I did a sloppy lab meeting presentation this afternoon on my inconclusive results so far with the lacZ fusion strains. We discussed whether I could just use the moderate constitutive expression from the CRP-S promoters as the baseline, and look for conditions that increase it (and test that the induction does depend on Sxy and CRP).
One of the post docs reminded me that we have a plasmid carrying an inducible version of the sxy gene, and made the excellent suggestion that I should introduce this plasmid into these strains and see if overexpressing sxy causes a dramatic increase in beta-galactosidase activity. If not, then the system is unlikely to be much use. But if it does (and it should) then I'll know the range of signal I can expect, and can get to work testing possibly-inducing conditions.
She already has a stock of the plasmid, so I just need to make the recipient cells competent and transform this plasmid in. I'll test both the strain with the chromosomal fusion and the two strains carrying the plasmid-borne fusions. She says that the origin of replication of the sxy-expression plasmid is compatible with (i.e. different from) the origin of the fusion plasmids.
End of summer &
10 hours ago in The Phytophactor