loss of Ecoli over-expression

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cbourne
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loss of Ecoli over-expression

Post by cbourne » Wed Jan 17, 2007 9:11 pm

We are expressing a 150 amino acid protein in pET11b vector in BL21 DE3 e coli cells. This over-expression is very leaky in LB media and we typically don't have to induce with IPTG. This protocol has been successful in this lab for at least 5 years now.

Suddenly, twice within 1 year we are having troubles that seem to be specific to LB powder stocks. They will work initially, but within a few months the over-expression is completely lost. The cells grow to roughly the same density and pellet weight.
We've tried:
-different clones
-different antibiotic bottles/stocks
-different water
- plus or minus IPTG induction
-different LB stocks/ powders - older bottles of LB work fine, but using our fresh powder stocks expression is again lost!

Anyone have any thoughts- what could be changing to specifically inhibit over-expression (T7 promoter) but not affect e coli growth?

Thanks for any help,
Christina

joveryant
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wow

Post by joveryant » Wed Jan 24, 2007 7:10 am

It's ver surprising!!

try different temperature & different competent cell

good luck

oppox
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Post by oppox » Mon Feb 05, 2007 8:23 am

to inhibit expression u could try tuner or rosetta cells (these are from novagen other companies have different ones) they contain an extra plasmid for expression of the pet-blue which lacks the T7 thingy. Though rosetta is also a rare codon cell strain so maybe tuner is the first to start with.

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