I know that there are many experts here, so could any one help me to figure out what kind of trouble I'm having in expressing a gene using tac promoter?
Originally, I successfully overexpressed this gene in BL21(DE3) using T7 promoter and the same RBS (the expression level was high).
Then I wanted to express this gene in MG1655, so I had to change the promoter to Ptac. I did PCR to clone the Ptac + lacO sequence to replace PT7 (in pET16b) as I described in the below sequence. I also calculated the strength of the previous RBS in MG1655 and I got the translation initiation rate of around 57.000 au (it was around 9.000 au in BL21(DE3)). I checked the sequence very carefully, everything seems correct, but I didn't get any expressed band after inducing the cell with 1 mM IPTG (cells grew normally as the control with blank vector).
I did the same thing with the other two genes that I already expressed in BL21(DE3) with PT7 and all of them failed under the control of the new Ptac.
Is that any problem in this sequence, or what should I do to find out the reason of my failure?
Any help will be appreciated, thank you so much in advance.
Edited by Quasimondo, 03 April 2012 - 12:24 AM.