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 UBC.ca 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, contentand 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 im...
Source: RRResearch - April 27, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
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 ...
Source: RRResearch - April 17, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
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 seque...
Source: RRResearch - April 17, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
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 f...
Source: RRResearch - April 4, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Big prep of MAP7 DNA
We're almost out of the standard DNA that we use in our transformation assays. It's chromosomal DNA of a strain called MAP7 (because it contains point mutations conferring resistance to seven different antibiotics).So I grew up a liter of cells and prepped DNS from them. Now I have 25 ml of nicely viscous DNA solution. It's transparent and colourless but I suspect it's not really pure yet, so I'm doing a second purification on 0.5 ml just to check if the apparent concentration or purity changes when examined with the Nanodrop spectrophotometer. I'll also do test transformations, with the last of the...
Source: RRResearch - March 26, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Getting ready for RNA-seq cell/RNA preps
The RA's missing notebook hasn't turned up, so I don't have her notes of how she prepared the samples for the RNA-seq analysis. Luckily the main procedures are ones she used in many experiments and are described in her earlier notebooks and in an email she sent me. The basic procedures:Collecting samples:Grow cells to desired state in rich medium (sBHI) or competence medium (MIV).Mix 2 ml with 4 ml RNAprotect reagent (Qiagen); leave 5 min at RT. We have 100 ml and can get more quickly through LSC Stores.Mix 2 x 1 ml with 0.25 ml 80% glycerol and freeze at -80°C. (for later competence assays).Pellet RNAprotect cells ...
Source: RRResearch - March 17, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
She lost me at the Central Limit Theorem
I've been saying for ages that I need to learn the statistical programing language R, so that I can work with all the bioinformatic data we're generating. So yesterday I looked through the Coursera offerings and found an introductory statistics course that taught R (Data Analysis and Statistical Inference, taught by Mine Cetinkaya-Rundel of Duke University. It started a few weeks ago, and I've spent yesterday watching the Week 1 videos and doing the Week 1 R lab. The labs are excellent. They use a web-based R learning platform called DataCamp - each lab is a long series of guided exercises: with each...
Source: RRResearch - March 16, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Does fructose inhibit development of competence?
I gave a seminar at Michigan State yesterday, invited by the Microbiology graduate students (Thanks guys!). While I was there I met with a research group that works on Actinobacillus succinogenes, a relative of H. influenze. They were interested in improving the competence levels of this species, and I explained that induction of the H. influenzae competence regulon was controlled by CRP and cAMP, with cAMP levels determined by the availability of external fructose to the phosphotransferase system’ (PTS) sugar-uptake system.The PI then asked me whether we’d tested the obvious prediction that adding frucose ...
Source: RRResearch - March 12, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Woohoo! RRResearch made a top-ten list!
If you've come from the list of The 10 Must Read #womeninscience Blogs, you might be a bit disappointed to see that the most recent posts are descriptions of day-to day work in my lab. That's typical for RRResearch, but here are a few posts you might find more interesting:Who really discovered Trisomy 21?Apple Academic Press: predatory publisher of scholarly booksArsenic-associated bacteria (NASA's claims)Heresy about ethidium bromide (Source: RRResearch)
Source: RRResearch - March 10, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Checking the hypercompetence mutants
Here's the results of the transformation time courses of the control strain 'K' (= KW20) and the hypercompetence mutants (RR735 and its backcross descendant RR735).The lower graph is just culture growth (CFU/ml). You can see that the two mutants (red and green lines) grew slightly slower than the control (blue line). The upper graph is the transformation frequencies. You can see that the two mutants behave identically, and that their transformation frequencies are 100-fold higher than wildtype in log-phase growth and 10-fold higher as growth slows. The dotted red circle indicates two samples that gave no ...
Source: RRResearch - March 9, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
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 (RR7...
Source: RRResearch - March 6, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Who really discovered trisomy 21? (righting an old wrong)
A few days ago a French student in my Useful Genetics Coursera course posted a link to an article in Le Monde (sorry, it's both in French and behind a paywall, but this link might get you a translation). It reported that a Jan. 31 award ceremony for the discovery of the cause of Down syndrome, part of the 7th Human and Medical Genetics Congress in Bordeaux, had been blocked by a Down syndrome support organization (Fondation Jerome-Lejeune). The back story is very depressing, an egregious example of a woman scientist being denied credit for her discovery.Photo source: Le MondeThe woman is Dr. Marthe Gautie...
Source: RRResearch - February 6, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Plans for RNA-seq analyses
I should have posted this after last week's lab meeting but am only now getting to it. I sensibly took snapshots of the whiteboard at the end of that lab meeting, so I could check what we'd decided.The issues: We have several Haemophilus influenzae mutants whose gene-expression profiles we want to examine, either during competence development in the MIV starvation medium or during normal growth in the rich culture medium sBHI. For most of these (i) we want samples from several timepoints over a few hours, (ii)we want wildtype controls done in the same experiment, and (iii) we want three replicate samples ...
Source: RRResearch - February 1, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
But it's MY figure!
The postdoc and I have a minireview coming out in an ASM journal, and we're at the 'permissions' stage.One of the figures is an explanatory diagram I drew for this article (figure on the left below). It's similar to an explanatory diagram I drew for an article we published in Genetics a few years ago (on the right below). Because one of the reviewers asked if our minireview figure was adapted from the published figure, in the Figure Legend we wrote '(adapted from (91)' even though it wasn't. .Now the Production Editor is asking us for the formal permission from Genetics to republish this...
Source: RRResearch - January 28, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
List of RNAseq analyses to do
In case we have money for lots of RNAseq runs, here's a list of every sample that might be useful:24 samples = 1 run:Replicates of the samples we've already done: MIV competence induction at t=0, t=10, t=30 and t=100:1 x KW20, 1 x sxy-, 2 x ∆HI0659, 2 x ∆HI0660.Miscellaneous samples (3 replicates of each?):KW20 in late-log (time of max 'spontaneous' competence)KW20 in log phase + hydroxyurea: t=0, t=30? t=60? HI0659/0660 double mutant, in MIV at t=0 (= log phase) and t=???murE749 mutant in log phase growth and stationary phaseOther hypercompetence mutants: murE750, 751, 752? in log...
Source: RRResearch - January 22, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
