I am planning an experiment where I will integrate an operon construct in the gfc locus E. coli using CRISPR-cas9 mediated homologous recombination. To insert my operon construct in the gfc locus, I have included a portion E. coli endogenous gfc sequence on either side of my operon construct so that homologous recombination become induced upon induction of Cas-9 mediated double-stranded break at the gfc locus. However, my worry is that presence of gfc locus in my operon construct might create problem during cloning as the gfc sequence should also be present in the E. coli strain commonly used for cloning. It may be that portion of gfc in my operon construct and one that is present in E. coli cloning strain might undergo recombination and make this construct unstable and difficult to made. Initially, I planned to synthesize them from a company, however, later I feel the company might face the same problem. I have only 5 months to complete my project and thus I am looking for an efficient and quick way of making my operon construct. Any suggestion on how can I design a better strategy to make this construct will be highly appreciated. For easy understanding, I have uploaded my construct design here. Thanks.
As Delin Liang said, the majority of common cloning strains are RecA- so recombination isn't an issue in those strains, this kind of situation is part of why it tends to be mutated in cloning strains in the first place. Though, even in a RecA+ strain it's probably not going to recombine with the chromosome at such a high frequency that you won't be able to isolate your plasmid by miniprep.
By the way as long as you're transforming a suicide plasmid (preferably one with a counter selection marker for double cross-over events so you can get stable recombination) and your destination E. coli strain is RecA+, you don't even need to bother with the CRISPR-Cas stuff unless that's specifically part of your experiment for some reason. Skipping out on that will probably save you at least a week or two, and everything you've designed up until now should still be functional.
Go ahaed and don't worry! As already stated E. coli strains used for cloning are specifically engineered to be RecA deficient.
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