Team:Penn/LightActivatedOverview

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<p style="color:black;text-indent:30px;">In order to develop a module for light activated cell lysis, we had to implement two elements:
<p style="color:black;text-indent:30px;">In order to develop a module for light activated cell lysis, we had to implement two elements:
<ol style="font-size:15px"><li><b>Construct a light-activation system that can express a downstream gene of interest.</b></li>
<ol style="font-size:15px"><li><b>Construct a light-activation system that can express a downstream gene of interest.</b></li>
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<li><b>Express a cytolytic protein that can be utilized as our therapeutic drug to lyse cancer cells.</b></li>
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<li><b>Express a cytolytic protein that can be expressed as our therapeutic drug to lyse cancer cells.</b></li>
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<b><div class="name" align="center">Objective 2: Expression of a Cytolytic Protein</div></b><br><br>
<b><div class="name" align="center">Objective 2: Expression of a Cytolytic Protein</div></b><br><br>
<b><div class="name" align="center">Cytolysin A (ClyA)</div></b><br>
<b><div class="name" align="center">Cytolysin A (ClyA)</div></b><br>
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<p style="color:black;text-indent:30px;">ClyA is a pore-forming toxin endogenous to <i>E. coli</i>,<i> S. typhimurium</i>, and other strains of enteric bacteria.</p><br><br>
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ClyA is a protein native to E. coli, Shigella flexneri, and Salmonella typhi that is capable of forming 13-mer pore complexes in a redox-independent manner. Expression of clyA in the absence of other hemolytic toxins is sufficient to induce hemolysis experimentally, and is therefore considered to be a potent cytolytic agent. Unlike a similar protein, HlyA, ClyA is not synthesized as a protoxin, which requires further posttranslational modifications to become active. ClyA is functional immediately following translation of mRNA to protein.
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ClyA is a 34kDa protein that is composed primarily of α-helical bundles that form a rod-shaped molecule. The membrane insertion domain is known as a β tongue (shown in yellow in Figure 2) and is critical for hemolytic activity. If the β tongue is mutated, the hemolytic activity of clyA is abrogated. </p><br><br>
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<b><div class="name" align="center">Mechanism of Action</div></b>
<b><div class="name" align="center">Mechanism of Action</div></b>
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<p style="color:black;text-indent:30px;">We selected ClyA as our cytolytic protein because its unique mechanism of action that makes it especially potent. ClyA is secreted from bacteria in outer membrane vesicles (OMV's), within which it forms pore assemblies. As shown below in Figure 3, these pore assemblies allow ClyA to latch on to the cell wall of other cells and through the use of its encapsulating pore assembly lyse the cell wall.  </p><br>
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<p style="color:black;text-indent:30px;">Our selection of ClyA for our hemolytic drug As shown below in Figure 3, ClyA's</p><br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2012/7/74/PDAWN.gif" width="500" height="300" />
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<div align="center"><img src="https://static.igem.org/mediawiki/2012/6/6c/ClyA-Pore-Assembly.jpg" width="500" height="300" />
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Revision as of 00:10, 27 October 2012

Penn 2012 iGEM Wiki

Image Map

Light-Activated Cell Lysis

Objectives

In order to develop a module for light activated cell lysis, we had to implement two elements:

  1. Construct a light-activation system that can express a downstream gene of interest.
  2. Express a cytolytic protein that can be expressed as our therapeutic drug to lyse cancer cells.
Objective 1: Light-Activated Sensor


Selection of YF1/FixJ Blue Light Sensor

After reading many papers to select an appropriate light-sensing system to use, we selected the YF1/FixJ blue light system. We had also considered the red light sensor Cph8 but ultimately decided on YF1/FixJ because of its high on/off ratio of gene expression and also because of its availability to us (we were fortunate enough to come across the YF1/FixJ system in the form of the pDawn plasmid from the Moglich lab in Germany).


YF1/FixJ System (pDawn)

As shown below in Figure 1, the YF1/FixJ system works through a "repress the repressor" concept. Upon 480 nm blue light illumination, YF1 (a fusion of a LOV protein domain and a histidine kinase) phosphorylates a FixJ response regulator that activates the pFixK2 promoter. The activation of pFixK2, promotes expression of the cI repressor that, in turn, represses the lambda promoter pR. The net result is activation of the gene in the downstream MCS.



Figure 1
Objective 2: Expression of a Cytolytic Protein


Cytolysin A (ClyA)

ClyA is a pore-forming toxin endogenous to E. coli, S. typhimurium, and other strains of enteric bacteria.



Mechanism of Action

Our selection of ClyA for our hemolytic drug As shown below in Figure 3, ClyA's


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