exposure had killed some of the cells in the lawn. We then decided to make pLPTa, a low power toggle that

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<a href="https://static.igem.org/mediawiki/2010/e/eb/Igem_uv_prelim.png" class="thickbox" title="Power Modulation using the Low Power Toggle and cells that fluoresce red with UV induction."><img src="https://static.igem.org/mediawiki/2010/e/eb/Igem_uv_prelim.png" width=300px></a><br>Here we see cells controlled by the Low Power Toggle. The cells fluoresce red with UV induction, but at higher UV levels cell death can be seen in the green field.</div> still provided bistability, but required less UV power to induce a toggle switch. <br>

&nbsp;&nbsp;&nbsp;&nbsp;By site-directed mutagenesis, we changed the lambda repressor (cI) gene in the Collins pTSMa to a cI that is more sensitive to cleavage by Rec-A, the enzyme activated by UV light exposure. Thus, we modified the Collins toggle to minimize cell death in the UV-exposed regions while still maintaining its switch-like behavior.