Difference between revisions of "Team:Pitt/3-hybrid/Project"

(Created page with "{{Pitt}} <html> <div> <h1>VEGF-A Sensor</h1> <p>This project uses a 3-hybrid approach to activate transcription of a reporter gene based on the presence of VEGF-A. The α-s...")
 
Line 1: Line 1:
 
{{Pitt}}
 
{{Pitt}}
 
<html>
 
<html>
−
<div>
+
<style type="text/css">
 +
.color {
 +
padding: 50px;
 +
background: #ffe88e;
 +
background: -moz-linear-gradient(top, #ffe88e 0%, #ffde6b 100%);
 +
background: -webkit-gradient(linear, left top, left bottom, color-stop(0%,#ffe88e), color-stop(100%,#ffde6b));
 +
background: -webkit-linear-gradient(top, #ffe88e 0%,#ffde6b 100%);
 +
background: -o-linear-gradient(top, #ffe88e 0%,#ffde6b 100%);
 +
background: -ms-linear-gradient(top, #ffe88e 0%,#ffde6b 100%);
 +
background: linear-gradient(to bottom, #ffe88e 0%,#ffde6b 100%);
 +
filter: progid:DXImageTransform.Microsoft.gradient( startColorstr='#ffe88e', endColorstr='#ffde6b',GradientType=0 );
 +
}
 +
</style>
 
<h1>VEGF-A Sensor</h1>
 
<h1>VEGF-A Sensor</h1>
−
<p>This project uses a 3-hybrid approach to activate transcription of a reporter gene based on the presence of VEGF-A. The &alpha;-subunit of E. coli RNA polymerase often recruits the polymerase to the appropriate DNA through its C-terminal domain. By replacing the C-terminal domain with an antibody fragment - known as single chain variable fragment (scFv) - specific to VEGF-A, the &alpha;-subunit will recruit the polymerase to a molecule of VEGF-A. By creating another fusion protein that consists of a DNA-binding protein and another scFv, the same VEGF-A molecule can be recruited to the DNA, thus completing the 3-hybrid system, and allowing the polymerase to transcribe the reporter. While this project is specific to VEGF-A, the system is incredibly easy to modify to work for other similarly-sized biological molecules. By choosing the scFv's used, and the spacing between the upstream promoter element and the transcriptional start site, the specificity of the system is easily customized. This method can potentially replace ELISA in some instances as a faster, cheaper, and more transportable alternative. </p>
+
<div class="color"><h4>Project Background</h4>This project aims to develop the full versatility that paper-based sensors can have. This system uses the idea of a three-hybrid system, where one part binds the promoter, another part is a subunit of <i>E. coli</i> RNA Polymerase, and the analyte of choice provides the bridge that recruits the RNAP to the DNA. For this system, we chose two analytes: VEGF-A, which is a small dimeric protein involved in many cancers, and anti-MUC1 antibodies, which are present in many cancer patients. Both of these sensors use the same DNA binding domains and RNA Polymerase domain, inspired by a bacterial two-hybrid system developed in 2000. (<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC16554/">Joung  2000</a>) In fact, the only difference between the sensors are the proteins fused to these domains. In the VEGF-A sensor, we used a single chain variable fragment antibody, which is extremely specific to its target. (<a href="http://www.sciencedirect.com/science/article/pii/S0022283699931923">Chen 1999</a>) In the anti-MUC1 antibody sensor, the bait is a portion of the MUC1 protein, as shown in the image below.<br/><img style="width:85%" src="https://static.igem.org/mediawiki/2015/5/52/Pitt6.png"/></div>
−
</div>
+
 
 +
<div class="color"><h4>Project State</h4>Currently, we are cloning the constructs using PCR tricks. Once we have the plasmids, it will be a simple matter to apply the 3-day process to create sensor extracts. Since we have worked out most of the kinks in the sensor extract protocol, we expect to get results within a month, which will be posted on this page.</div>
 +
 
 
</div></div> <!--These are the closing tags for div id="mainContainer" and div id="contentContainer". The corresponding opening tags appear in the template that is {{included}} at the top of this page.-->
 
</div></div> <!--These are the closing tags for div id="mainContainer" and div id="contentContainer". The corresponding opening tags appear in the template that is {{included}} at the top of this page.-->
  
 
</html>
 
</html>

Revision as of 01:26, 19 September 2015

VEGF-A Sensor

Project Background

This project aims to develop the full versatility that paper-based sensors can have. This system uses the idea of a three-hybrid system, where one part binds the promoter, another part is a subunit of E. coli RNA Polymerase, and the analyte of choice provides the bridge that recruits the RNAP to the DNA. For this system, we chose two analytes: VEGF-A, which is a small dimeric protein involved in many cancers, and anti-MUC1 antibodies, which are present in many cancer patients. Both of these sensors use the same DNA binding domains and RNA Polymerase domain, inspired by a bacterial two-hybrid system developed in 2000. (Joung 2000) In fact, the only difference between the sensors are the proteins fused to these domains. In the VEGF-A sensor, we used a single chain variable fragment antibody, which is extremely specific to its target. (Chen 1999) In the anti-MUC1 antibody sensor, the bait is a portion of the MUC1 protein, as shown in the image below.

Project State

Currently, we are cloning the constructs using PCR tricks. Once we have the plasmids, it will be a simple matter to apply the 3-day process to create sensor extracts. Since we have worked out most of the kinks in the sensor extract protocol, we expect to get results within a month, which will be posted on this page.