Field of Science

UBC's new website

Here's the email UBC sent everyone the day before they launched their new web presence:
"To the UBC Community,

We are pleased to announce that a significantly redesigned website will launch on April 25th, 2014.

Consultation and research showed that the current site could do a better job reflecting the true nature and scope of our university. The navigation, content
and functionality of the current site makes it difficult for our visitors to find the information they are looking for and the overall look is considered conservative and dated. The redesign addresses these challenges; it is bold and experiential, offering improved design, navigation and content. A few highlights of these changes are:

1. Moving from a solely internally-focused navigation structure to one that is also audience-based

2. Expanding the opportunity for faculties and units to share their content through a new homepage section called “UBC Now”

3. Creating innovative and in-depth stories on the homepage that illustrate the impact UBC is making in the world

4. Implementing secondary pages that provide a stronger introduction to internal UBC partner sites to improve site navigation and hand-off

We are encouraged by the positive feedback we have received from those who have seen the site while in development and will continue to monitor its performance and fine-tune as required following launch.

We wish to thank the large number of contributors from across our campuses for bringing their energy and ideas to the project.

We invite you to explore the site in the days ahead:

Kari Grist
Managing Director
Communications & Marketing

UBC a place of mind"

 And here's the home page:

Actually there are several other versions with different photos, all of people evidently thrilled by what they're discovering at UBC.  The little dots in the ring around his head are links to pages about specific aspects of UBC's wonderfulness.

And here's the obligatory xkcd cartoon.  Rich FitzJohn found this and points out how obedient UC remains to its precepts.

A new mutation causing hypercompetence

I described last month how we were revisiting on old hypercompetent mutant whose causative gene was unknown.  I rechecked its phenotype and prepped DNA to sequence, from both the original EMS-induced mutant strain (strain RR735) and from a 'backcross' strain where the unknown mutation had been transferred to an unmutagenized genetic background by transformation (strain R753). 

Here's the phenotype again.  The lower graph shows that it grows slightly slower than wildtype, and the upper graph that it has a 10-200 times higher transformation frequency in the rich medium sBHI.

The postdoc just emailed me the sequencing results.  Both the original and backcross strains have the same single mutation, an amino acid substitution in the rpoD gene, which encodes the sigma-70 transcription factor.

This is a surprisingly clear result.  We had expected to find many EMS-induced mutations in the original strain, and probably several mutations in the segments of DNA transferred in the backcross transformation, and were planning another series of analyses to sort out which mutation causes the phenotype.  But both strains have only the one rpoD mutation, suggesting that our EMS mutagenesis wasn't nearly as heavy as we had thought.  As controls we had sequenced the original and backcross strains of another hypercompetence mutant (RR749, known to have a mutation in I, and both these strains also had only the single known mutation.

A hypercompetence mutation in rpoD fits very nicely into my thinking about how competence is regulated.  I'll write a separate post about this.

Cell preps for RNAseq are all done

I'm pretty sure that I've now done all of the cell preps for our big planned RNAseq analysis, more or less as diagrammed in the previous post. 

Instead of a cya knockout mutant as a negative control (pink in the diagram I used a crp knockout.  cya encodes the enzyme that synthesizes cyclic AMP (cAMP), and crp encodes the transcriptional activator CRP, whose ability to induce transcription is entirely dependent on cAMP, so the two mutants have the same phenotype - inability to induce both the competence genes and the energy-balance genes in the CRP regulon.  I decided to use the crp mutant partly because that strain grew up first and partly just in case there are traces of cAMP in our sBHI medium.  I did one MIV-competence time course with this mutant (4 samples) and one sBHI time course (3 samples rather than the 2 in the diagram).

I also did 3 sBHI-timecourse samples of the sxy knockout as another negative control (also pink in the diagram).  I think I now have two more samples than will fit in 3 lanes of sequencing (24 samples multiplexed per lane), so I'll probably omit the OD=0.6 samples of the negative control sBHI timecourses.  But I'll process the RNA from them just in case something goes wrong with another sample.

I found my missing DNase.  I hadn't lost it, just ordered the wrong kind.  So now I have $250 worth of a high-quality DNase I don't really need, and will need to order the right kind.

I'll get to the RNA preps once I get some teaching responsibilities under control - partly the grading for my face-to-face Human Ecology course but mainly the need to rerecord ~150 lecture videos for Useful Genetics/Genetics for Life.

RNA-seq progress, problems and plans

I've been growing the cell preps for the RNA-seq analysis, as shown in the planning figure below.

I did Day C's cultures, freezing 1 ml of cells for later transformation testing if needed, and 2 ml of cells for RNA purification.  The first snag was the invisibility of the cells!  Following instructions from the former RA, I mixed 2 ml cells from each of the first samples with 4 ml of the magic 'RNAprotect' reagent, let the mixture sit for 5 min at room temperature, and spun down the cells (2 ml of mixture in each of three 2 ml tubes) in our mini-spin microcentrifuge.  The plan was to discard the liquid and freeze the cell pellets at -80 °C, for later RNA preps.  But there was no visible cell pellet!  This amount of cells typically forms a small but easily visible pellet when centrifuged, but the bottoms of the plastic tubes looked perfectly clean.

I spun the tubes again - still no pellet.  So I pretended a pellet was present, discarded the liquid, and froze the apparently empty tubes anyway.  I checked the RNAprotect instruction booklet which reassuringly said that sometimes the pellet might be invisible, but I also contacted the RA, who said that her preps had given clearly visible pellets.  Later I thawed out one tube and did a wilful-suspension-of-disbelief RNA prep using the RNeasy kit, which produced the same concentration of (high-quality) RNA as the RA's original preps.  So I'm now assuming that there are invisible cell pellets in all the tubes, and I've done Day D's preps.

The RNAeasy kit also had some surprises, a solution that was supposed to be clear (or with a bit of particulates that could be removed by centrifugation), instead was very cloudy, and centrifuging it raised the cloudy material to the top (as a diffuse scum) rather than pelletting it.  I couldn't get rid of the scum (it just redispersed when I tried to pipette it), so I went on to the next step (adding 100% ethanol), which eliminated the cloudiness completely.  (Maybe this cloudiness came from the presence of too much residual RNAprotect in my tubes - because I couldn't see any pellet I didn't thoroughly drain the tubes before freezing them.)

I would have also tested the DNA-elimination step, which uses Turbo Dnase and a DNase-inactivator chemical.  But my brand new box of TurboDNase ($250) has gone missing.  I've searched the freezer a couple of times, and racked my brain in case I put it somewhere special for 'safekeeping', with no success. My next step is to go down to Stores and have them show me exactly what the TurboDNase box looks like, so I know I have the right search image.

I've also revised my plans for the cells I'll test.  As I wrote earlier, I'm only going to do one replicate of the ∆hfq mutant in MIV, since we really should use special precautions to avoid losing small RNAs from the prep (and maybe to do strand-specific sequencing).  Since we planned on three full lanes of Illumina sequencing, each 24-fold multiplexed, this change opens up space for 8 additional samples. Four of these will come from a MIV time-course using a crp or cya knockout strain (in Day E, which will be tomorrow).  This is an excellent control since it lets us identify all of transcripts dependent on the transcription factor CRP.  I'll also include both crp (or cya) and sxy knockout strains in the rich medium cultures on Day H; taking two time points for each will give the four additional cultures to complete the first two lanes of sequencing.