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
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To clone three modules in the 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 E.coli<.i> 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 .
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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.
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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.
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