Negative results (no transformants)

The results of my big transformation experiment are clear but not what I had hoped.

There's no evidence of transformation of either strain by any of the markers. As expected, the C600 strain produced Lac+ revertants (frequency 10^5 - 10^6) and Leu+ revertants (frequency 10^6 - 10^7). I know these are revertants because the control cells (no DNA or no sxy insert or no inducer) produced just as many Lac+ and Leu+ colonies as the cells given DNA, sxy and inducer. The thr-1 mutation didn't revert detectably (frequency <10^8) style="font-style: italic;">lacZ gene.

I know the plates used for the selection were OK because the donor strain (w3110) grows fine on all of them but the recipients don't. This also confirms that the donor strain carries the expected wildtype alleles of lac, leu and thr.

So what next? First the RA and I need to discuss her latest results checking out the crp::kan marker she's been using for her (apparently successful) transformations. Then I'll probably do another set of transformations and controls, this time with that marker.

Waiting for the colonies to grow

OK, I did my assigned E. coli transformation experiment, though it took a couple of days.  I did find out that cells with the sxy plasmid grow just as well as those with the no-insert plasmid, at least in the absence of the inducer IPTG.  I still need to have someone show me how to use the nano-drop DNA-concentration spec, and before using it I need to further clean up my DNA prep to get rid of the RNA.  (I know there's RNA in it because I estimated its concentration and quality (fragment length) by running it in a gel.)

When I was in grad school we always added tetrazolium tetrachloride to our minimal plates because it makes the colonies red and easy to see.  I couldn't find any instructions for using it in minimal plates but I found old (Lederberg-old) instructions for using it in nutrient agar, so I used that concentration.

Now I just need to wait for the colonies to grow.  That will be slow because they're on minimal agar, so they probably won't be countable until tomorrow.  I can spend the intervening time helping grade my last exam question and entering the numbers into my spreadsheet, and then doing the Excel-wrangling to get the final grades for my ~400 freshman biology students.

Experiment plans

OK, my assignment (from the RA) is to test transformation of E. coli with nutritional markers rather that the crp::kan cassette she's used so far.

The first step is to inoculate strain W3110 into, say, 10 ml of LB broth and grow it overnight, and then tomorrow morning do a DNA prep. This will be the donor DNA for my transformations.

I'll be transforming two strains, C600 and BW25113. C600 is a work-horse strain from my grad-school days; genetically it is lacY (it can't take up lactose), thi (it needs the vitamin thiamine in its medium), and thr leu (needs the amino acids threonine and leucine in its medium). Its full genotype is F- tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1 λ-. F- and λ- means it differs from the ancestral K-12 strain in lacking the conjugative plasmid F and the lambda prophage. The tonA21 mutation removes a protein used as a receptor by phage T1, the glnV44 mutation (also called supE44) changes a glutamine tRNA gene so it inserts its glutamines at what would be UAG stop codons, rfbC catalyzes a step in synthesis of the outer-membrane sugar rhamnose, and fhuA helps cells take up ferrichrome (an iron-scavenging siderophore). I've listed all these because I want to make sure I've thought about all the factors that might confound my experiments - I see none here.

The other strain, BW25113, comes from Barry Wanner by way of the fantastic Keio group in Japan who have generated many of the E. coli clones and cassette mutants we've used. Its full genotype is F- ∆(araD-araB)567, ∆lacZ4787(::rrnB-3), lambda-, rph-1, ∆(rhaD-rhaB)568, hsdR514. So it has deletions of the araBAD operon (can't use arabinose) and the rhaBAD operon (can't take up rhamnose). rph-1 helps processing of tRNAs, and hsdR is a restriction nuclease that cuts DNA lacking a specific methylation. 

I'm only interested in the lacZ mutation; this is an insertion of 4 rrnB terminators that block transcription of the lac operon. I'd better check for an associated deletion, and for the size of the insertion, as these factors may influence the efficiency of recombination. Well, after quite a lot of searching I've come up with not much. This allele has an unspecified deletion and an insertion of 3 (or 4) copies of the rrnB transcriptional terminator in the lacZ promoter. I've emailed Barry Wanner asking for more details that would let me estimate the length of the heterology between this allele and a wildtype allele. (Prompt reply! The heterology is about 1 kb.)

What controls will I want to do? No DNA, to confirm that I'm seeing transformants and not new mutants. Cells without the inducing sxy gene (with a no-insert version of the plasmid). Cells with the sxy plasmid but without IPTG induction.

What will I select for? In BW25113 I can only select for Lac+, so I'll need minimal plates with lactose as well as minimal plates with glucose. BW25113 doesn't need any supplements. In C600 I can select for Lac+ by providing lactose as the only sugar, but the plates need to be supplemented with thiamine, threonine and leucine. I can also select for Thr+ and for Leu+ by not adding these amino acids to the medium.

So I'll need stocks of glucose and lactose (20%, I think - it's been a very long time since I did simple genetics in E. coli) and of threonine and leucine (10 mg/ml is standard for amino acids, I think). Thiamine I might as well put into all the agar. And minimal salts, and autoclaved agar.

Because I'll be growing up cells with both the inducing plasmid and a no-insert plasmid, I can also follow their growth in enough detail to see whether the insert slows growth.

No real results yet

The first experiment didn't produce much in the way of results.  Mostly because of a strain-name mixup that had me trying to transform kanamycin-resistant cells with a kanamycin-resistance marker...  (certainly not the first time I've made that kind of mistake).  The other transformations gave somewhat-unexpected results, but I hadn't done the proper controls for them so it's all still inconclusive.

I'm going to switch to transformations with simple nutritional markers (sugar use, amino acid requirements), but I'll need to first make some DNA from the donor cells (W3110), and then make lots of minimal plates with glucose or other sugars.  I think I can just spread the required amino acids on the plates as needed, rather than pouring plates with particular amino acids in the agar.

But first I have to deal with the problem of too many freshman biology exams to grade and too few graders to do the grading.  Then some Excel-wrangling, then I'm done with teaching for a long time.

Today's experiment

Today I'm planning to do the first of what I hope will be a long series of natural transformation experiments in E. coli.  The research associate has already done a number of these, but I didn't trust her positive results because they happened in the supposedly recA- strain DH5alpha as well as in another recA+ strain.  But now I've found that our DH5alpha stocks have the UV-sensitivity typical of recA+ strains I'm more optimistic that the transformations really are working.

What strains will I test?  The two strains that she has previously tested, plus a control strain that doesn't carry the plasmid we use to induce the chromosomal competence genes.  (I could not bother with the DH5alpha strain, but I think its unexpected phenotype may provide useful information.)

What DNA will I use?  The RA has made a stock of DNA from a strain carrying a kanamycin-resistance insertion in the crp gene.  She uses it at 2 micrograms/ml.  She's now making a better strain whose DNA we'll use for future experiments; it carries this insertion and easily-selectable wild-type alleles of other genes.

How will I prepare the cells?  I've grown the two plasmid-carrying cultures overnight from single colonies (over two nights really as I inoculated them on Friday night).  I'll dilute these 1/100 in LB and grow them to OD=0.2  The other strain I'll need to inoculate directly from a single colony this morning.  When the cells are at OD=0.2 I'll add IPTG and then the DNA.  After 2 hours I'll plate the cells with and without kanamycin.  I'll use a wide range of dilutions on the kanamycin plates, because the RA has found 'bald-spot' problems when dense cultures are plated directly.  I'll include a negative control with no DNA, and another with DNaseI added at the same time as the DNA.

What do I need to do to prepare?  Pour lots of LB plates, especially kanamycin ones.

Results of UV-sensitivity tests

My tests of UV sensitivity have found something odd about strain DH5alpha. I know it's supposed to be recA1, but both our standard lab strain and an independent DH5alpha derivative obtained from a European lab are no more UV-sensitive than the Rec+ strains we've tested (W3110, C600, BW25113), and much more UV-resistant than our NM554 strain (recA13).

This suggests that our DH5alpha strains are not really RecA-. That would be consistent with the RA's results in her transformation assays, but it seems unlikely that both our stocks are not what they're supposed to be.

Another weird result is that when DH5alpha is carrying a low-copy sxy expression plasmid it becomes as UV-sensitive as NM554. But the UV-sensitivity of the Rec+ strain BW25113 isn't altered by the same plasmid. (sxy expression in these cells wasn't induced with IPTG, but that may not matter because DH5alpha is deleted for lacZYA and maybe also lacI.)

I guess I should check other components of the genotype of our DH5 alpha strains before I start emailing the RecA experts for advice. It's supposed to be F-, φ80dlacZΔM15, Δ(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk-, mk+), phoA, supE44, λ-, thi-1, gyrA96, relA1. Easy to check for Lac- (the RA just made some MacConkey plates), but many E. coli strains are Lac- so that's not very diagnostic. Hmmm....

What I love about doing E. coli genetics

Sometimes you can do one simple experiment in the morning, check the plates late that afternoon, and do a follow-up experiment before you go home!

Photo documentation

 This is an iphone snap of my test plate after overnight incubation.  The first and third streaks are a recA+ control strain and the second and fourth are of a known recA- strain.  Because I was working at an odd angle, the green lines I drew to guide the exposures aren't lined up with the actual exposures.   The recA+ cells grew fine after 5 seconds of UV, and gave a few colonies even after 1 minute (probably cells that were shielded in some way from the UV).  The recA- cells grew fine when they were not exposed to any UV, but didn't grow at all even after only 5 seconds UV.

So today I've streaked all the cells I want to test, each on at least 2 different plates.  I also reduced the UV dose.  Yesterday I used a high dose range (5, 15, 30 and 60 seconds) to make sure I had a dose that would kill the recA- cells; today I used 2, 5 10 and 15 seconds.

Doing an experiment at last!

Only a tiny experiment, but it's a start.  I need to test some E. coli strains to find out whether or not they have recA mutations.  Because RecA regulates DNA repair, the easy phenotypic test is UV sensitivity.

Here's a diagram of how it's done.  First find a piece of cardboard and a short-wavelength UV lamp (I use the handheld one we sometimes use to look at DNA in gels).  Lightly streak each strain you want to test onto an agar plate, as shows in the Before picture, being sure to include both recA+ and recA- controls.  Then expose different parts of the streaks to the UV, using the piece of cardboard to shield the rest as shown in the middle picture.  (Be sure to take the lid off the petri dish because the plastic is UV-opaque.)  Then incubate the plate overnight.  If your UV dose is appropriate, you'll see the growth shown in the After picture.

This is very quick and easy, but it's not foolproof. If the UV dose is too high none of the strains will grow in any of the exposed areas, and if it's too low all the strains will grow everywhere.  If you make the mistake of streaking the cells too thickly the cells on top will shield the cells underneath them and all the strains will grow everywhere.  Because this is the first time I've done this with E. coli since I was in grad school, I'll need to try several different combinations of lamp distances and exposure times.  

What's done, what's not

OK, now I guess I'd better go through these parts of the UV-variation manuscript, figuring out what's done and what still needs to be done:

• Analysis of the true consensus and variation in uptake sequence motifs in all the bacterial genomes that have uptake sequences (= the family Pasteurellaceae (USS) and the genus Neisseria (DUS)).

I've done whole-genome Gibbs analyses and logos for all the species with uptake sequences. (Hang on, better check that no new ones have appeared since I did this.) Good thing I did; they've finished the Haemophilus parasuis genome. A quick count of canonical USSs (MS-Word bioinformatics) finds only 99 Hin-type USS cores (AAGTGCGGT and reverse) and 450 Apl-type (ACAAGCGGT and reverse). The genome has only 12 of a predicted novel USS core GAGTTCGGT), nicely confirming our prediction that another group's assignment of this as the H. parasuis uptake sequence was an error (Redfield et al. 2006). Now I need to remember how to do the Gibbs analysis and do it on this new genome.

• Analysis of variation in DUS and USS motifs across different location categories (orientation wrt replication, in coding sequences, in non-coding sequences, in terminator positions).

We're only describing this for N. meningitidis and H. influenzae. The H. influenzae work is all done, but I still need to do at least some of the N. meningitidis analysis. My notes say I haven't done the direction-of-replication analysis but I think I have - maybe I didn't finish it up.

• Analysis of covariation between the different positions of the DUS and USS uptake sequence motifs (e.g. does having a particular base at one position correlate with having a particular base at another position).

I've done this for both N. meningitidis and H. influenzae, and prepared the figure.

• Additional experimental data on how variation in uptake sequence affects uptake by H. influenzae. (This will just be a paragraph as it only modestly enriches a previously published dataset.)

Done, figure prepared.

• Development of a computer-simulation model of uptake sequence evolution, and use of it to investigate the roles of key factors in maintaining uptake sequences in the non-coding parts of genomes.

Now this is the biggie. The model is all developed, and we've done a lot of work with it. But I need to remind myself of what we'd found (my recollections are all muddled withthe confusing interim results and changes we made to the model). Luckily, before the former post-doc left she put together a good summary of where things stood, so my first task is to use that to restore my brain to its previous understanding.

The Perl-model manuscript (and the data) still need lots of work

I guess it's not surprising that I'd overestimated how close to completion the work is for the manuscript that, among other things, describes results from our computer simulation (Perl) model of uptake sequence evolution . As it's the only uptake sequence manuscript we still have under way, I think I'll start by giving it a simpler title. But first I'd better summarize what it now contains.

1. Analysis of the true consensus and variation in uptake sequence motifs in all the bacterial genomes that have uptake sequences (= the family Pasteurellaceae (USS) and the genus Neisseria (DUS)).
2. Analysis of variation in DUS and USS motifs across different location categories (in coding sequences, in non-coding sequences, in terminator positions).
3. Analysis of covariation between the different positions of the DUS and USS uptake sequence motifs (e.g. does having a particular base at one position correlate with having a particular base at another position).
4. Additional experimental data on how variation in uptake sequence affects uptake by H. influenzae. (This will just be a paragraph as it only modestly enriches a previously published dataset.)
5. Development of a computer-simulation model of uptake sequence evolution, and use of it to investigate the roles of key factors in maintaining uptake sequences in the non-coding parts of genomes.

In its present incarnation the manuscript is titled Evolution of DNA uptake sequences under molecular drive, but that title really only refers to the simulation model part. The real title should probably include the word 'variation', because that's what's been missing from previous work. For now, in this blog, I think I'll refer to it as the US-variation manuscript. (I reserve the right to come back and change the previous sentence if I find a better name.)

Excavated documents

Well, the big pile of documents has been excavated and the results are i) a few minor contributions to the recycling bin; ii) a smaller pile of printouts of articles; and iii) a similar pile of assorted research notes and records. Not the Perl-model manuscript drafts and notes I was looking for - they turned out to have been neatly filed in the file box labeled "Manuscripts in progress", which sits on a shelf right next to the file box labeled "Manuscripts failing to progress".

I now remember that the stuff in the pile on the floor was there for a reason. It's all sources of important ideas that I keep forgetting about - either papers I've read that tell me things I really want to remember, or notes from previous research that should someday be followed up on. Filing them would have almost the same effect as just throwing them out. This way I periodically go through the pile hoping to tidy it away but instead discovering that I need to keep these things where I'll see them now and then.

So what did I find? (Maybe if I blog about them I can decide how to use some of them?) Starting from the top of the research notes pile:

1. The table of contents of part of a former tech's lab notebook, indexing the DNA uptake experiments she had done.
2. A table listing the H. influenzae strains in a 'tiling-path' collection that a colleague had given us, with a map of the large-insert plasmid they're in. These are cloned in E. coli, and I think I was planning to use them to test whether H. influenzae competence genes work in E. coli (maybe make E. coli competent). I should mention these to the RA when we sit down on Tuesday to discuss the E. coli transformation experiments.
3. The abstract of a paper reporting that RadC (competence-induced in S. pneumoniae, H. influenzae and E. coli) does not contribute to transformation or DNA repair in S. pneumoniae. This belongs in the pile of articles, next to one showing that RadC contributes to replication-fork stabilization (highly relevant to our ideas about what else the 'competence' regulons control).
4. A very old (c. 1992?) folder containing restriction maps of some plasmids we made with the H. influenzae cya gene. I think these could be filed away.
5. A table summarizing 'Next-Generation Sequencing Informatics', printed four months ago and probably already out of date. But highly relevant to the new post-doc's research and our planned NIH proposal.
6. A list of the research questions we hoped to answer by microarray analysis of H. influenzae gene expression (also c. 1992). We've certainly answered most of these, but perhaps not all. Certainly I can't remember the answers to some.
7. An unpublished summary of research some colleagues did into the distribution of transformability in H. influenzae strains. I think it was presented as a poster about 4 years ago. They sent us the strains, and a former post-doc included some of them in her more detailed analysis of the distrobtuion of competence and transformability (now in press in Evolution).
8. Notes from my analysis of PTS genes in Pasteurellaceae, particularly the glucose and fructose transporters. Of interest because the PTS regulates cAMP which regulates competence, and the H. influenzae PTS has only the fructose transporter.
9. The reviewers' comments on a manuscript that the former post-doc is revising.
   
10. A page of notes from last summer (or the summer before last) when I was planning to test conditions that might induce E. coli sxy by assaying for expression of two lacZ fusions (to comA and ppdA). I did this and saw no induction.

That's enough for today, even though it's only about 10% of the pile.

One manuscript accepted, another resubmitted

Our manuscript on the coevolution of uptake sequences and bacterial proteomes has been accepted for the inaugural issue of Genome Biology and Evolution.  This is the one that's been ten years in the making!

And our manuscript on the CRP-S regulon of E. coli has been resubmitted.  It was rightfully trashed by the editors in its previous incarnation (mostly for claiming what it didn't deliver), but now we think it's very good.

And classes are over!  Still lots of teaching stuff to deal with - I have 24 term papers discussing whether intelligent design is a scientific alternative to natural selection, and a final exam to compose - but I've started excavating the stack of research-related paper on my office floor.  Today I expect to reach the layer of drafts and data for our Perl-model of uptake sequence evolution.  As I recall, much of the draft manuscript is already written, and only bit more simulation data is needed.  

And maybe by Monday I can get into the lab...

NIH programs

I spoke yesterday with the Program Director for the NIH Evolution of Infectious Diseases program.  She suggested that the analysis we're planning might fit better in a different program, Prokaryotic Cell Growth, Differentiation and Adaptation.

She gave me contact information for the director, so one of today's tasks is to compose an email that introduces the research I want to propose and asks when would be a good time to call him and discuss it.

CMYK - the saga continues

I found a grad student in the lab next door who's a graphics whiz, and she converted my PowerPoint files to CMYK for me.  But I forgot about the need for 300dpi resolution and for reducing the size to approximately that of the final printed image, so the CMYK versions are both about twice as big as they should be and much too low in resolution (72 dpi, says the digital image analyzer thoughtfully recommended by the journal).  

Unfortunately the size discrepancy isn't large enough compensate for the resolution discrepancy.  So I've sent the PowerPoint file and jpgs and tiffs derived from it to my collaborator, hoping she can do the conversion we need.  She's sent me the other information I need for the final revisions, so maybe we can still get it all submitted before Monday.

Tne Sxy in E. coli manuscript

About 2 weeks ago (maybe even longer) the RA gave me what was supposed to be the final version of our manuscript about what Sxy does in E. coli. She and the former post-doc had laboured over the revisions (we had convinced the editor to treat it as a 'revise and resubmit' rather than an absolute never-darken-our-door-again rejection), and I was just supposed to suggest a few minor improvements in the writing.

Instead I found lots of problems, so she and I have spent much of the last 10 days revising and re-polishing the sentences, and changing parts of the text, and completely replacing the Discussion, and making the figures clearer. Finally we have a version to send to the former post-doc for final approval. We think it's now quite good, and we certainly don't want to d any more writing, so he's been strictly warned that he's not allowed to make any substantive changes to the text, except maybe to the Discussion, which still needs a final paragraph.

Here's hoping we can submit this one on Monday.

CMYK!

The uptake sequences vs proteomes manuscript is almost ready for resubmission. The bioinformatics author is just checking whether she can do any statistical analysis for one simple graph, and I'm trying to prepare the figure files for submission.

Unfortunately the journal wants the files in CMYK format, but we made the figures with PowerPoint, which doesn't do CMYK. I spent much of yesterday evening looking for a way to convert them that didn't involve PhotoShop. I don't want to buy PhotoShop because it's far too sophisticated and complex for our needs, and it's very expensive.

So first I Googled the problem, but didn't find any easy free solutions. Then I tried my test version of the program Acorn, which costs less than 10% of what Photoshop costs but claims to do most of the same things, only easier. But Acorn can't convert files to CMYK. Then I Googled some more, and found that Macs come with a utility application called ColorSync that claims to do this. So I spent quite a while figuring out how, and doing it, only to discover that the CMYK files became their negatives every time they were saved. Everything goes black except the text, which goes white. The colours don't exactly go black, but they become very dark.

So then I emailed the former post-doc who had mastered these file conversions, and he said he'd had the same problem with ColorSync, but had done CMYK conversions using an old copy of Photoshop on one of our computers. I found our old copy of Photoshop Elements on that computer (it came free with a scanner), but it refused to open. The RA said she has Photoshop (no, wait, it's on the home computer), and that it's also on another old computer. That was again Photoshop Elements, and it did open. But when I tried to use it on my files, there was no CMYK option, and further Googling revealed that Photoshop Elements doesn't support CMYK at all.

I'd really like to get this done today, because the journal wants the resubmission by today so they can include it in their inaugural issue. So I think I'll ask around to see if someone else has a copy of Photoshop I can use for a little while.

Should I go to the SMBE meeting?

I've been debating whether to attend the Society for Molecular Biology and Evolution meeting in June.  It's in the same place (University of Iowa) as and right after a meeting I will attend (John Logsdon's Evolution of Sex and Recombination), so practically it would be easy and I'm paying the airfare anyway.  But I'm also going to the American Society for Microbiology meeting in May (Philadelphia), and because I'm combining this with a visit to family in Florida I'll be home for only 2 days between this and the Iowa trip.  So I'll be pretty burned out from meetings and traveling by that point.

I've never been to Iowa, so few days there sounds interesting, but a week in a small college town surrounded by cornfields?  I suppose I could get a lot of work done.  Dates:  Fly in May 31, Sex meeting May 31-June 3, SMBE June 3-June 7, fly home Sunday June 7.

Also, registration for the SMBE meeting is $450.  But accommodations are cheap, especially if I stay at the Motel 6 ($36/night).  But staying there would require renting a car, which would be fine if I wanted to do a lot of sightseeing, but I don't think there are many sights to see (though I'd like to drive over and look at the Mississippi).  I could stay on campus for about $75/night, which is still far cheaper than what I'm paying for my ASM accommodation.

On the other hand, I just got an email announcement that the SMBE meeting will include a symposium on the impact of next-generation sequencing methods for evolution.  As the CIHR and NIH proposals I'm planning will include massive sequencing, this sounds like something I should attend.  And other symposia look excellent too.

I need to decide by April 5 to get the early registration discounted fee - otherwise I'd pay $50 more.

Starting to plan proposals

I'm planning to write two grant proposals this summer, to the Canadian Institutes for Health Research (CIHR) and the US National Institutes of Health (NIH).  They'll be on similar topics, aspects of how H. influenzae and E. coli take up DNA.  NIH has way more money to give out, so I'll be asking them to support the really expensive work involving massive amounts of DNA sequencing.

The first (practical) issue is when grant proposals are due.  CIHR proposals are due Sept. 15, and NIH proposals Oct. 5.  (Both actually have to be in the hands of the UBC research administration a few days before, because the streamlined electronic submission process takes a lot longer than the old paper submissions, which just had to be in the hands of the courier by midnight on the due date.)

These dates work out well, because I won't have to tell CIHR all the details about the megabucks we're asking NIH for (you have to include with your application a copy of the summary page of any related proposals you have submitted), but can just casually mention that we plan in the future to apply to NIH for sequencing money to address related questions.  This is good because Canadian grant panels aren't always comfortable with giving their limited funds to people who have lots from other sources.  They also may not be happy if they see that you have already applied elsewhere for funds to do overlapping work.  But NIH doesn't mind (see my previous post), so having already applied to CIHR won't faze them.