I am trying to clone 2 genes from bacillus subtilis within a triple strong promoter ( amyQ, amyL,amyE) in E.coli ( both EPI400 and JM109 )with PUC57 and pgD1662 vectors, but I failed for several attempts. as no colony which including my construction survive. I want to ask if the triple promoter is leaky in E.coli and is there a problem with triple strong promoter? as its constitutive? (like changing the E.coli metabolism because of my genes expression?) or the construction ,includes triple promoter and 2 bacillus genes, is not stable in E.coli?
or do I need to change my E.coli strain? do you have any idea of how can I clone my full construction in E.coli? I did not find the problem yet. does anyone has the same experience in this area?
regards
I think it is unlikely the promoters are the problem. You can clone a very strong E. coli in an E coli plasmid so having multiple BS promoters (which generally don't work well in E. coli) are probably not the problem. I also would not think those genes are problematic in E. coli. Are you sure that all steps in your cloning are working well?
Thanks for your reply Dr Benedikt, I cloned my genes with others commercial promoter in E.coli and it was succesful, but when I assemble them with my triple promoter the bacteria does not survive, so I guess there is problem with my promoter, although it is a bacillus promoter but I guess it make problems in E.coli when I clone my whole fragment on it.
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