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

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)P_T7-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 .

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.

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.

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.