Field of Science

Planning the experiments with our new knockout mutants

In an overview post last week I described some experiments we should add to the paper describing our new collection of knockout mutants.  Now I want to think more about these, laying out what we should do and maybe how the work might be divided up.  We don't want to take on a lot of work at this stage, just enough to make the paper more than a list of mutant phenotypes.  I mentioned in the earlier post the three mutants that have specific points of interest, but now I also want to consider the mutations that have no phenotype at all.

A.  The ATP-dependent periplasmic DNA ligase:   Repeat the undergrad's experiment testing the sensitivity to nicks, using DNase I and confirming presence of nicks by running the DNA with and without denaturing.  Test the ability of wildtype and mutant cells to ligate a plasmid cut at one site by a restriction enzyme, by recovering periplasmic DNA from rec2- and rec2- lig- periplasms and transforming the DNA into E. coli.

B.  The periplasmic protein ComE1:  Repeat the uptake assays of comEI- and comE1- rec2- (and comE1- recF-?) mutants, to see if the comE1- residual uptake is dependent on translocation of DNA across the inner membrane.

C.  The potential regulator HI0659:  Test the effect of added cAMP on this mutant - if this increases competence then we know HI0659 is needed for cAMP production.  Test competence of HI0659- in sxy1 (and murE749?) hypercompetent mutants - if this increases competence then we know... what?  That HI0659 helps with induction of gene expression?

D.  The proteins that don't appear to contribute anything to competence or transformation (HI0660, HI1631, RadC, and again the ligase):  Look carefully at growth properties (from the Bioscreen time courses we're doing for all the mutants) - are they at all different from wildtype cells?  The genes may not be expressed in log-phase growth, so look at growth properties in sxy1 mutants with added cAMP (so the genes are always on).  Make double mutants - do these still grow and transform normally?  Are they UV sensitive (this would imply a repair deficit.

How much work would all this be, and who would do what?  I could do most of it, although I probably shouldn't because I've got a lot of other stuff to do.  I could easily make the double mutants (rec2- lig- and various sxy1), selecting for the SpcR cassette in the marked knockouts.  (The RA might even have DNA from these mutants already made.)  And I can make new competent preps of the comE1and rec2 combinations for the postdoc to test DNA uptake.  I can also do the competence assays of HI0659 with sxy1 and with cAMP.  The postdoc needs to do the uptake assays.  The Bioscreen assays are easy to set up but take time to analyze - this is something the RA will do better than me.  The ligase assays should probably be a group effort - I can do the transformations with nicked chromosomal DNA, and the postdoc should do the H. influenzae transformations and plasmid-recovery, passing the recovered DNA on to the RA for E. coli transformation assays.  We can discuss these experiments at Tuesday's lab meeting and divide up the work.

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