Back to the bench

I think I'm about done with manuscript writing for a while.

• The RA's manuscript on natural competence in E. coli, has been published in PLoS One.
•  The visiting grad student's paper on natural competence in Gallibacterium anatis is in press at Applied and Environmental Microbiology.
• The RA submitted her manuscript on all her H. influenzae competence knockout mutants last week; it's now under review at J. Bacteriology.  
• The post-doc's DNA uptake manuscript will be ready to submit tomorrow or Monday to Nucleic Acids Research.  
• On Thursday I submitted what I hope are the final revisions for my PLoS Biology opinion piece on genetics teaching.  
• And, we're still waiting for the word from Science about our revised #arseniclife manuscript (it was sent back to the reviewers).

So it's time to do some experiments.  In fact I hope that the whole summer will be time to do some experiments.  I think I'll start with something easy, checking the knockout mutant of HI0659 for effects of known competence inducers.

HI0659 is a competence-induced gene that is needed for both DNA uptake and transformation.  Lots of genes are needed for these processes, but what's surprising about HI0659 is that the protein it specifies doesn't appear to be part of the DNA uptake machinery.  Not only does it lack any features of known DNA uptake proteins, it's small, cytoplasmic, and has a helix-turn-helix domain (these usually function as DNA binding elements).

I'm going to check whether any of the known competence inducers can turn on competence when HI0659 is knocked out.  Two of the inducers I'll test are genetic - the sxy-1 mutation and the murE749 mutation, both of which cause cells to be competent when they normally wouldn't be (hypercompetent).  The third is the small molecule cyclic AMP.

I'll need to make double mutants (sxy-1 HI0659::spc and murE749 HI0659::spc).  This should be straightforward - I'll just transform the hypercompetent mutants with DNA from HI0659::spc and select for spectinomycin resistance.

First I need to streak the cells out - no time like the present.  Then on Monday I'll do a quick DNA prep of HI0659 and transform the hypercompetent mutants.  No need to make them competent - they'll be competent enough straight from the plate, or in log phase growth.

Monday:  The cells all grew up nicely but I'm not going to proceed until the RA has confirmed (by email, she's on leave) that the HI0659 strain in the freezer stocks is the correct new one, not the incorrect old one.  The label looks old, and there's no note in the strain list saying that the vials of the incorrect strain were discarded and replaced..