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 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
