Back in the lab

I am currently planning my experiments for the next couple of months so that I know exactly what I want to be doing each day. My first goal is to resolve a problem that arose just before my departure to Inuvik. I was repeating an experiment to quantify the relative levels of sxy mRNA in strains that have regulatory mutations in the sxy gene.

There are several steps required to do this. First, cells are cultured and all RNA is extracted. The RNA samples are treated with DNase, then random hexamer-primed reverse transcription is used to convert the RNA into cDNA. The cDNA is then used as template in a SYBR green real time PCR assay. In this assay, more starting template results in the synthesis of more amplicon early in the PCR cycles. Amplicons are bound by the SYBR green fluorophore and fluorescence increases; fluorescence is measured each thermocycle. There are many steps where problems can arise, but appropriate checks and controls are used throughout. First, a gel is run to test the quality of the extracted RNA. Later in the final PCR reactions, non-DNase treated RNA is included as a template to ensure 1) that there is more template in reverse transcription reactions, confirming that the RT step worked, and 2) that chromosomal DNA is not a significant contaminant. Also, chromosomal DNA is used to confirm that the PCR Master mix can amplify target genes.

In the past we have had problems with chromosomal DNA contamination during the RNA extraction/purification, but that has long since been remedied. On Friday Oct. 13, I encountered a whole new problem: no amplification was detected in any of the real time PCR reactions. One odd result is that the fluorescence levels were very high throughout the run, suggesting that there may have been amplification as usual, but that the Master Mix fluorophores or the detector are not working properly. There are other possible suspects: 1. The least probable problem is that the primers have gone bad (I know they were added to the Master Mix), but they worked beautifully a couple of months ago. 2. I used a new batch of SYBR Master Mix, and it may have bad Taq enzyme or bad fluorohpores. 3. The machine is not thermocycling or its detector is not working properly.

My first troubleshooting step is to run some of the PCR reactions on a gel to see if amplification did occur. If yes, fluorescence is the problem: either the detector or the MMix is not working. If no, Taq or the thermocycle is not working. Either way, the second step will be to run a couple of reactions comparing the new Master Mix to older leftover MMix. Also, I will include another primer set to test whether my primers are the problem. If I again see no amplification, I will try the reactions again on a different realtime PCR machine.

On the positive side, my cDNA samples are already prepared. Once I solve the PCR problem, it will be relatively quick to measure sxy mRNA levels as I had originally set out to do.

Yesterday's graph is wrong

I awoke early this morning with a nagging feeling that I had made a bad graph yesterday. The second graph in "Musing on Sxy", which outlines my predictions about how adding cAMP to log-phase cells will affect transformation frequency, is missing data we already have. What would help is if my imaginary graph became more concrete with some actual numbers and data (again from Redfield 1991). First, a reminder that our simple model for the regulation of competence currently involves two activators (or two “on” switches), CRP and Sxy. CRP levels are fairly constant throughout different growth stages; by supplying 1mM of it’s allosteric effector cAMP, we are turning that switch full on.

This graph is now based on real data - the four data points are roughly the average transormation frequencies measured in Redfield 1991. Here we see moderate competence development in KW20 when CRP is fully activated, even though KW20 has very little Sxy during log-phase growth. Thus even very low levels of Sxy are sufficient to allow some competence, in contrats to what I said yesterday. Also, this helps clarify what I was already planning for today’s blog: I want to take a reciprocal approach to yesterday and look at what limits maximal competence when both the CRP and Sxy switches are turned on. (A subsequent blog will discuss the role of stochasticity and competition/interference between regulators - but that’s trickier and needs clear groundwork thinking. I’ll stick with the simple on/off switch analogy for now).

Maximal competence (ie. a transformation frequency just over 0.01 with good, fresh transforming DNA) develops when cells are incubated in MIV medium for 90 minutes. Redfield 1991 found that the sxy-1 hypercompetent mutant achieves the same high transformation frequency in MIV and during late log-phase growth. When I repeated this experiment to test all hypercompetent mutants, I observed a ~5 fold lower maximal transformation frequency in MIV , and transformation frequency in late log was 7-10 fold lower again (see graph). However, I used old transforming DNA and found that with fresh DNA, transformation frequencies in MIV were the same as Redfield 1991 (not shown on graph. Also, I did not test late log growth with fresh DNA). This raises the possibility that in Redfield 1991, using very potent, fresh transforming DNA obscured a difference in transformability between MIV and late log conditions, which only became apparent when using old (sub-saturating?) DNA. If so, why don’t hypercompetent sxy mutants achieve maximal transformation in late log-phase? One possibility is that the cells are maximally competent, but the transformation machinery within cells is more efficient in MIV conditions. However, I will explore the alternate hypothesis that hypercompetent sxy mutants are not maximally competent in late log growth.

Hypercompetent sxy mutants in late log-phase have equal amounts or more Sxy than does KW20 when it is maximally competent in MIV, therefore Sxy is not limiting. The cAMP data described yesterday and at the beginning of this post shows that cAMP is not limiting. Thus, I can elaborate the first graph, and include a possible role for a third regulator. We have good evidence that PurR, a protein that represses gene expression in the presence of the purine bases hypoxanthine or guanine, is a third regulator of competence. To date, we have been testing for PurR activity by supplementing growth media with purine nucleotides, but this has been problematic partly because cells don’t like having high levels of these nucleotides in their growth medium. If all of my speculation presented here has some validity, we can test for PurR activity in late log-phase by measuring transformation frequency in double hypercompetent sxy/purR- mutants. If the hypothesis is correct, then when transforming with sub-saturating DNA, hypercompetent purR- cells will have higher transformation frequencies than their hypercompetent parents.

Musing on Sxy

In my last blog posting I said that both Sxy and cAMP-CRP are limiting to competence development during growth in rich medium. However, I would like to make a more precise statement about how these limitations may change as culture conditions change. Here I expand my previous graph showing the correlation between increasing Sxy levels and increased transformation in both KW20 (blue) and hypercompetent mutants (red) during log (OD 0.2; filled circles) and late log (OD 1.0; empty circles) growth in rich medium (see "A strong positive correlation"). The black line plots the correlation, and the dashed black line is a hypothetical leveling-off of transformation frequency when Sxy levels become saturating. The dashed green line is a prediction of how the plot would look if cAMP were added to the culture medium.
Redfield (1991) showed that adding cAMP to hypercompetent sxy-1 cells increases transformation frequency in early log but has no effect in late log growth. Therefore, cAMP is not limiting to competence development at ~OD 1.0. The green line is not predicted to have the same slope as the black line because cAMP is only limiting during log growth; presumably, as PTS sugars are depleted in late log growth, cells are making their own cAMP. Thus, the two lines converge and level-off when Sxy becomes saturating. We know that Sxy isn’t saturating during late log because some hypercompetence mutants have more Sxy than others, and this correlates directly with increased transformation frequency.

This predicts that titration of cAMP during log growth will have no effect in KW20, but will increase transformation frequency in hypercompetent mutants such as sxy-1.
This hypothetical graph shows that due to insufficient Sxy levels in KW20 during log growth, no competence develops even at high cAMP levels. On the other hand, there is sufficient Sxy in sxy-1 to activate competence genes, thus adding cAMP increases transformation frequency. Hypercompetent strains are transformable in log growth, so there must be some cAMP produced by cells in rich medium. In the graph, the cyaA- mutants cannot make cAMP thus even the sxy-1 cyaA- double mutant cannot become competent until cAMP is added.

A strong positive correlation

My time in Inuvik so far has consisted primarily of making figures. In order to stop procastinating, here's a blog post to show a satisfying result. (A "Where is Inuvik?" post will follow soon). Plotting Sxy abundance in cells growing in sBHI against transformation shows a positive correlation: as Sxy levels increase, cells become more competent.
This tells us that Sxy is limiting to competence development during gowth in rich medium, as is cAMP-CRP. In the figure, blue=KW20 and red=hypercompetent mutants, while filled circles are OD 0.2 and empty circles are OD 1.0. This plot includes our latest protein data - with this new data, the corellation between Sxy levels and transformation frequency is very strong (and satisfying).

"A quick one while he's away"

The title is an homage to The Who, who it turns out have just released a new single after all these years, but refers more to the state of my brain while writing this post. And this has just reminded me of an article I skimmed over in (an old) Nature today describing how new neurons in adult brains only become established if they are stimulated. Thus, our everyday activities and thoughts get hardwired to a certain extent by selecting for neuronal pathways that contribute to or fit with our brain processes that day - budding new neurons that aren't stimulated die off. So Cliff Claven was right that there is selection for neurons in our brain, though not as he imagined it (ie. beer kills slower and weaker neurons, thus making the remaining herd of neurons faster and the brain smarter... I think he explains it with an analogy of lions killing sick and old wildebeest).

This weekend I had hoped to repeat some old experiments conducted by a former grad student in the lab, however there are no freezer stocks of the strains he made, and worse yet, used. In other words, he used up the last of some older strains in the lab. I bow my head in sorrow at the extinction of some unique and special bacterial species. Oh well, I'll just have to make them again. Also this weekend, I almost wrapped up my NSERC application - that will easily be done tomorrow. On the dock this week: review a manuscript, measure some sxy mRNA, start to put together a manuscript on CRP binding to CRP-S sites, make some strains and repeat the old experiments to test whether PurR regulates rec2 expression, and probably all sorts of other odds and ends (or, "Odds and Sods" as The Who would say)...