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

Line 66: Line 66:
 
<h4>Module 3- It activates in presence of salicylate</h4>
 
<h4>Module 3- It activates in presence of salicylate</h4>
 
</section><br />
 
</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>
+
     <center><table><tr><td><img src="https://static.igem.org/mediawiki/2017/4/4a/T--IISER-Mohali-INDIA--GD1.png" alt="Figure1. Genetic circuit for detection of salicylate" width="100%" height="100%"></td></tr></table></center>
 
         <br/>
 
         <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/>
 
         <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/>

Revision as of 23:42, 30 October 2017

gEco
Genetic Design






The circuit for detecting the salicylate is shown in diagram 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- It 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 nymber plasmid for immediate detection. The module 3 is already present in the E.coli chromosome.


Module 1 :

It comprises two constructs :

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

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


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 .

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 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.


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. 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.