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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: 1%;" 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: 1%; padding-top: 1%;"></a> | ||
+ | <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: 1%;"></a> | ||
+ | <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: 1%;"></a> | ||
+ | <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: 1%;"></a> | ||
+ | <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: 1%;"></a> | ||
+ | </header> | ||
+ | <div class="page_logo"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/4/4d/T--IISER-Mohali-INDIA--Design.png"></div> | ||
+ | <article> | ||
+ | <div class="page-title"><h5>Genetic Design</h5> | ||
+ | </div> <br /><br /><br /><br /><br /><br /> | ||
+ | <section><h3>The circuit for detecting the salicylate is shown in diagram 1. It comprises of three modules :</h3> | ||
+ | <h4>Module 1- Produces yellow color constitutively in bacteria</h4> | ||
+ | <h4>Module 2- Produces blue color in bacteria in presence of ToxR</h4> | ||
+ | <h4>Module 3- It activates in presence of salicylate</h4> | ||
+ | </section><br /> | ||
+ | <center><table><tr><td><img src="https://2017.igem.org/File:T--IISER-Mohali-INDIA--GD1.png" alt="Figure1. Genetic circuit for detection of salicylate" width="100%" height="100%"></td></tr></table></center> | ||
+ | <br/> | ||
+ | <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 nymber plasmid for immediate detection. The module 3 is already present in the <i>E.coli<.i> chromosome.</h3><br/> | ||
+ | <h3>Module 1 :</h3> | ||
+ | <h4>It comprises two constructs :</h4> | ||
+ | <h4>1)Ptet- RBS- T7 RNA polymerase -terminator 1- terminator</h4> | ||
+ | <h4>2)P<sub>T7</sub>-RBS- Chromoprotein II-RBS- tetR-terminator</h4> | ||
+ | </section><br /> | ||
+ | <section><h3>Both the constructs are cloned in low copy number plasmid, pZS21MCS. In presence of T7 RNA polymerase , chromoprotein II is transcribed and translated in bacteria and it gives yellow color to bacteria. Construct 1 is cloned at AatII and ApaI site, and construct 2 is cloned at SalI and BamHII site .</h3></section> | ||
+ | <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/a/a5/T--IISER-Mohali-INDIA--GD2.png" alt="Figure 2: Vector map of pZS21MCS with cloned module 1" | ||
+ | width="100%" height="100%"> </td></tr></table></center> <br /> | ||
+ | <section> | ||
+ | <h3>Module 2 :</h3> | ||
+ | <h4>It comprises two constructs :</h4> | ||
+ | <h4>1)Pmar-RBS- ToxR-terminator</h4> | ||
+ | <h4>2)Pctx-RBS- chromoprotein I- RBS-TetR- terminator</h4> | ||
+ | </section> | ||
+ | <section><h3>Both constructs are cloned in intermediate copy plasmid, pACYC177. Construct 1 is cloned at XhoI and XmaI site and construct 2 is cloned at BamHI and AatII site.</h3></section><br /> | ||
+ | <center><table><tr><td> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/e2/T--IISER-Mohali-INDIA--GD3.png" alt="Figure 3: Vector map of pACYC177 with cloned module 2" width="100%" height="100%" ></td></tr></table></center> <br/> | ||
+ | <section><h3>Module 3 :</h3> | ||
+ | <h4>Module 3 contains mar promoter which is native to E. coli strain and present in mar operon. MarRAB operon is present in E. coli. It has three genes- marR, marA and marB. marR promoter is present upstream of marR and has two pair of direct repeat elements nearby.</h4> | ||
+ | </section> | ||
+ | <br/> | ||
+ | <center><table><tr><td> | ||
+ | <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%" > | ||
+ | </td></tr></table></center> <br /> | ||
+ | <section><h3> | ||
+ | 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. </h3></section> | ||
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Revision as of 23:36, 30 October 2017