Prok. expression vectors; anything that is not based on Lac operon and Amp R?

Mikhail Alexeyev 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>
wrote:

> 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 
> one.
> 
> 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.

M. Alexeyev





































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