Where'd the regulation go?

I wanted to find out why the plasmids with fusions of CRP-S promoters to lacZ (hofM::lacZ and ppdA::lacZ) give such high lacZ expression. Because CRP-S promoters require both CRP and Sxy for high activity, I transformed the plasmids into strains with crp or sxy knocked out. But I also needed crp+ sxy+ cells of the same genetic background (frozen as RR1321; I forget its original number).

So yesterday I made cells of this strain 'chemically competent' to take up plasmids by incubating them in a cold solution containing rubidium chloride. (The cells didn't become 'naturally competent' - that's the long-term goal of these experiments.) I scaled down the cumbersome procedure described in the methods manual. This made it much faster because the cells could be collected by filtration and further concentrated by microcentrifugation, rather than using a big slow centrifuge. I wasn't sure this would work well but it did - this morning I found about about 100,000 transformants on my ampicillin plates).

So now I had all the strains I needed to test whether expression of the fusion depends on CRP and Sxy. Here's the beta-galactosidase 'activity' produced by each strain (in 'Miller units'):

Parent with no plasmid: 3 units
Parent plus either plasmid: 194 and 230 units
crp knockout plus plasmid: 301 and 406 units
sxy knockout plus plasmids: 361 and 463 units.

The differences between the various plasmid-containing cultures are probably not significant, as in this quick experiment I didn't take pains to make sure the cultures were all at exactly the same growth stage. This is unlikely to be important because my previous analyses found no little dependence on growth stage.

The conclusion? Expression of these fusions is independent of both CRP and Sxy. This is a troublesome result. The most likely explanation is that the inserts that carry the CRP-S promoters also contain quite a bit of sequence upstream of the promoter, and these sequences may be responsible for the high basal expression.

I will first repeat the experiment, this time having all the cultures at the same growth stage. Then I'll email the researchers who kindly gave us these plasmids to ask if they found high basal expression too. And I'll start looking the sequences of the promoter-containing inserts, in preparation for engineering them to remove extraneous sequences.