Planning more RNAseq experiments
The postdoc and former RA generated some great RNAseq data, which I'll write about in another post. But we have some money that needs to be spent on sequencing in the next couple of months, so we need to decide which additional RNA seq runs we should do. And then I'm going to grow the cultures and prep the RNAs.We have data sets showing how RNA levels change after transfer to competence-inducing MIV medium for several Haemophilus influenzae strains: wildtype (2 expts), sxy- (2 expts), HI0659- (1 (antitoxin?, 1 expt) and HI0660- (toxin?, 1 expt). For each we have samples at t=0, t=10, t=30 and t=100 m...
Source: RRResearch - January 19, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
What I've done lately
('Lately' being the 20 months since I last updated the Table of Contents of my lab notebook.)I've been keeping a Table of Contents of my lab notebooks since I was a grad student, initially on paper but for the past 20+ years as a Word file. As can be seen from the screenshot above, each experiment has a number, and I record the date and a few words about what I was trying to do and what I found. Before I kept this blog, searching it was the easy way to find experiments on a particular problem, and it's still a very valuable resource.One good thing about keeping a Table of Contents is that updating it forces me to go back o...
Source: RRResearch - January 19, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Research direction and goals (pondering)
Depending on when I count from, I've spent the past twenty or thirty years trying to get people to think rigorously about whether bacteria have any processes that evolved to promote random recombination of chromosomal alleles or genes.And I've largely failed at this. A few people think my ideas are reasonable, but the great majority of microbiologists and evolutionary biologists continue to comfortably assume that genetic exchange happens in bacteria because it's an evolutionary good thing. I'm still pretty sure they're wrong, but I think I've done almost all I can to change their minds.I'm at a decision point....
Source: RRResearch - January 12, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
An important paper about the interactions between influenza and H. influenzae infection
Wong SM, Bernui M, Shen H, & Akerley BJ (2013). Genome-wide fitness profiling reveals adaptations required by Haemophilus in coinfection with influenza A virus in the murine lung. Proceedings of the National Academy of Sciences of the United States of America, 110 (38), 15413-8 PMID: 24003154Haemophilus influenzae is a bacterium that causes respiratory tract diseases, but it's often confused with the virus that causes influenza (also a respiratory tract disease). The modern confusion arises from the similarity of the names, but the name similarity arises from an older confusion about the cause of influenza.The in...
Source: RRResearch - January 11, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Back to blogging
I haven't posted anything in ages, partly because I haven't done an experiment in ages. I'm not planning any experiments right now, but I do need to do a lot of thinking and writing about research because it's once again grant proposal time. March 3 is the deadline for CIHR Operating Grant proposals. I've been struggling to get started, mainly because I couldn't identify an angle that reviewers would find persuasive. The more I learn about grant writing the more I realize how important it is to get the reviewer excited about your proposal; enthusiastic reviewers will overlook problems that bored reviewers would...
Source: RRResearch - January 9, 2014 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Thinking about the fraction-competent issues
The post-doc is polishing up has latest manuscript, and on reading it over yesterday I realized that we have data to clarify a very old issue. Here's a link to an old post on this topic: http://rrresearch.fieldofscience.com/2011/05/fraction-competent-problem.htmlIn the old days, the best way to estimate the distribution of competence among the cells of a 'competent' culture was to measure the proportion of cells that became transformed by selectable markers that were on two separate DNA fragments (usually markers carried by a single donor strain, but far enough apart on the chromosome that they were never taken up on...
Source: RRResearch - November 18, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
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If I have lots of KanR colonies from yesterday's hypercompetence-enrichment step...
Today:
Pool the colonies (from each of 6 original mutagenized cultures).
Freeze part.
Make chromosomal DNA preps from the rest. These will be used for sequencing and for the backcrosses (steps 5 & 6)
As controls also make DNA from the pooled NovR colonies from yesterday.
Use the 2 DNAs from the StrR parent and the 2 DNAs from the CmR parent to transform KW20 to StrR and to CmR.
Maybe use the DNAs from the wildtype parent to transform KW20 to KanR.
(Incidentals: make lots more BHI agar, pour plates, use frozen competent KW20, te...
Source: RRResearch - October 5, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Yet more on-the-fly experiment planning
Hm, yesterday's attempt to do two rounds of enrichment for hypercompetent mutants yielded very few colonies (about 10 total). That's probably because I didn't let the NovR cells grow to a high enough density before transforming them with KanR DNA. There were so few cells present with the KanR DNA was added, 5 x 10^5/ml - 6 x 10^6/ml based on the numbers of colonies that grew on the plain plates after the second round transformation).
But I can do the second round enrichment again, by pooling all the NovR colonies that grew on these plain plates. I have between 1000 and 60,000 colonies to pool, depending ...
Source: RRResearch - October 4, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
AAACCCKKK!!! - I screwed up the selection
I did Day 1 of the big experiment yesterday. But because the NovR DNA fragment the postdoc gave m hadn't transformed very well in the test, and I didn't have enough of it for the large volumes I was transforming, I used MAP7 DNA for the transformations that select for hypercompetent mutants. At the time I thought the only disadvantages were a slightly lower overall transformation frequency and the 1-2% risk (for each new mutant) that transformation would also replace the mutation itself.
I forgot that, because the MAP7 DNA carries resistance to all the antibiotics we commonly select for, the pool of NovR cells...
Source: RRResearch - October 3, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Timing for the big mutagenesis experiment
In the previous planning post I ended with the following breakdown:
Day -1: Streak out the cells. (3 strains)
Day 0: Inoculate single colonies overnight. (3 strains)
Day 1: Dilute and grow, mutagenize, wash, dilute and grow,
freeze, dilute and grow, transform with NovR, wash,
grow with nov, freeze, grow with nov, maybe plate. (9 cultures)
Day 2: (Pool), dilute and grow, transform with KanR, plate.
(6 cultures (not the controls))
Day 3: Pool KanR, make DN...
Source: RRResearch - October 2, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Timing of competence development in a new murE749 transformant
In the previous post I realized that I don't know how quickly competence will develop in cells that have just acquired a murE hypercompetence mutation. In that post this was a practical problem (how long should the mutagenized cells be incubated for), but it also has research-grade implications. That's because we still have no idea how these mutations turn on the competence regulon. If they take a long time to have an effect, I would suspect some sort of gradually developing imbalance that eventually changes Sxy expression, whereas if they act quickly I'd suspect a more direct effect.
How could I find th...
Source: RRResearch - September 23, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
The EMS mutagenesis mutant hunt - progress and plans
I wrote about this general experimental plan here, and described the preliminary test of the NovR selection here. The EMS mutagen has been ordered and should arrive within the next few days. The experiment isn't as urgent as we originally thought, because the DNA sequencing plans have changed, but it still needs to be done soon, so I'd better be ready to get started. Here's the plan:
Do the initial mutagenesis in three different strains:
Wildtype H. influenzae Rd
H. influenzae Rd carrying the normal sxy gene and a streptomycin-resistance mutation. StrR is only about 50 kb from sxy, so we can use i...
Source: RRResearch - September 22, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
A better strategy for finding new hypercompetence mutations in murE
If we did decide to buy some EMS (it's cheap and readily available from Sigma) and repeat the mutagenesis, I think we should use a different strategy to select for hypercometent mutants. Specifically, we should focus on getting more strains whose hypercompetence is caused by mutations in murE (see this old post for a description of the murE results). We have four such mutants now, which change two different amino acids, and we don't understand how they work. Having more independent mutants would help clarify the situation, whether we get new mutations or just more of the same mutants.
How to do ...
Source: RRResearch - September 14, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
Selecting for rare NovR cells by enrichment in broth - would it work?
In the previous post about searching for new hypercompetent mutants I mentioned that our ability to find rare NovR transformants in a log-phase culture is limited by the need to put relatively small numbers of NovS cells (less than 5 x 10^7) on each plate. If we use more cells we see large 'bald spots' where the NovR cells are unable to form colonies; we speculate that this is because of toxic effects of too many NovS cells dying around them. When the problem is severe we see no NovR colonies at all even though hundreds were plated.
One solution is just to distribute the cells over more and bigger plates.&n...
Source: RRResearch - September 14, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
A new way to make money from researchers?
Basically, you give World Biomedical Frontiers $38 and they list your paper's Abstract on their website along with whatever supplementary explanation of the work you provide. I gather that "cutting-edge biomedical research" means "research by people who gave us $38".
A bit of surfung suggests that you might then add a note like this to your publication list:
"This paper has been selected to be featured in World Biomedical Frontiers (http://biomedfrontiers.org/cancer-2013-may-2-5/) because
of its innovation and potential for significant impact. World
Biomedical Frontiers [ISSN: 2328-016...
Source: RRResearch - September 13, 2013 Category: Medical Scientists Authors: Rosie Redfield Source Type: blogs
