Field of Science

A provocative sxy result?

Provocative in the sense that it may require that we do something.

A few days ago, in sxy continued I wrote
8. The sxy-6 mutant isn't just not hypercompetent, it's much less competent than wildtype cells (10-fold to 500-fold, depending on the assay conditions), even though it is predicted to have the same number of base pairs when folded. This was especially surprising because its mutations replace a strong G:C basepair with a weaker A:T basepair. This may be telling us that in wildtype mRNA the G and C bases interact with bases at other positions as well as with each other, and that these unknown interactions enhance expression of sxy.
Now one of the lab people has checked the amount of Sxy protein in this mutant, and found that in rich medium there is a little more than wildtype, as we would have originally expected, not less than wildtype as predicted by its less-than-wildtype transformation frequency.

One possible explanation for the discrepancy between Sxy protein and transformation frequency is that the sxy-6 mutant could have some other genetic change that we're not aware of, that reduces its ability to take up DNA. We can test this by resequencing its sxy gene to check if other mutations are present, and by doing either of two genetic tests. First, we could use transformation to move its sxy gene into a 'clean' genetic background and see if the transformation frequency stays the same. Second, we could move a wildtype sxy gene into it (again by transformation) and see if its transformation frequency becomes wildtype. Either test may be a pain to do because we can't directly select for the desired recombinants, but have to rely on selecting for a linked antibiotic resistance and then checking the genotype by sequencing.

So before doing any of these I'll carefully recheck the PhD thesis of the student who originally made the mutant and studied its properties. If she made and tested a lacZ fusion of this mutant, her results may clarify things.


  1. The lack of selectable markers linked to sxy and sxy-6 is unfortunate. How did Laura and other members of the lab screen for transformants using the sxy-1 to -7 clones generated by site-directed mutagenesis?

  2. Three markers are linked to sxy+ (str, nov, kan) in MAP7 and other strains, but the sxy-6 strain has the sensitive alleles.

    We mostly used colony-competence screening, but that won't work if competence might be near-normal....

    So it would be easy to transform sxy-6 to sxy+, selecting for strR and then screening by PCR. Competence of the strain could then be tested - if it's not wildtype then we suspect the strain has another mutation.

    I haven't heard back from Laura yet.


Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="">FoS</a> = FoS