Field of Science

I'm finally testing a hfq mutant

The RA's gone off on a short but well-earned trip to the sun, and before she left she made the H. influenzae hfq knockout I've been waiting for.  So yesterday, after clicking 'Submit' on our NSERC grant proposal, I did competence time courses of wildtype cells and the mutant, hoping to see a difference in transformation frequencies (preferably a decrease).

You can read all the background in this post from last March.  And here's a hexameric structure for Hfq (from Wikipedia):

The colonies are still too tiny to count, but it looks like the transformation frequency is down about 10-fold in the mutant!

Next step, make some DNA from the mutant so I can introduce the mutation into my hypercompetent mutant backgrounds.

Of HQPs and KM plans

I don't know why this latest round of grant proposal writing sapped my drive to write blog posts.  But the last of the three proposals (to NSERC, Canada's equivalent of NSF) is pretty much done, and I'm turning my mind to doing some experiments.

But first, some NSERC acronyms:

NSERC:  (I have to check every time...)  Canada's Natural Sciences and Engineering Research Council.

HQP:  NSERC is obsessed with training of 'highly qualified personnel'.  Fully one third of the proposal's score is determined by our past production of HQP and our plans for producing HQPs from whatever trainees our successful grant might support.  Both have to be meticulously documented, on penalto of having your proposal dumped in the recycling bin.

KM:  I had to Google this one.  The 'NSERC Tips and Tricks' document kindly provided by our research services people  recommends that our research plan includes comments on open access (for sure!), and follows this with:
If results are appropriate for open dissemination, what is your KM plan? If results are not appropriate, mention why.
Google says that KM is knowledge mobilization (this cracked the post-doc up).  So now my HQP training plan says that "Everyone is encouraged to keep an open research blog, providing both writing experience and knowledge mobilization."

Erudite Journals: Yet another predatory journal strategy

I just got an email invitation to publish in the Erudite Journal of Microbiology and Biodiversity.  This is a group; they list ten other Erudite journals.
Their publication fee is only $300, but they offer a special Language-Editing service for authors.  For $150, they will edit your manuscript's writing (though not the equations), independent of its length, with a turnaround time of one week.  After the editing is done you can decide whether to proceed with the submission. 

At first I wondered whether I might be unfair in treating Erudite Journals as predatory.  Maybe the publishers genuinely want to help researchers from non-English speaking countries publish their work.  However, the Erudite Journal of Microbiology and Biodiversity has no Editor, and its archive has no papers.  Neither does the Erudite Journal of Biotechnology, though it does claim that a first issue is coming soon (hopefully not before they find an Editor).  Neither does the Erudite Journal of Law and Jurisprudence, or most of their other journals.  The Erudite Journal of Business Administration does have an Editor, as do a couple of others, but the Erudite Journal of Social Science Research appears to be edited by a page full of advertisements.

Note added Sept. 24, 2013:  I got another email, "Introducing Erudite Journals".  Still no papers in the Erudite Journal of  Microbiology and Biodiversity or the Erudite Journal of Law and Jurisprudence, but there's one in the Erudite Journal of Biotechnology!  I don't see how they can possibly be making enough money to even pay for their website and spam.

I've already done Expt. C once; need to repeat

In my last post I described four experiments/series of experiments I wanted to do.  One series (= Expt. C) I'd blogged about back in June; at that time I wrote that I was all set to do the experiments (strains frozen in log phase and ready to go).  I hadn't blogged about any results, so I jumped to the conclusion that I hadn't yet done the experiments, but yesterday I discovered I had done them, with the results all neatly analyzed and graphed in my notebook.

Here are the results.  The top graph shows the culture densities and the bottom graph shows the transformation frequencies.  The purpose of the experiments was to find out if any of the cell-wall-peptide recycling mutations the 4 different colours of bars, plus the turquoise wildtype control) alter transformation frequency under conditions that do or don't induce competence.  I had to control for culture density because this has a strong influence on competence induction.

The first two sets of bars are from the first experiment, testing cells in log phase (not inducing, left set of bars) or in log phase plus cyclic AMP (partially inducing, second set of bars).  There's very little difference in culture density or transformation frequency between any of the mutants and the wildtype control.  But the values for the log phase transformation frequencies (bottom graph, left set of bars) is not very reliable because the total numbers of antibiotic resistant colonies I counted were 1, 5, 1, 1, 4.  To get better numbers I need to repeat this experiment using cells at a slightly higher density (OD = 0.2 rather than 0.1 (still log phase) and plating larger volumes of cells (using more plates). 

I did the second experiment twice because the culture densities were too low the first time.  There appear to be some (small) consistent differences in transformation frequency between the different mutants.  But some of the numbers are still unreliable (too low, or too many colonies to count), so I need to do this experiment again too.

The problem with the low cell densities was partly because my plan of starting each experiment with cells that were already in log phase (grown and frozen in a previous experiment) didn't work very well.  Apparently cells in log phase don't survive being frozen and thawed very well.  I started each culture by thawing cells, diluting them into fresh medium, and checking the OD (measure of culture turbidity), but when I checked the OD an hour later it usually had gone down instead of increasing 3-4 fold.  Apparently most of the cells contributing to the initial OD were not viable.  So then I'd have to allow the culture an extra 2 or more hours for the surviving cells to reach the desired density, or close enough that I thought I could use them.  And because these cells are the survivors of whatever trauma the freezing and thawing may have induced, they might not yet have settled down into a reproducible physiological state, which might have influenced their levels of competence.

So this time (first experiment repeat tomorrow, second one repeat on Tuesday) I'm going to allow lots of time for the cells to recover from freezing and get comfortably into log phase before I transform them.

Oops, RRResearch has become RRProposals andRRPublishing

It's been four months since I posted about an experiment!  (I discovered this because I looked back through old posts to see what experiment I should start with.)  I had one experiment (A below) underway, one (B below) planned (and blogged about ) but not actually started, and one (C below) that was waiting for the RA to make a mutant.

After consulting with the RA, here are some plans:

Experiment A:  The goal is to make a H. influenzae mutant strain that has two genes knocked (HI0659 and HI0660).  On its own, a HI0659 knockout eliminates competence; a HI0660 knockout has no effect.  Because both genes have homologs in toxin-antitoxin systems, I hypothesize that HI0659's job is to prevent HI0660 from doing something toxic when it is induced in competent cells.  This predicts that a double knockout will have normal competence.

I had been going to make the double mutant myself, while the RA was on leave, but now she's back she's got this underway.  Next week she is going to create the double-mutant plasmid in E. coli (she has everything ready except the electro-competent cells)  I'll then transform this mutant segment into H. influenzae and test competence, with both single mutants and wildtype cells as controls.

Experiment B:  I want to carefully recheck the competence phenotypes of all our hypercompetent murE mutants, under several conditions.  This is basically a long series of competence assays; I  just need to streak out the various strains and get to work.

Experiment C:  The secondary structure of the sxy gene's mRNA regulates its expression, and the Hfq protein contributes to gene regulation by helping small regulatory RNAs (sRNAs) find and bind to their target mRNAs.  So we're going to make a H. influenzae hfq knockout and test its effect on competence.   The RA is making the knockout - it's at the same stage as the double-mutant knockout described above.  Once she's made the knockout in E. coli (next week), I'll transform it into H. influenzae and test competence.

Experiment D: I don't think I've ever done a blog post about this - it arises out of the post-doc's experiments transforming fragments of the clinical strain 86-028NP into the lab strain Rd and sequencing the recombinants.  (He doesn't seem to have posted about it either.)  86-028NP transforms about 100-fold less well than Rd.  None of the recombinants acquired the full transformation defect with their segments of 86-028NP DNA, but one of them transforms about tenfold worse than Rd.

The only known Rd competence gene acquired by this recombinant is comM, and the post-doc has hypothesized that its lower competence is due to replacement of the Rd comM allele with its 86-028NP homolog.  ComM increases transformation frequencies by protecting incoming DNA strands from degradation in the cytoplasm.  Knockouts have normal DNA uptake but about 50-fold lower transformation frequencies.  (In other species the transformation defect is more severe.)  Under the post-doc's hypothesis, the 86-028NP allele would be less active than its Rd homolog.

One way to test this is to restore the Rd comM allele to this transformant (replacing the one from 86-028NP but not the other 86-028NP sequences) - if differences in comM are responsible for the differences in transformation, this should increase transformation frequencies back to the Rd level.  The RA has tried to do this but the construction didn't work - I may try it again.  As an alternative she's put plasmids carrying the Rd or 86-28NP alleles into the recombinant strain, and I'm going to test these strains for differences in transformation frequency.

OK, about that Tawfik paper on arsenate resistance

Nice work.  But I think it should be of interest only to people who care about the biochemistry of arsenic resistance, a set that doesn't include me.

GFAJ-s level of arsenate resistance is not all that exceptional.  What was important about the Wolfe-Simon et al. paper was the claim that GFAJ-1 actively incorporates arsenic in place of phosphorus.  We already know that this claim was an error (the sum of many errors) by the authors.

I'm returning my attention to the much more interesting question of whether bacteria have any functional parallel to the meiotic sex of eukaryotes.

Sunshine and benchwork = bliss?

I'm back...  (I don't know why I haven't been posting while I've been grant-writing.)

Anyway, to start off easy, here's today's weather forecast:
Nothing but sunshine for the next week.  (Normally most days in October are rainy.)  And nothing but sunshine for the past 2 months and more!  Since July 24 Vancouver has had a total of only 8 mm of rain (normal is more than 100 mm).  Anywhere else people would be worrying about the drought, and about climate change, but here we're just glorying in all the sunshine.

Two grant proposals have been submitted.  You can get the CIHR one on the 'What we're planning' page of our website (link in the left sidebar); the CFC one will appear there soon.  The first is a big one to CIHR (Canadian Institutes for Health Research, Canada's NIH), proposing to develop the information base and algorithm needed to predict transformational recombination in the respiratory tract.  The second is a smaller one to Cystic Fibrosis Canada, proposing to use H. influenzae's ability to extract H. influenzae DNA from complex mixtures as a tool to characterize H. influenzae populations in respiratory tract samples from children with cystic fibrosis.  The post-doc (blessed be his name) wrote this one, with minimal input from me.

A draft of a third proposal, to NSERC (Natural Sciences and Engineering Research Council, Canada's NSF) has been passed on to UBC's internal-review system.  It proposes to test the hypothesis that the self-biased uptake systems of the Pasteurellaceae and Neisseria species are due to mechanistic biases in the uptake process rather than to selection for optimal recombination, by looking for similar biases in bacteria that don't preferentially take up their own DNA. The final proposal doesn't need to be submitted until the end of the month.

So I might have time to do an experiment or two.   I'll have to ask the Research Associate what I should do.