I'm trying to convert my vector into a Gateway destination vector so that I can insert a large section of DNA without relying on restriction enzymes. However, the only site available has a T3 promoter and not a T7 promoter. The DNA I'm inserting isn't for bacterial expression, but for use as a homology directed repair template, so it doesn't need a promoter as one would for bacterial protein expression.
I need a promoter solely for selection of recombinant clones with Chloramphenicol resistance and later with ccdB toxicity. Will the T3 promoter result in constitutional expression for this purpose? Or should I insert a T7 promoter prior to the chloramphenicol resistance gene in the gateway sequence?
T3 & T7 promoters will only be expressed if either T3 phage or T7 phage RNA polymerases, they're not recognized by E. coli's native RNA polymerase and aren't expressed in its absence.
I'm not actually aware of any common E. coli strains that express T3 RNA polymerase, so I'm guessing the main purpose of the T3 promoter is as a sequencing primer site. There are some strains that express T7 RNA polymerase, but they have it under an inducible promoter (usually some variant of the lac operon promoter).
Probably an easier way to get constitutive expression would be to either use the chloramphenicol resistance gene's native promoter or attach a different constitutive promoter to it.
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