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
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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.
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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 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.
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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<\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.
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