Field of Science

the new purH mutant

The ever-efficient RA has used recombineering in E. coli to make a knockout of the purH gene, and has transformed it into H. influenzae.  So now we can start investigating its phenotype, alone and in combination with other mutations.  We should probably confirm the purine defect by showing that it can't grow in a defined medium unless a purine is provided.

The purH gene encodes the protein that carries out the last common step in de novo purine biosynthesis, so knocking it out prevents the cells from synthesizing any purines.  If cells in rich medium indeed get all their purines by salvage, then the purH cells should grow normally in sBHI and develop normal late-log competence.  They may also have normal MIV competence, depending on whether de novo synthesis affects induction of sxy or survival in MIV.

We can also use the purH mutant to cleanly test whether PurR represses the rec2 gene, because we won't have to consider confounding effects of purine pools on sxy-regulated expression of rec2.  If it does, then there should be more rec2 mRNA in a purH purR double mutant than in a purH mutant.  We can measure this by quantitative RT-PCR and maybe using our rec2:lacZ fusion strain (if the antibiotic resistances aren't the same).  This is an important experiment because finding that PurR represses rec2 would be the first clean implication of purine pools in competence regulation.  It also would let us do cleaner analyses of how purine pools affect sxy expression.

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