Back to the bench

OK, the RA has sent in the revisions of her E. coli paper, the former visiting grad student is working on her G. anatis revisions, and the phosphate measurements of the #arseniclife culture medium are underway.  But we still need to do quite a bit of work for the manuscript about our new collection of H. influenzae competence-gene knockout mutants, which we want to submit before the RA goes on a few months' parental leave in six weeks.

First, we want to use the lab next door's BioScreen machine to do growth curves on all the 'unmarked' (clean deletion with no insertion) mutants.  The plan is to dilute cells directly from fresh colonies into culture medium that's out into the wells of the BioScreen plate, but before we set up big screens with lots of mutants I need to check that this will work as planned.  So this morning I'm going to dilute a few wildtype colonies and measure the cfu/ml (by plating); we'd like to start with about 10^6 cfu/ml, as this should give dense growth overnight if the growth rates in the BioScreen are like those in our incubator.   On Thursday I'll streak out a few mutants and on Friday set them up in the BioScreen.

Second, we need to do a few more transformation assays and a lot more DNA uptake assays.  For these I've promised to make all the competent cell preparations - I have a big list above my bench.  I should be able to get these done over the next week or so, if I get my act together.

Third, we'd like to be able to include some follow-up analysis of interesting mutants - otherwise the paper is just a dry list.  One interesting mutant is comE1.  Homologs of this protein are present in all competent species, and it's always found to be essential for DNA uptake and transformation.  But the H. influenzae mutant has only a 5-10-fold defect.  (Tenfold sounds big, but transformation can usually be measured over at least several orders of magnitude.)  We don't know why we see so much residual DNA uptake.  We'll present a detailed analysis of the homologs in the different species, and an experiment testing whether the Rec2 function is responsible for the residual uptake.  The postdoc and I did one experiment testing this last month, so I'll make more competent cells and we'll replicate it. 

Another interesting mutant is the ATP-dependent DNA ligase that's predicted to be in the periplasm (see http://rrresearch.fieldofscience.com/2006/08/ligase-puzzle.html and http://rrresearch.fieldofscience.com/2009/03/progress-on-ligase-puzzle.html and http://rrresearch.fieldofscience.com/2009/03/that-periplasmic-ligase.html.  We have some data from experiments done by a former student in the Honours program, working with a mutant that had been created by another lab.  She tested whether transformation of the ligase mutant was more sensitive to nicks in the DNA (it isn't), and did all the preliminary work for testing whether ligation occurs in the periplasm.  In this experiment competent H. influenzae rec2-mutant cells are given a USS-containing E. coli plasmid that's been cut with a restriction enzyme, and the plasmid DNA that's been taken up is then recovered from the H. influenzae cells and used to transform E. coli.  Only plasmid that's been ligated will give E. coli transformants.   She didn't have time to do the final experiment, testing the effect of the ligase mutation, and we can probably make the experiment much more sensitive by using the postdoc's periplasmic DNA prep and using very competent E. coli.