Tightly regulated E. coli Promoters
peterg at rnaworld.bio.ukans.edu
Mon Sep 26 04:51:53 EST 1994
In <1994Sep18.045414.72840 at kuhub.cc.ukans.edu>, peterg at rnaworld.bio.ukans.edu (Peter Gegenheimer) writes:
>In <zjons-150994113819 at 126.96.36.199>, zjons at vetbio.unizh.ch (Zophonias O. Jonsson) writes:
>>In article <3586ad$98e at mark.ucdavis.edu>, ez001427 at dale.ucdavis.edu ()
>>> Dear Philip-
>>> Have you tried using the T7 RNA polymerase promoter, and inducing
>>> by addition of phage lambda CE6? In E. coli strains without the T7 RNA
>>> polymerase, you will get no expression of your target gene, because of
>>> dissimilarities between E. coli promoters and the T7 promoter.
>> This is unfortunately not entirely true in all cases. At least some T7
>>expression vectors are leaky even in cells that lack T7 RNApol. I had
>>problems propagating a pET23 derivative with a moderately toxic (but also
>>essential) gene inserted, in E. coli TG1. The plasmids kept loosing the
>>inserts. I then transformed a ts E. coli strain (again with no T7 RNApol)
>>with the vector and found that the inserted gene was expressed enough to
>>complement the mutation completely. My guess is that the terminators
>>behind the bla gene on the vector are leaky enough to let some
>>transcription pass on down.
>> The T7lac promoter will probably do a better job, but there are other
>>possibilities like the lambda repressor system that might be even better.
>>That's all !
Another, and more definite follow-up: Read Fletterick's account of regulation of
lac-controlled systems (pTAC and pET), both for a thorough analysis of how to get
good regulation and for an excellent (or lucky) example of production of soluble
protein by regulation temperature and expression level. See Browner et al., Protein
Engineering 4(3), 351-357 (1991).
| Peter Gegenheimer | pgegen at kuhub.cc.ukans.edu |
| Departments of Biochemistry | voice: 913-864-3939 |
| and of Botany | |
| University of Kansas | FAX : 913-864-5321 |
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