What else are my experiments telling me?
The previous post discussed what I can call Problem area #1: the evidence that my plasmid prep results have been unreliable - that the absence of plasmid in the prep didn't mean that the cells I started with didn't contain plasmid. So now I need to go back through the other experiments, to check if my conclusions are still solid.Problem area #2: Can the specR PCR fragment be ligated into a blunt-cut plasmid, when phosphorylation isn't needed? Answer: NO.My first experiment said 'No', but it was flawed by using too high a ratio of plasmid to insert. So I repeated it using much more specR fragment than plas...
Source: RRResearch - November 12, 2015 Category: Molecular Biology Authors: Rosie Redfield Source Type: blogs
Spot 42 RNA doesn't explain our ∆hfq competence phenotype
Gisela Storz from NIH gave a great seminar here yesterday about the many roles that small RNAs and very small proteins play in E. coli gene regulation. It gave me an idea about the role the small-RNA-accessory protein Hfq might be playing in competence regulation. One example she discussed is the abundant Spot 42 RNA, so named because it was discovered as an RNA chromatography spot long before any function was discovered. This small (109 nt) RNA acts in a feed-forward regulatory loop with the cAMP-dependent transcriptional activator CRP. This regulation fine-tunes the control of the CRP-activated genes th...
Source: RRResearch - October 13, 2015 Category: Molecular Biology Authors: Rosie Redfield Source Type: blogs
Is there DNA in oreos?
I have to weigh in on this.I spend a lot of time discussing the idea that bacteria can use DNA as a source of nutrients, and audiences are always surprised when I show this graphic and point out that DNA is ubiquitous in our foods. And these are scientifically sophisticated molecular biologists and microbiologists.So it's not at all surprising to me that 80% of the general public would check 'Yes' when asked, in a set of survey questions about food labelling and regulation, whether foods containing DNA should be labelled as such. Instead of laughing at their ignorance we should think about how much expert ...
Source: RRResearch - January 19, 2015 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Transformations with dirty DNA
The previous post considers ways to test the effects on transformation of chromosomal proteins bound to donor DNA, by gently lysing donor cells and transforming recipient cells with the crude lysate. We've now done one experiment trying out some methods.We used donor cells resistant to novobiocin. We tried freezing the cells without adding the usual 16% glycerol as cryoprotectant, vortexing them with a drop of chloroform or in 0.0001% SDS to disrupt the membranes, with or without pretreatment with EDTA and lysozyme (0.1 mg/ml, 1/10 the normal concentration) to break down the cell walls.One question was how effi...
Source: RRResearch - January 7, 2015 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
RNAseq success!
The sequences are back for our big RNAseq project, and the big good news is that the RNA preps were of good enough quality to give useful sequences for all the samples! Thos was very much in doubt, because the Bioanalyzer characterization of the RNA samples showed almost no detectable mRNA-sized molecules in the samples we tested.Now we have all this data, we need to decide how to analyze it. It's not just a big dataset but a very rich one since the samples differed in what can be considered to be three independent directions, all of which are underlain by rich sets of biological information about phenotypes an...
Source: RRResearch - January 7, 2015 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Do chromosomal proteins on 'donor' DNA affect transformation?
I've started polishing my not-quite-good-enough CIHR proposal for what's called the 'Transitional Open Operating Grant Program' competition. This is the last-chance-under-the-old-system competition; any future proposals will be evaluated under the new system, which doesn't have much use for pure science or small labs. Proposals are due March 2 2015.Before then I'd like to do some preliminary experimental work on one of the studies I'm proposing. I expect that the H. influenzae DNA available to H. influenzae cells in the host environment will still have bound to it many of the normal chromosomal proteins (HU, H-NS. Fi...
Source: RRResearch - December 4, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Effects of cAMP and AMP on competence development by the rpoD mutant
This is a continuation of yesterday's post on the phenotype of our hypercompetent rpoD mutant strain RR753. Yesterday we wrote about its behaviour under 'normal growth conditions, and now we're going to consider two new factors, cyclic AMP (cAMP), which induces competence under what are otherwise non-inducing conditions, and AMP, which inhibits competence development under what are normally inducing conditions.First the effect of adding cAMP: We tested this by adding 1 mM cAMP to cells growing exponentially at an OD600 of 0.1, and measuring transformation 60 min. later. At this growth stage, normal c...
Source: RRResearch - October 23, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Interaction of a ∆hfq mutation with the rpoD mutation
Here's the last part of the summary of what our senior co-op technician has been doing. The last set of experiments tested the interaction between a new ∆hfq mutation we've been studying, which reduces competence) and the rpoD mutation (which increases competence). The Hfq protein binds small regulatory RNAs, helping them to form base-pairs with the mRNAs that they regulate. In other bacteria we know that this base pairing can either reduce the mRNA expression (usually mediated by RNase E degradation) or increase it (by reducing the effect of otherwise-inhibitory mRNA secondary structure). Our ∆hfq mu...
Source: RRResearch - October 23, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
What should our 'senior' co-op tech do next?
We have two co-op (undergraduate) technicians at present (paid from the last of our leftover CIHR funds). Each is with us for 8 months; one started in May and the other in September, so there's a 4 month overlap.The senior one has done almost all of the work preparing the samples for the RNA-seq project, and lately she's also been doing competence time courses to characterize the phenotype of our hypercompetent rpoD mutant. She's looked at growth conditions, at the effects of added cAMP (competence up) and added AMP (competence down), and the effects of knocking out the small-RNA regulator Hfq (down). Wri...
Source: RRResearch - October 22, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Phenotype of the rpoD mutant
This mutation causes H. influenzae cells to become competent prematurely, and to reach levels of competence in rich medium that are about 100 times higher than normal cells. The mutation causes a single amino acid substitution in domain 3 of the 'housekeeping' transcription factor called 'sigma 70'. rpoD is an essential genes, needed for transcription of most housekeeping genes. Since the mutant strain (named RR753) shows only a very slight decrease in exponential growth rate we think the mutation causes only a very minor change in the protein's functionMy earlier post this morning said I had never e...
Source: RRResearch - October 22, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
So long since I've posted...
But here's an update on the RNA-seq saga.We've been struggling to get evidence that our RNA preps are of good enough quality for library preparation and sequencing. Because the Ribo-zero kits that we use to remove the rRNA from the samples are so expensive, we wanted to be sure the final mRNA concentrations and integrity would be good enough to make the equally expensive sequencing worthwhile.Unfortunately my attempts at using a Bioanalyzer have been very frustrating (and it's not cheap - several $ per sample), with odd smeary/spiky patterns and poor recognition of the size standards. This is partly because the conce...
Source: RRResearch - October 13, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Possible work with the new rpoD mutation
I'm up to my ears preparing materials for the new version of Useful Genetics, but we have a great new technician through UBC's Science Co-op program. She's nearly finished the work preparing the RNA samples for our big RNA-seq project, so it's time to consider what she should work on next.One possibility is characterizing the newly identified rpoD mutant strain. This strain (RR753) has a point mutation in HI0533, which encodes the sigma factor that regulates initiation and early elongation of transcription of 'housekeeping' genes, especially during exponential growth.One analysis we need is BioScreen growth cur...
Source: RRResearch - July 10, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
CIHR results...so depressing
The results have been released for the latest competition for operating grants from the Canadian Institutes for Health Research (Canada's equivalent of NIH). Our proposal was ranked 13/72 by its assessment committee (Microbiology and Infectious Disease), but they only funded 11.This is our 7th failed CIHR proposal in a row. They all had good scores, and several, like this one, were very close to being funded. The scores show that our proposals do keep getting better (this time 4.5/5), but the funding cutoffs also keep rising and we're never quite good enough.Will I try again? Perhaps not. This...
Source: RRResearch - July 4, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
More cell preps for RNA-seq analyses
Unfortunately we have to repeat most of the cell preps I made for the big planned RNA-seq analysis , because our RNA preps used up all the cells without producing any RNA. The main problem was that we were using a Qiagen RNeasy Plus kit (designed to remove contaminating DNA) instead of the usual plain RNeasy kit. I had bought the Plus kit because of a cheap introductory price, planning to just leave out the final 'on-column DNase digestion step, since this hadn't been very effective in the past. However Qiagen had changed the kit without changing its name, replacing the final 'on-column' digestion with an ...
Source: RRResearch - June 11, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Why isn't competence regulated by the availability of DNA?
Most bacteria tightly regulate the genes that enable them to take up DNA from their surroundings. This makes sense, since the uptake machinery is complicated, probably expensive to produce, and may interfere with other membrane functions, and since the benefits of DNA uptake may arise only under particular circumstances.The regulatory signals include diverse physiological and environmental cues. In other posts I've discussed the signals that regulate H. influenzae competence, and here's a couple of recent reviews for Gram-negative and Gram-positive bacteria (http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2012...
Source: RRResearch - May 3, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
