Shine Dalgano and T7 promoter

Dominic-Luc Webb molmed domweb at mbox.ki.se
Thu Jul 12 12:18:53 EST 2001


Could someone please answer a couple questions I have...
I'll bet more than 50% of the people in here have a
good idea. This regards protein expression in E coli.

1) Is the Shine Dalgano sequence obligatory for a
plasmid that only has one ATG when expressing in E
coli? That is, can I get high protein expression
or is this just not realistic?

2) How far can the T7 promoter be from ATG? Is 100
bases too much (I have a lot of restriction sites)?

3) Is mutation of the T7 promoter a real problem
such that during the course of overnight growth
trying to raise protein the plasmid will mutate to
yield only plasmids with a deletion in the T7
promoter such that I get no protein? Can this be
prevented?


Cheers,


Dominic




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