Q. Shang (qshang at u.washington.edu) wrote:
[various tried methods deleted]
: anybody have suggestions as of what other factors I can change to have
: the bacteria work harder for me? I don't necessarily need them to express
: soluble protein, I just need them to express...and express...
Well, Tanya, I would take a serious look at your promoter. What vector
are you using for expression, and what promoter is your gene under? Some
plasmids are just not that stable, or are low copy in coli, but I assume
that you're using drug selection to maintain plasmid, and that you're
using a high copy expression vector. Also, as was suggested earlier,
try varying the length of your induction time. Are you adding
chloramphenicol to your inductions? Rifampicin? Finally, have you taken
a look at the expressed protein? Is it expressed into inclusion bodies or
is it soluble? It may be that your protein is toxic to coli and that's
why you only get limited expression...
"The worst monotonous drone coming from a lectern or the most eye-splitting
textbook written in turgid English is nothing in comparison to the
psychological Sahara that starts right in your bedroom and spurns the
-Joseph Brodsky, from "In praise of Boredom"
delivered as a commencement address at Dartmouth College.
Hans T.H. Beernink, Department of Biochemistry, University of Vermont
hbeernin at protein.med.uvm.eduhbeernin at zoo.uvm.edu