The post-docs have finished the first-pass analysis of how E. coli gene expression is affected by both the E. coli Sxy and the H. influenzae Sxy proteins. I suppose I shouldn't be surprised that it's more complicated than I had hoped. For example, unlike the situation in H. influenzae, in E. coli there are also groups of genes whose expression goes down when Sxy is present.
One complication is that these cells are probably seriously OVER-producing Sxy. Unlike H. influenzae, where we've only done arrays of cells expressing a single-copy sxy gene under its natural promoter, these E. coli studies used a sxy gene on a high-copy plasmid and under a highly inducible promoter. We know that prolonged expression of Sxy from this plasmid produces large quantities of denatured Sxy (in inclusion bodies) and we don't know the extent to which even the 30-minute expression used for the array studies might create a situation unlike that of natural sxy expression.
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I can't remember what you've said about sxy in the past. Can you make a null version of the protein (mutated active site/binding site/something essential) and overexpress that to test for effects of pure overexpression?
ReplyDeleteNice idea. But we're in a bit of a bind with respect to binding sites or active sites as we still don't know what Sxy binds to or how it acts, and the only null mutants we have are insertional knockouts. We still need to do a screen for point mutations that affect function.
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