Difference between revisions of "Team:IISER-Mohali-INDIA/Design"

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{{IISER-Mohali-INDIA}}
 
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<div class="column full_size">
 
<h1>Design</h1>
 
<p>
 
Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
 
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This page is different to the "Applied Design Award" page. Please see the <a href="https://2017.igem.org/Team:IISER-Mohali-INDIA/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
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</head>
</p>
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<body>
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<div class="container">
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<a href="index.html"><img src="https://static.igem.org/mediawiki/2017/7/7b/T--IISER-Mohali-INDIA--logogeco.png" style="float: left; width:8%; padding-top: 2%;" alt="gEco"></a>
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<a href="https://www.facebook.com/igemiiserm/"><img src="https://static.igem.org/mediawiki/2017/5/5f/T--IISER-Mohali-INDIA--FB.png" style="float: right; width: 4%; margin-right: 4%; padding-top: 2%; position:relative;" /></a>
  
<div class="column half_size">
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<a href="https://www.instagram.com/igem_iisermohali/"><img src="https://static.igem.org/mediawiki/2017/c/ce/T--IISER-Mohali-INDIA--Insta.png" style="float: right; width: 4%; margin-right: 1%; padding-top: 2%; position:relative;" /></a>
<h5>What should this page contain?</h5>
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<ul>
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<a href="#"><img src="https://static.igem.org/mediawiki/2017/d/d7/T--IISER-Mohali-INDIA--G%2B.png" style="float: right; width: 4%; margin-right: 1%; padding-top: 2%; position:relative;" /></a>
<li>Explanation of the engineering principles your team used in your design</li>
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<li>Discussion of the design iterations your team went through</li>
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<li>Experimental plan to test your designs</li>
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</ul>
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<a href="https://twitter.com/iGEMIISERM"><img src="https://static.igem.org/mediawiki/2017/0/04/T--IISER-Mohali-INDIA--Tw.png" style="float: right; width: 4%; margin-right: 1%; padding-top: 2%; position:relative;" /></a>
 
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<div class="column half_size">
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<h5>Inspiration</h5>
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<ul>
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<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
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<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
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<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
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</ul>
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</div>
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<a href="https://www.youtube.com/channel/UCXhpctjIXTHUpVCwq_ctgDg"><img src="https://static.igem.org/mediawiki/2017/3/3a/T--IISER-Mohali-INDIA--You.png" style="float: right; width: 4%; margin-right: 1%; padding-top: 2%; position:relative;" /></a>
  
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<article>
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<div class="page-title">Genetic Design
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</div> <br /><br /><br /><br /><br /><br />
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<section><h3>The circuit for detecting the salicylate is shown in figure 1. It comprises of three modules :</h3>
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<h4>Module 1- Produces yellow color constitutively in bacteria</h4>
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<h4>Module 2- Produces blue color in bacteria in presence of ToxR</h4>
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<h4>Module 3- Activates in presence of salicylate</h4>
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</section><br />
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    <center><div class="data-img"><img src="https://static.igem.org/mediawiki/2017/9/99/T--IISER-Mohali-INDIA--circuit2.png" alt="Figure1. Genetic circuit for detection of salicylate" width="100%" height="100%"></div></center>
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        <br/>
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        <section><h3>To clone three modules in the <i>E.coli</i>, they should have different copy number plasmids. The module 1 will be active even in the absence of pollutant. So, we decided to put it on low copy number plasmid. The module 2 will get activated only after pollutant is present. So, we decided to put it on high copy number plasmid for immediate detection. The module 3 is already present in the <i>E.coli</i> chromosome.</h3><br/>
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        <h3>Module 1 :</h3>
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<h4>It comprises of two constructs :</h4>
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<h4>1) Ptet - RBS - T7 RNA polymerase - terminator 1 - terminator</h4>
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<h4>2) P<sub>T7</sub> - RBS - Chromoprotein II - RBS - tetR - terminator</h4>
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</section><br />
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<section><h3>Both the constructs were cloned in low copy number plasmid, pZS21MCS. In the presence of T7 RNA polymerase, chromoprotein II is transcribed and translated into bacteria giving it yellow color. Construct 1 is cloned at AatII and ApaI site, and construct 2 is cloned at SalI and BamHI site.</h3></section>
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<center><div class="data-img"><img src="https://static.igem.org/mediawiki/2017/a/a3/T--IISER-Mohali-INDIA--GDsa.png" alt="Figure 2:  Vector map of pZS21MCS with cloned module 1"
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width="100%" height="100%"></div></center> <br />
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    <section>
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    <h3>Module 2 :</h3>
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<h4>It comprises two constructs :</h4>
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<h4>1) Pmar - RBS - ToxR - terminator</h4>
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<h4>2) Pctx - RBS - chromoprotein I - RBS - TetR - terminator</h4>
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</section>
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<section><h3>Both constructs were cloned in intermediate copy plasmid, pACYC177. Construct 1 was cloned at XhoI and XmaI site and construct 2 was cloned at BamHI and AatII site.</h3></section><br />
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<center><div class="data-img">
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<img src="https://static.igem.org/mediawiki/2017/6/6e/T--IISER-Mohali-INDIA--GDsb.png" alt="Figure 3:  Vector map of pACYC177 with cloned module 2" width="100%" height="100%" ></div></center>  <br/>
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    <section><h3>Module 3 :</h3>
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<h4>Module 3 contains <i>mar</i> promoter which is native to <i>E. coli</i> strain and present in mar operon. MarRAB operon is present in <i>E. coli<\i>. It has three genes - <i>marR</i>, <i>marA</i> and <i>marB</i>. <i>marR</i> promoter is present upstream of marR and has two pair of direct repeat elements nearby.</h4>
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</section>
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<br/>
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<center>
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<div class="img-data"><img src="https://static.igem.org/mediawiki/2017/3/35/T--IISER-Mohali-INDIA--GD4.png" alt="Figure 3:  Vector map of pACYC177 with cloned module 2" width="100%" height="100%" ></div></center>
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  <br />
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        <section><h3>
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        marR repressor is constitutively produced in <i>E. coli</i> and represses transcription of marRAB operon. Presence of salicylate inhibits marR repressor protein and induces expresssion of marRAB operon.  </h3></section>
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Latest revision as of 04:13, 14 December 2017

gEco '
Genetic Design






The circuit for detecting the salicylate is shown in figure 1. It comprises of three modules :

Module 1- Produces yellow color constitutively in bacteria

Module 2- Produces blue color in bacteria in presence of ToxR

Module 3- Activates in presence of salicylate


Figure1. Genetic circuit for detection of salicylate

To clone three modules in the E.coli, they should have different copy number plasmids. The module 1 will be active even in the absence of pollutant. So, we decided to put it on low copy number plasmid. The module 2 will get activated only after pollutant is present. So, we decided to put it on high copy number plasmid for immediate detection. The module 3 is already present in the E.coli chromosome.


Module 1 :

It comprises of two constructs :

1) Ptet - RBS - T7 RNA polymerase - terminator 1 - terminator

2) PT7 - RBS - Chromoprotein II - RBS - tetR - terminator


Both the constructs were cloned in low copy number plasmid, pZS21MCS. In the presence of T7 RNA polymerase, chromoprotein II is transcribed and translated into bacteria giving it yellow color. Construct 1 is cloned at AatII and ApaI site, and construct 2 is cloned at SalI and BamHI site.

Figure 2:  Vector map of pZS21MCS with cloned module 1

Module 2 :

It comprises two constructs :

1) Pmar - RBS - ToxR - terminator

2) Pctx - RBS - chromoprotein I - RBS - TetR - terminator

Both constructs were cloned in intermediate copy plasmid, pACYC177. Construct 1 was cloned at XhoI and XmaI site and construct 2 was cloned at BamHI and AatII site.


Figure 3:  Vector map of pACYC177 with cloned module 2

Module 3 :

Module 3 contains mar promoter which is native to E. coli strain and present in mar operon. MarRAB operon is present in E. coli<\i>. It has three genes - marR, marA and marB. marR promoter is present upstream of marR and has two pair of direct repeat elements nearby.


Figure 3:  Vector map of pACYC177 with cloned module 2

marR repressor is constitutively produced in E. coli and represses transcription of marRAB operon. Presence of salicylate inhibits marR repressor protein and induces expresssion of marRAB operon.