Planning the GTA work

My goal for the rest of my time in Andrew Lang's GTA lab  is to gather data that constrains estimates of the efficiency of GTA transduction.  I have lots of ideas but they're not very well organized, and I keep getting distracted by the minutiae of GTA biology (and our general ignorance of same).  So this post is an attempt to get a sensible plan written out.The bottom line for efficiency is how many transductants…

Continue reading


Why doesn’t all the GTA get taken up?

I've been modelling the production and uptake of GTA particles in a culture, hoping to understand the cause of the surprising GTA-accumulation curve I described in the previous post.  But this has led me to a more fundamental surprise.Only a very small fraction of the cells in a GTA+ culture produce GTA particles and lyse, and all the other cells are able to bind GTA particles and take up their DNA.  So why doesn't all…

Continue reading


Marc Solioz’s 1975 PhD thesis on GTA

PhD students, don't assume that your thesis will moulder unread in the library.  More than 40 years after he submitted it, I'm reading Marc Solioz's PhD thesis (The Gene Transfer Agent of Rhodopseudomonas capsulata).  I want to understand the kinetics of GTA production, and his is the only good data I can find.Here's what he reported:A. Stability of and transduction by GTA in various solutions:  He tested a wide range of solutions.  In these studies…

Continue reading


Summary of R. capsulatus Bioscreen growth curves

The previous post (GTA competition experiments) described the results of the follow-up set of R. capsulatus growth curves that I planned at the end of the previous experiment (R. capsulatus growth curves in RCV medium).  But it didn't pull together the results of all the Bioscreen growth curves, nor integrate them with what was previously known/thought).  So here goes:First, what's already been reported about growth in liquid culture?  Not a lot.  The graphs below are…

Continue reading


GTA competition experiments

I'm in St. John's for the 'summer'*, doing GTA-related experiments in Andrew Lang's lab at Memorial University of Newfoundland ('MUN').The first experiments I'm going to do are growth competitions between GTA-producing strains and otherwise-identical non-producer strains created by deleting the GTA genes.  Because GTA production requires cell lysis, we predict that the non-producers should outcompete the producers.While I was still in Vancouver I did detailed growth curves of the various strains.  Preliminary ones are described…

Continue reading


Wait, there’s a much simpler explanation! (For CRISPR-Cas, not for GTA)

I'm in Halifax for a couple of weeks, visiting Ford Doolittle and his philosophical colleagues,  We've spent much of the time considering the extent to which CRISPR-Cas systems can or should be considered 'Lamarckian'.  I started with the simplistic perspective that of course it is, because an acquired character (immunity to future phage or plasmid infection) becomes inherited because the Cas proteins insert short phage- or plasmid-derived DNA sequences as a CRISPR 'spacer' into the…

Continue reading


R. capsulatus growth curves in RCV medium

My upstairs GTA colleague and I were surprised that the Bioscreen growth curves in the previous post didn't show a dip in OD600 of the GTA-overproducer strain like that seen in manual (non-automated) growth curves.  This dip is thought to be caused by the lysis of GTA-producing cells as GTA production peaks when cells hit stationary phase.We thought part of the problem might be that I used the standard YPS medium which is based on…

Continue reading


What can we learn from growth curves?

Here's the results of the Bioscreen growth curves I ran for Rhodobacter capsulatus strains:Each dot is the mean OD600 of 15 replicate wells, each containing 300 µl of culture, with ODs read every 20 minutes for 45 hours.  The cultures all grew up at about the same times, but I've shifted the X-axes so the curves don't overlap.  OD values below about 0.015 are not significantly above the backround absorption of the culture medium. The…

Continue reading


growth time courses

In a few weeks I'll be headed for the Maritimes, for the final part of my sabbatical work on Gene Transfer Agent.  But before I leave here I want to run some detailed growth time courses on GTA-producing strains, taking advantage of the BioScreen machine belonging to the lab next door.I'll first do a trial run with all the strains I have,  to check the basic growth kinetics under the Bioscreen growth conditions.  Then I'll…

Continue reading


Phage plaqueing still sucks – what to do now?

I feel like I've been sucked down a hole of trying to get consistently countable plaques from the Rhodobacter capsulatus phage I'm testing.  After seven weeks of plaqueing with various combinations of strains and agar concentrations and cell densities, I'm no closer to having a well-behaved phage I can use to test the GTA-as-vaccine hypothesis.Along the way I've eliminated various sub-hypotheses:1.  The plaques are tiny/faint/blurry/invisible because the phage capsids have long fibers that reduce diffusion…

Continue reading