Been a while...

And, sadly, after an exhausting day I'm not going to post much to make up for it. Tomorrow is dedicated to composing pieces of our sxy manuscript, including: crunching today's data, getting figures together, writing my and Laura's Material and Methods sections, finishing the strain list, and (time premitting, which I doubt) thinking about elements of the discussion.

Now, a little bit of thinking on MurE hypercompetence mutants: Inspired yesterday by handing Rosie a figure showing that addition of AMP or GMP to MIV cultures results in reduced Sxy production. A neat feature of the data is that AMP has a more dramatic effect than does GMP on Sxy levels, and this is exactly what we see in terms of repression of transformation frequency. It got me to thinking that I saw the same thing when treating murE749 cells with nucleotides, which got me wondering if murE749 induces competence through the standard Sxy + CRP mechanism, does it do so by specifically inducing sxy? Caixia describes testing one of Laura's sxy::lacZ fusions in the murE749 background, but the strain described in that paper is misslabeled in the paper's strain list (ie. our records show the strain number from the paper to correspond to a xyl mutant of some sort). I sifted through Caixia's notebook and could find most of the other murE experiments, but not this one... So I don't know what type of fusion Caixia tested: protein or operon? It's possible she tested an operon fusion, which we now know doesn't acurately reflect Sxy abundance in the cell. I could directly measure Sxy levels in the murE749 strain, and if we found that they were increased, we could be fairly confident that murE749 induces competence through Sxy. This would be interesting, but also just another piece of the complex and unresolved story of how sxy is regulated.

RNA vs protein

The results are in: sxy transcript levels vary much less dramatically between mutants and between growth conditions than do Sxy protein levels. This suggests that RNA secondary structure has it's greatest effect post-transcriptionally by influencing translation.

Yesterday's real time pcr experiment gave nice results consistent both with expectations (ie. more RNA in hypercompetent mutants and less in "hypocompetent" mutants) and with microarray results (ie. RNA levels increase in WT cells as cell growth slows at the onset of stationary phase). Today I have been crunching yesterday's RNA results, reanalyzing old protein data, and learning to use the new fluorescence scanner in our department (which will be used this week for western blot detection). Tomorrow I start the latest round of western blots, and then some beautiful gel pictures for our manuscript.

RNA becometh DNA day

Today I destroy some DNA and make some more (ie. DNase treat my RNA samples and then reverse transcribe them). I can then do the real time pcr tomorrow, and then western blots to measure Sxy levels during the rest of the week. I am very eager to get this sxy manuscript together, so I will bust my ass to get the results we need before the long weekend. At the same time, I'm going to work more on writting the manuscript - it's hard in the absence of the key experiments I'm doing now, but I will try write outlines to address the possible outcomes and begin to flesh out the discussion. I look forward to writing the discussion because it will require delving into some good ol' RNA regulation work from the microbiology community.

Also, if my real time pcr shows that hypercompetent mutants have higher levels of full-length sxy transcripts, I want to draft up an experiment to test whether WT cells generate just as much 5' UTR RNA, which would suggest that the the hypercompetence mutations allow for full transcript reads, while in WT the transcript is prematurely terminated by formation of the loop. The outcomes of this experiment could be complicated by many things (ie. rapid degradation of 5' ends when they are not followed by a full transcript), so I hope to mine the trp attenuation literature to see how that model was established. A full discussion of attenuation will also be necesarry for the discussion of the paper as we work to solve how sxy is regulated.

Cell lysis (or lack thereof)

My poor RNA recovery has resurfaced, and it is exclusive to cells in MIV for 30minutes, with improved (but still lower) yields from cells in MIV for 90minutes. I went back through my old notes to find that previously when working with Hflu, I had speculated that MIV treated cells require more intense lysozyme treatment. For many months I have been working with E. coli and had continued on with the same protocol when switching back to Hflu, with the standard thought that "Hflu is a wimp - it falls apart if you look at it long enough". But maybe I'm wrong, maybe MIV treated Hflu is a tough nut to crack. I have been visually cheking my cell suspensions for clarity, which is usually a good indicator of lysis, so I really don't know what's up.

Today I will add more lysozyme and also split my pellets so as to load less cell material onto the RNA isolation columns. It's possible that a tough cell wall is not the problem, but that something else about the cell constituents of MIV treted Hflu is negatively impacting column performance. By diluting the cell prep and loading less material, I hope to dilute the inhibitor and ultimately improve RNA yield.

I had a good visit with the Sigma rep yesterday. We really like their DNA purification and isolation columns (half the price and twice the performance of Qiagen = 4x better), and he said they now have RNA isolation columns, so he's sending a sample for us to try. It turns out that Sigma makes the buffers that Qiagen sells in their kits, so it's not surprising that Sigma can make a column and undersell their competitors (who they also sell to)... Sigma really can't lose.

More RNA isolation

Since submission of the CRP-S manuscript Monday evening, the past two days have been spent back in the lab. A fair amount of sorting, tidying, ordering, cleaning, and other boring stuff - but the tedious RNA preps should be done today. I had poor yield from two samples yesterday and there is no rhyme or reason to their poor quality. The cell pellets were ample in size and they were prepared concurrently with good yield samples. I'm tempted to blame Qiagen for poor column consistency. Their name usually strikes a sour cord with me as they sell very expensive stuff, yet have some of the most useless tech support. To Qiagen's credit, even with low yields, the RNA still looks great and the sample is not infiltrated by RNases. Hopefully, better yields will result today.

Aleeza successfully defended her thesis yesterday. She did well, and I took mental notes to try and remain as relaxed and composed as she when my own defence comes around. I was particularly frustrated by two things: 1) the audience gets their only chance to ask questions immediately following the intro presentation, while their minds are still swirling from the tonne of info just presented. Instead, the audience should get to ask questions after the committee (who have all just read the thesis) ask their paced and thought-out questions. I think that this would do much to stimulate the audience into asking more pointed questions. 2) (this is not to be missconstrued as sucking up to the boss) The questions were all the same and rather boring. They were exactly what you would expect based on the material presented. Defences benefit from Rosie-style questions, which probe different and accessory (but equally relevant) issues. At least the examining committee did sound genuinely interested in the thesis material, even if they all fixated on the same stuff.

Manuscript is done!

Well, besides a few i's that need dotting. Unfortunately, it took the weekend, mostly to get the citations all sorted (77 in total now) and the citation manager Bookends to put them all together. This means that this afternoon I can get down to sxy - both some RNA work and filling in some pieces (like Methods) in the 2º structure manuscript. Maybe soon I'll have something more scientifically stimulating to add to this blog - I think my brain was bored this weekend doing menial things like replacing ( ) with { } at all citations in the CRP-S manuscript.

Of scientific interest, may I didrect your attention to a recent article in Science "Depletion, Degradation, and Recovery Potential of Estuaries and Coastal Seas" (23 June, p. 1806-1808), which tracks human impact on coastal ecosystems across the ages of human expansion (the cultural periods make for a neat X-axis). There is currently much press on estimating the future impacts of global warming, but what strikes me is how we rarely stop to think about how our everyday activities, like fishing and pissing in the river, have, over the ages, caused more global damage in terms of depletion of species richness and abundance than global warming is predicted to accomplish in the next few hundred years.

Manuscript be done

Today I get the CRP-S manuscript done.
1. Finish figure legends, 2. Finish table of contents and strain list, 3. Get bookends working, 4. Make sure everything is up to NAR snuff, 5. Finish fig.8 (make a nicer tree - include sxy evolution) 6. Do a final read-through... Then, on to Sxy - and I can commit myself wholeheartedly to studying "the other" regulator.

On a sad note, today I have a final visit with a dear friend (and co-founder of our long-running Darwin reading group) before he moves off to the States to work for the Department of Home Land Security (or at least be funded by them) to model the spread of terrorist seeded diseases. He comes from a long line of politically active communists, which meant a childhood of moving great distances and living in exile. He's had trouble visiting the states before, but now they want him to come work the front line against a new evil breed of people. But what happened to communists? Aren't they (and their children) still evil? Did Patrick Swaze make Red Dawn for nothing? If I were a super bad guy, I don't think I'd like it if my fickle nemesis decided to drop me from the radar.

Today is an RNA day (with a few dashes of manuscript work thrown in for flavour).

The Sxy protein is required for the induction of competence genes in H. influenzae. We are working to dissect the mechanisms by which RNA 2º structure regulates sxy expression, to ultimately identify the signals that stimulate competence. I have already found that mutations that destabilize sxy RNA 2º structure result in increased Sxy protein production. Is this because the mutations allow for increased sxy transcription or increased translatability (or a bit of both)? I have prepped cell pellets from sxy mutant strains and now I will extract the RNA (26 samples in all)... tedious, but the results from quantifying sxy RNA levels will be most rewarding.

In the beginning...

Hello lab gang,

I was hoping to support UBC's blog host, but it's not particularly well maintained and it's much easier to use this Blogger site.

Before the science of the day, an intro to my site:

"Duckfeet" and "Duck in the muck" are both references to the observations and experiments described in Darwin's "On the origin...". Darwin, in his supremely subtle brilliance, draws parallels between his simple experiments with ducks in a pond and the biogeography of plants and small animals that range across and between continents. I love this passage because it is both brilliant and funny. I'm including it here, but as I'm working in Safari, I can't readily use formatting to highlight it.

"Although the beaks and feet of birds are generally quite clean, I can show that earth sometimes adheres to them: in one instance I removed twenty-two grains of dry argillaceous earth from one foot of a partridge, and in this earth there was a pebble quite as large as the seed of a vetch. Thus seeds might occasionally be transported to great distances; for many facts could be given showing that soil almost everywhere is charged with seeds. Reflect for a moment on the millions of quails which annually cross the Mediterranean; and can we doubt that the earth adhering to their feet would sometimes include a few minute seeds? But I shall presently have to recur to this subject.

Almost every year, one or two land-birds are blown across the whole Atlantic Ocean, from North America to the western shores of Ireland and England; but seeds could be transported by these wanderers only by one means, namely, in dirt sticking to their feet, which is in itself a rare accident.

When a duck suddenly emerges from a pond covered with duck-weed, I have twice seen these little plants adhering to its back; and it has happened to me, in removing a little duck-weed from one aquarium to another, that I have quite unintentionally stocked the one with fresh-water shells from the other. But another agency is perhaps more effectual:

I suspended a duck's feet, which might represent those of a bird sleeping in a natural pond, in an aquarium, where many ova of fresh-water shells were hatching; and I found that numbers of the extremely minute and just hatched shells crawled on the feet, and clung to them so firmly that when taken out of the water they could not be jarred off, though at a somewhat more advanced age they would voluntarily drop off. These just hatched molluscs, though aquatic in their nature, survived on the duck's feet, in damp air, from twelve to twenty hours; and in this length of time a duck or heron might fly at least six or seven hundred miles, and would be sure to alight on a pool or rivulet, if blown across sea to an oceanic island or to any other distant point."
On the origin of Species Chapter 12 - Geographical Distribution