E gene that I customized was inserted in pUCIDT-amp which carries lac promotor. After transformation and induction with 1mM IPTG there were no lysis at all. both induced and control cultures showed the same OD and CFU count after 3, 6,18 hours post-induction. Now I am considering its expression using pET40 plasmid which has T7 promotor and is a high expression vector. In this plasmid T7 promotor is followed by ribosomal binding site and UTR region and then DsbC gene which encodes for a periplasmic protein. If I insert my E gene in MCS there will be a194 nucleotides long intergenic region consisting of sequence for S. tag and His. tag. Now I have 2 questions.
1) if I simply express my gene, the E lysis protein will be in fusion with DsbC protein, will that be okay for my protein as E lysis protein also act on membranes and form transmembrane tunnel sealing the periplasmic space ???
2) or should I add separate RBS before start codon of my gene by adding Shine-Dalgarno sequence and 5' UTR in my forward primer ???? and can I use the same RBS as already present in this plasmid before DsbC gene ???
If you are trying to express a lysis protein like E, you do not need high level expression. I would guess your problem is that the expression plasmid you already have is lethal/toxic to the cell and you just picked up some mutations that inactivate the E gene so nothing is expressed. This happens very readily with toxic proteins and by definition a lysis protein is about as toxic as it gets. Secondly, it is really difficult (and often toxic) to overexposes membrane proteins as well. So I would very much suggest to NOT use pET40.
Why are you wanting to co-express with DsbC? It probably will be fine and would work, but I'm not sure why you would want to do that since you won't really know what impact it might have on the lysis event.
Does your clone of the E gene already carry the natural RBS? If so then you probably don't really need to engineer another, but yes you could add it to your forward primer or you could clone into an expression vector that already carries one.
Thank u so much for your kind response..
First of all coexpression with DsbC Is not desirable. But expression of gene in pET40 results in the desired protein fused with DsbC. And I am not sure about its effect on my lysis protein. Secondly my cloned gene doesn't carry RBS naturlly but my expression vector does.
You should be able to clone your gene in such a way that it is not fused to Dsb such that it is out of frame or dsb is deleted. It may be that the fusion protein is preventing the lysis protein from normal activity and hence permits expression.