I am currently cloning codon-optimized I-SceI gene (from S. cerevisiae) into pET vector using E. coli DH5a, but I am keep failing cloning. Even if I assume a leaky expression of I-SceI cloned into pET vector (harboring T7 expression system) in E. coli DH5a strain--that I do not understand, either--I do not understand this phenomenon as there is no I-SceI recognition site in DH5a, as I understand. Is there any special factor required for cloning I-SceI gene in E. coli?
Before assuming toxicity of I-SceI on DH5a are you certain your insert is ligating into the vector? Are you using T4 Ligase? LIC? OE-PCR? Have you run your ligation reaction on a gel? Do you see a shift in band size?
@Marcus Hanley: Thank you for your answer. I am currently using T4 DNA ligase to clone the gene in a single restriction enzyme site. Yes I am sure it is ligating since I can detect I-SceI genes cloned in opposite direction.
Hmmm......difficult one. I'm wondering whether the fidelity of I-SceI, considering the size of the restriction site, is 100% within the cellular milieu and and that perhaps you're observing some kind of star activity. In parallel to your trouble shooting maybe clone I-SceI into a non-pET related vector (with T7 promoter) and transform into a DH5a-unrelated non-DE3 host strain. Extra work perhaps but withtout understanding the mechanism of toxicity you could spend an awful lot of time trying to optimise something which is intrinsically flawed.
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