I want to use two promoters to express two gRNA in a single plasmid and wish them to have same number(i.e. two promoter with nearly same strength). I am not sure whether I should insert a constitutive promoters in the plasmid twice, which would have some problems in PCR steps, or I could just modify several sequence between -10 and -35 region and then create a artificial one similar to the constitutive one?
Also, I assume if I use some promoter that is natural to the bacteria cell I want to use, it will interfere with the gene express by this promoter, probably I should avoid that?
You can use the same promoter in two locations in your plasmid, however if you intend to do so, then you should be sure the host strain you are using is recA- so that it is defective for homologous recombination. Otherwise you are likely to loop out one of your inserts. Having said that, if you vary the sequence sufficiently you can probably diminish the probability of recombination.
Regarding interference with the endogenous promoter. I would only expect this to happen if the promoters are regulated by a transcriptional activator protein (ie positive gene regulation) which might become limiting. But if the promoters are truly constitutive then this implies no regulation and it should not be a problem.
However I would suggest you circumvent the problem entirely and just clone the two genes next to each other. In bacteria you can transcribe multiple genes from the same promoter, this is the same thing as an operon in bacteria. So long as there is minimal space between the genes, you should get them both transcribed on the same mRNA, so a 1:1 ratio. However you WILL need to have a ribosome binding site for each of the genes. Although transcription will be linked, translation will not be linked unless you have the stop codon immediately adjacent to the start codon of the second gene.
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