Field of Science

More hypercompetent mutants Part 1

Well, I did submit a CIHR proposal after all.  Not on the regulation of competence as I had originally planned, but a resubmission of our last proposal (from 18 months ago) to develop the knowledge base to predict transformation in vivo.  Now that's done I'm planning some research to identify new mutations that cause hypercompetence.

One project will start right away, to identify the mutation causing hypercompetence in a strain we isolated about 10 years ago.  I wrote about this a few months ago, but we didn't do anything then.  We're now going to do sequence both the original EMS-mutagenized strain (RR735) and its backcrossed derivative RR753.  As controls we'll sequence the original and backcrossed versions of a parallel isolate whose mutation is well-characterized (RR727 and RR749).

My job is to prepare the DNA preps for sequencing.  I've streaked out the strains from the freezer stocks so I can grow up the cells - making DNA is easy.  If I inoculate the cultures this morning I can make DNA later today.  (Later: inoculated them in the afternoon; will make DNA tomorrow.)

But I'll also repeat the transformation time courses for RR735 and RR753, with wildtype cells as a control. I should try to get that done tomorrow (in between appointments) so I can get the data analyzed over the weekend, since I'll be out of town Monday to Wednesday (giving seminar at Michigan State!). (Later: too many appointments tomorrow so will set things up for Saturday.  If the colonies aren't ready to count on Sunday I'll have the grad student put them in the fridge on Monday so I can count them when I get back.)

Fortunately all the strains are NovS, so I can select for NovR (that's the easiest marker to work with because it gives a low background and doesn't need expression time).

There's a chance that this sequencing will discover something interesting about EMS mutagenesis.  As far as I know nobody has used genome sequencing to examine the effects of EMS in bacteria (I surveyed the community via Twitter but nothing was known), though the post-doc thinks this may have been done in Drosophila.

Part 2 of this post will be on strategies to isolate large numbers of new hypercompetence mutations.

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