Prok. expression vectors; anything that is not based on Lac operon and Amp R?
malexeyev at thi.tmc.edu
Wed Mar 6 18:26:37 EST 1996
In article <4hkfru$nl at news1.ucsd.edu>, Hedrick Lab <lkane at jeeves.ucsd.edu>
> I'd like to do cotransfections of genes into E. coli for some enzymatic
> studies, but I've run into a little problem. It seems that all of the
> prokaryotic expression systems utilize the lac operon and are therefore
> dependent on IPTG inducibility. Also, so far, I've only seen expression
> plasmids with Amp resistance. Currently, I have one of my genes of
> interest cloned into pRSET, which has a T7 promoter at the 5' end prior
> to a transcriptional start site and the polyhistidine tract and
> enterokinase domain. When these are transfected into BL21 (E coli
> transformed with IPTG-inducible T7 polymerase expression), I get good
> IPGT-inducible expression of my gene.
> So, what I'd really like is a plasmid that has a kanamycin or
> tetracycline resistance marker, as well as an inducible promoter (that
> is not contain the lac operator) that I can use to get expression of my
> second gene. I could also use a system where the second gene is not
> inducible, and that would work too, although I would prefer an inducible
> Finally, I am slightly concerned that such a plasmid would require a
> different origin of replication (other than the ColE1 origin commonly
> found in a variety of cloning and expression vectors). Do you think
> that it is very important to have a different origin in this plasmid for
> coexpression of my genes?
> Thanks for all of the information.
> Craig Walsh
You may consider trp or recA promoters. Both are inducible and both are
leaky. Lambda Pl and/or Pr promoters are quite tightly regulated by cI
repressor. Sorry, I am not aware of any particular vector with Kan and
these promoters. Finally, you MUST use vector based on another
(compatible) replicon. The choice is quite broad: pSC101, p15A, R6K,
RSF1010, RK2, etc. depending on copy number that you need.
Hope, this will be of some help.
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