pET, lysE, lysS, bacterial expression questions

Peter Gegenheimer peterg at rnaworld.bio.ukans.edu
Mon Jun 6 14:13:47 EST 1994


In <ma.42.000E7520 at ABC.univie.ac.at>, ma at ABC.univie.ac.at (Manuel Simon) writes:
>In article <Cqs4LF.4y5 at ucdavis.edu> jfh <jfhess at ucdavis.edu> writes:
>>From: jfh <jfhess at ucdavis.edu>
>>Subject: pET, lysE, lysS, bacterial expression questions
>>Date: Thu, 2 Jun 1994 17:00:51 GMT
>
>
>>Howdy gang,
>[....]
>>Our major problems are 1) expression of the protein in UNinduced cells
>>and 2) not major increases in exression upon induction. 
>
>>My questions are 1) any better strains to try with my existing pT7
>>construct?
>>2) anyone have experience using rifampicin to shut down the e colis?
>>3) anything else to try?
>
>These strains in my hands worked nicely. You ought to add some glucose to the 
>medium and make sure that your cells are in a early logarthmic phase before 
>shifting to the induction.
>
>manuel
>
Also, make sure that you are using a pET vector or similar plasmid with NO LAC 
OPERATOR! (if the host is BL21(DE3) -- any plasmid with Lac operator will induce the 
chromosomal T7 RNA polymerase gene). One additional trick, which we haven't yet needed
to try, is to use the pET11a..d series, which are like pET3a..d  but contain a lac 
operator at the T7 promoter to really shut it down, and then the complete lacI gene 
to provide enough Lac repressor!

Since several people have posted queries about the sequences of these vectors, I will
put the updated sequences on my Gopher server later this week. I'll send an note out 
with details when I do.

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