Blunt end cloning

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Thu Jul 7 00:59:21 EST 2011


Hi Matt,

As Irit suggested, growing your bugs at different temperatures is an
option that is possibly easy to test and quite straightforward.

I don't think that the vector is the problem (as long it has a
promoter). Your protein simply might disturb membrane integrity or do
some bad signaling. Thus, you'll select for clones with non-functional
(at least: non-toxic) protein.

Do you have any inducible vectors, eg one of those that used for blue-
white selection? Maybe you can get hold of an E.coli strain that is
suitable for blue-white screening (eg XL1-blue, check the tech section
in the NEB catalogue or the internet). While growing the bacteria,
include glucose in the medium for efficient repression. Then you may
induce expression with galactose. In that scenario, you should at
least get some protein expression before your bacteria die.

Are there any 'inhibitors' that may interfere with your protein's
signaling pathways (if any) that you could add to the growth medium?

If your protein has one or more known partner(s), you also might try
co-expression. Cell-free expression systems might yield protein but
may lack any posttranslational processing (you might add something
like a His-tag for recovery). Before you switch to e.g. yeast, check
for homologous proteins first by BLASTing your gene of interest to
minimize the risk of running into the same problem again.

HTH

Wo


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