We got the CIHR proposal in about an hour ago. And the postdoc submitted his paper on recombination tracts this weekend. But he has to do it again, because he bypassed the PLoS Pathgpgens formatting system (because it made the figures almost illegible).
Next week, we both need to do posters for the International Human Microbiome meeting, happening here in Vancouver.
Field of Science
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Revised outline
Introduction
Why H. influenzae and competence are important.
Background
Introductory background
Specific Aims: (5 questions, perhaps grouped into 'Aims')
Methods
Q. 1: Which genes affect which steps of DNA uptake?
Q. 2: Which genes cause the different uptake specificities of H. influenzae and A. pleuropneumoniae?*
Q. 3: What are the uptake specificities of H.influenzae and A. pleuropneumoniae?*
Q. 4: What are the physical properties of the H. influenzae and A. pleuropneumoniae USSs?
Q. 5: What forces do cells exert on DNA during uptake, and which genes are needed for these forces?
* The reader will ask why Q. 3 comes after Q. 2 when Q. 3 should be answered before Q.2 can be answered. It depends on how/if the questions are organized into Aims. (Otherwise we could just change the 'Specific Aims' heading to 'Specific Questions to be answered'.
Why H. influenzae and competence are important.
Background
Introductory background
- Gap 1: Which proteins affect which steps in uptake? [Here make the point that the steps themselves are not defined yet.]
- Gap. 2: What is the real sequence specificity and how does it contribute to DNA uptake?
- Gap. 3: What proteins exert forces on the DNA?
Specific Aims: (5 questions, perhaps grouped into 'Aims')
- Q. 1,
- Q. 2,
- Q. 3,
- Q. 4,
- Q. 5
Methods
Q. 1: Which genes affect which steps of DNA uptake?
Q. 2: Which genes cause the different uptake specificities of H. influenzae and A. pleuropneumoniae?*
Q. 3: What are the uptake specificities of H.influenzae and A. pleuropneumoniae?*
Q. 4: What are the physical properties of the H. influenzae and A. pleuropneumoniae USSs?
Q. 5: What forces do cells exert on DNA during uptake, and which genes are needed for these forces?
* The reader will ask why Q. 3 comes after Q. 2 when Q. 3 should be answered before Q.2 can be answered. It depends on how/if the questions are organized into Aims. (Otherwise we could just change the 'Specific Aims' heading to 'Specific Questions to be answered'.
Working on the DNA uptake grant proposal
Our solution the problem of lacking preliminary data for the crosslinking experiments is to eliminate these experiments from the proposal. But now I'm concerned that the proposal lacks scientifically interesting experiments. Here's a summary of the work we're proposing:
Q. 1: Which genes/proteins are required for DNA uptake:
We have made marked knockouts (= antibiotic-resistance cassette in a deletion) of all but one of the 25 genes in the competence regulon, and of the two other genes implicated in competence. Most of these have been tested for transformation frequency, but 10 haven't. Converting these marked mutants into unmarked mutant (by deleting the cassette) has been much more difficult than expected. Seven have been made and tested for transformation defects, the rest haven't been made yet.
The work of Q. 1 is to make the rest of the unmarked mutants and test them all for both transformation and DNA uptake. This will be quite boring work and would be best done by a junior technician.
Q. 2: Which genes contribute to uptake specificity:
Any cell-envelope mutants that retain substantial DNA uptake will be tested for decreased uptake specificity using 200 bp fragments with ideal or randomized uptake sequences.
Strains containing combinations of H. influenzae and A. pleuropneumoniae competence genes will be tested for uptake specificity with 200 bp fragments containing the Hin and Apl USSs. Any with altered specificity will be tested using deep sequencing of degenerate USS pools before and after uptake.
Q. 3: What are the uptake specificities of H. influenzae and A. pleuropneumoniae?
(This used to be Q. 1 and maybe we should restore this order so Q. 2 can follow this work.)
This will be more sequencing of degenerate USS pools before and after uptake. It's more of the same (though a lot more work and money), so we need to emphasize how preliminary the preliminary results are. The post-doc now has NIH funding for other work, so we could designate this as a grad-student project.
Q. 4: Does DNA conformation or flexibility contribute to uptake?
This work examines the physical properties of the USS and tests the effects of various physical permutations of it. It's a short section and should be built up a bit more.
Q. 5: Which genes power pseudopilus retraction and prevent backsliding?
This is an extension of the analysis in Q. 1. Mutants found in Q. 1 to have completely blocked uptake (~15 mutants?) will be tested for the ability to bind DNA to the cell surface - any mutants able to bind DNA but not take it up are candidates for defects in retraction of the pseudopilus. Mutants with some uptake (~5 mutants?) will be tested for the ability to take up very short fragments (~50 bp?) better than long ones, and maybe for the ability to retain bound DNA under shearing stress. Mutants that can take up short DNA but not long DNA will be candidates for defects in the hypothesized anti-backsliding function.
This section seems weak to me. I think we should find a way to go beyond the basic uptake experiments. Maybe we could reintroduce the crosslinking analysis as a way to test them?
Q. 6: What forces do competent cells exert on DNA?
This is the optical tweezers work. This time we explicitly propose the work needed to get the system functioning well, and then the measurements that will tell us about the uptake forces exerted by wildtype and mutant cells. The plan is to frame this as a way to test the candidates identified by the work in Q. 1 and Q. 5.
Q. 1: Which genes/proteins are required for DNA uptake:
We have made marked knockouts (= antibiotic-resistance cassette in a deletion) of all but one of the 25 genes in the competence regulon, and of the two other genes implicated in competence. Most of these have been tested for transformation frequency, but 10 haven't. Converting these marked mutants into unmarked mutant (by deleting the cassette) has been much more difficult than expected. Seven have been made and tested for transformation defects, the rest haven't been made yet.
The work of Q. 1 is to make the rest of the unmarked mutants and test them all for both transformation and DNA uptake. This will be quite boring work and would be best done by a junior technician.
Q. 2: Which genes contribute to uptake specificity:
Any cell-envelope mutants that retain substantial DNA uptake will be tested for decreased uptake specificity using 200 bp fragments with ideal or randomized uptake sequences.
Strains containing combinations of H. influenzae and A. pleuropneumoniae competence genes will be tested for uptake specificity with 200 bp fragments containing the Hin and Apl USSs. Any with altered specificity will be tested using deep sequencing of degenerate USS pools before and after uptake.
Q. 3: What are the uptake specificities of H. influenzae and A. pleuropneumoniae?
(This used to be Q. 1 and maybe we should restore this order so Q. 2 can follow this work.)
This will be more sequencing of degenerate USS pools before and after uptake. It's more of the same (though a lot more work and money), so we need to emphasize how preliminary the preliminary results are. The post-doc now has NIH funding for other work, so we could designate this as a grad-student project.
Q. 4: Does DNA conformation or flexibility contribute to uptake?
This work examines the physical properties of the USS and tests the effects of various physical permutations of it. It's a short section and should be built up a bit more.
Q. 5: Which genes power pseudopilus retraction and prevent backsliding?
This is an extension of the analysis in Q. 1. Mutants found in Q. 1 to have completely blocked uptake (~15 mutants?) will be tested for the ability to bind DNA to the cell surface - any mutants able to bind DNA but not take it up are candidates for defects in retraction of the pseudopilus. Mutants with some uptake (~5 mutants?) will be tested for the ability to take up very short fragments (~50 bp?) better than long ones, and maybe for the ability to retain bound DNA under shearing stress. Mutants that can take up short DNA but not long DNA will be candidates for defects in the hypothesized anti-backsliding function.
This section seems weak to me. I think we should find a way to go beyond the basic uptake experiments. Maybe we could reintroduce the crosslinking analysis as a way to test them?
Q. 6: What forces do competent cells exert on DNA?
This is the optical tweezers work. This time we explicitly propose the work needed to get the system functioning well, and then the measurements that will tell us about the uptake forces exerted by wildtype and mutant cells. The plan is to frame this as a way to test the candidates identified by the work in Q. 1 and Q. 5.
And he already has a blog!
infoenzae.wordpress.com
Hmmm... I see that it's high time I updated the Recent Papers and other links on this blog.
Hmmm... I see that it's high time I updated the Recent Papers and other links on this blog.
New grad student!
We have a new graduate student. He's been accepted by the Microbiology and Immunology program. He won't be able to officially register until May, but he's on the payroll and hard at work.
Maybe I can persuade him to keep a blog...
Maybe I can persuade him to keep a blog...
Did you read the post that said your model of journal publishing is a 'pyramid scheme'?
I've just received an invitation to be an Associate Editor of a new journal in the 'Frontiers of' family.
I did some investigation into this enterprise a while back, in response to an invitation to review a paper for Frontiers in Antimicrobials, Resistance and Chemotherapy. That post concluded that the enterprise is basically a pyramid scheme, drawing in scientists to create ever more journals and staff their editorial boards. A few journals are quite successful (the spammer Frontiers in Neuroscience is one), but most attract very few papers - usually there's an opinion piece by the Editor, and maybe a paper from an Associate Editor's group, but that's about it. The editors are (of course) unpaid, but the publication charges applied to any papers that do get published make money for the enterprise.
Ironically, the email I just received starts with this:
I did some investigation into this enterprise a while back, in response to an invitation to review a paper for Frontiers in Antimicrobials, Resistance and Chemotherapy. That post concluded that the enterprise is basically a pyramid scheme, drawing in scientists to create ever more journals and staff their editorial boards. A few journals are quite successful (the spammer Frontiers in Neuroscience is one), but most attract very few papers - usually there's an opinion piece by the Editor, and maybe a paper from an Associate Editor's group, but that's about it. The editors are (of course) unpaid, but the publication charges applied to any papers that do get published make money for the enterprise.
Ironically, the email I just received starts with this:
Dear Dr. Redfield:Perhaps he meant my post about NASA's arsenic debacle.
I came across your blog post and was very impressed with it! I am now writing to request your assistance: I have accepted an invitation to serve as Specialty Chief Editor of a new journal entitled Frontiers in...
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