I transformed K011 with the vector pet28A containing an enzyme. That enzyme was expressed successfully in BL21 (de3) strains. Moreover, PCR analysis showed that the transformation worked.
Although I could not detected the protein expression in LB media + IPTG even after the protein purification protocols. In addition, I could not see the expected response in the strain physiology.
Therefore, my doubt is…Is it a problem with the expression system or is it a problem with my enzyme itself?
I do not know what other vector should I use, nor, how to integrate this gene in the bacteria genome.
Kindest regards,
Hi there,
KO11 strain is not encoding for any T7 RNA polymerase therefore there is no chance to get any expression from pET vector in this host.
Hey Robson,
Try your induction in Terrific Broth. It's easy to make and I've had great success in the past using it instead of LB broth which usually led to low expression levels for me. And if that doesn't work, try your expression at 25 C instead of 37 C.
Best,
David
Try different temperature from 16 to 37 C. it should work. overnight at 16-18 C work for me.
Hi there,
KO11 strain is not encoding for any T7 RNA polymerase therefore there is no chance to get any expression from pET vector in this host.
Thank you very much Dominique!
Do you know another commercial vector should I use? Or, how to integrate this gene into the bacteria? Kindest regards.
Hi there,
Well there are plenty of vectors for bacterial expression. Among these, pGEX serie (tac promoter), pBAD serie (arabinose promoter), pUC serie (lac promoter), pQE serie (T5 promoter)... Depending on what you are actually looking for.
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