I needed an image of wt yeast in DI YPD to compare with the morphology of later strains. As I ramp up the D2O concentration, I expect to see some differences and this is good information to have in general.
I don’t think there is much to infer from the 20% D2O comparison image, but if it’s worth anything there are a few cells in that image that are elongated, and I never saw that morphology in DI water.
These images and the final comparison image (and ALL the original raw files from the camera) are available via the figshare link above. These images were also taken after this post, ie the conditions are the same.
Images were acquired at 10x magnification and the scale is 1um/px. The largest image is 1959×1925 px (the larger D2O yeast on normal agar) and the smallest image is 1462x1749px (the D2O yeast on D2O).
As you can see there is quite the interesting phenomenon here. The interesting thing is that there is something morphologically different about the D2O adapted yeast. The reason for that thinking is because the yeast colony retains its brainy shape when placed on normal media, as compared to the wt yeast on the normal agar (smooth circle). Before I make any bold claims about what may be happening here, I need to read some papers about yeast morphology.
Also if anyone wants a glycerol stock of this strain of yeast to run some tests on it, I’d be happy to send it to you. I personally don’t have the means to perform any advanced tests so any experimentation is welcomed!
K. Unno, T. Kishido, and S. Okada, “Effect of over-expressed Hsp26 on cell growth in yeast,” Biol. Pharm Bull., vol. 21, pp. 631–633, 1998.
In an attempt to further understand the poorly written paper I summarized yesterday I’m going back to read the paper they cited pretty frequently. Hopefully this paper will answer some of the questions I had yesterday. Note time:
The motivation for this study is that there has been no observation of the effects of the over-expression or depression of hsp26. Apparently hsp26 is induced during the transition of log-phase growth to stationary phase in yeast growth.
As they mentioned in yesterday’s paper (this one is earlier by a few years), the yeast cells used were ssa1ssa2 and have a slower growth rate than the wt.
Like the methods from yesterday, they picked clones of ssa1ssa2. However, this time the clones they chose had different doubling times from each other. Yesterday they chose clones whose doubling times were comparable to the wt.
cells were incubated at 25C in a minimal dextrose medium (YPd? hahaha) supplemented with nutrients but lacking uracil. WHY???
They plot the doubling time of the clones of ssa1ssa2 and the wt. It didn’t occur to me that the growth rate of clones could be different. This may be interesting to explore for my D2O variant strain. It should be noted that the doubling time of the wt strain had little variance amongst clones. What is the mechanism for this?
Exactly what is the difference between clones of the wt and clones of ssa1ssa2???
they then relate the expression of hsp26 to doubling time and compare that to other proteins (hsp70, hsp90, hsp104, Ssb, and Kar2). They find that hsp26 is more closely correlated to doubling time than the other 3 hsp’s and make no claim about Ssb and Kar2. But they do state that the amounts of Ssb and Kar2 in the clones is similar to the wt. This is sort of true.
So based on subtle information in this paper, it seems the mutant ssa1ssa2 lacks the genes Ssa1 and Ssa2 which make the hsp70 enzyme. But this paper mentions that Ssa4 is inducible and also produces hsp70 when induced. So in yesterday’s paper when they said there was no hsp70 but then they got hsp70, I’m guessing they induced the Ssa4 gene to get some hsp70. Good thing they mentioned that!
In this paper the induced hsp70 levels are less than the wt, and their data shows that hsp70 levels increase with increased doubling time. So yeast that lack Ssa1 and Ssa2 but have Ssa4 have higher levels of hsp26 (than wt) and almost normal levels of hsp70 are at risk for longer growth rates (doubling time). So the proteins may be linked in the extreme case that the Ssa1 and 2 genes are off and the Ssa4 gene is on (both produce hsp70).
But how does this information relate to the paper yesterday? It doesn’t really, but it may explain why they were seemingly contradicting the amount of hsp70 they had in their cells.
Ok just quickly rereading the first few pages of yesterday’s paper and I still have no idea what is going on. No matter. Their results claim to show that some clone of ssa1ssa2, which is beyond arbitrary, is D2O tolerant. I’m not too sure that their methods apply to what I’m doing, since they are just looking for deuterium resistance and not necessarily an organism that is made of D instead of H. I will go over yesterday’s paper again later tonight/early tomorrow to make sure things still make no sense (but maybe I’ll have a moment of clarity?).
I had a meeting this morning regarding the IGERT I speak so frequently of. That means the timed data wasn’t so consistent in the beginning. I took my first time point about 25 minutes after I set up the experiment. And then I took my second time point 2 hours later. The raw data will show that I took the first time point about 20 minutes later, but I assure you the time difference was more than that.
I did take the rest of the time points an hour apart.
I spilled a considerable amount of the D2O sample (which was in the beaker), maybe like 30-40% of it after I took the first time point.
In data news, I find it really strange that the yeast in D2O didn’t grow nearly as much as it did in the past. I’m wondering if the new setup had anything to do with that.
Also it looks like yeast in DDW grew more than the yeast in DI. Again I’ll have to do another run to verify, but I feel like this could be a real result.
The added volume definitely improved the results, as the yeast remained suspended in the flasks. In the previous trials I would get considerable settling because the test tubes wouldn’t swirl as much. Much better setup.
So the moral of this story is, I’ll have to do another trial of this setup. Fine by me! Whatever it takes to get good results that are repeatable. That’s the nature of open notebook science!
I set up the starter cultures yesterday. Today is the day to track the growth over time. The only differences between this experiment and the ones I’ve done in the past is that this time I’m: (1) using higher volumes of YPD (total of 25ml) and (2) only doing three samples (DI, DDW, and 99% D2O) instead of the normal five (DI, DDW, 30%, 60%, and 99% D2O). I just didn’t have enough made YPD to do the other two samples.
Here is my setup:
I’m using 2 small flasks and 1 small beaker. I only have 2 flasks so the beaker is an improvisation. I think they are all 50-80ml beakers/flasks but I’ll have to verify this. (Update: They are all 50ml glassware.)
I added 22.5ml of each YPD type to the glassware.
I added 2.5ml of each YPD starter culture to the glassware + YPD
Put in the incubator/shaker at 125rpm and 30C.
Record absorbance every hour.***
blank with each type of ypd (ie measure the DI YPD culture after blanking with DI YPD, etc).
I’m using semi-micro cuvettes and measuring with a NanoDrop 2000c (see experiment products page)
***I had some meetings this morning so my first time point is a half hour after I started incubation. And my second time point is 2 hours later. Every point after that is 60 minutes apart. See data for explanation.
Today I’m starting another starter culture for what will be the 5th experiment in this series of experiments. I’ve been getting mildly consistent data so I’m amping up production (in the form of volume) to see if the results change or become more consistent. I say mildly because the results across all experiments are similar (DI and DDW grow the most and then there is gradual declines in growth as D2O is added). But on a deeper level, time points typically cross paths or absorbance numbers decline temporarily. I’m hoping that it is the nature of the setup of the experiments as they are now, and that by increasing the volume of YPD for growth, there won’t be these minor inconsistencies. We’ll find out though!
Prepare 3 autoclaved and cleaned test tubes for YPD addition.
Add 10ml of DI, DDW, and D2O YPD (that is YPD diluted in each water type) to each test tube.
Inoculate a cell culture in each test tube.
Place starter cultures in incubator/shaker at 30C and 150rpm.
A quick note: It seems the growth increases steadily over time, but the accumulation of cells on the bottom of the test tubes is both annoying and startling. This affects the readings and explains why the growth increases dramatically across the board between hours 3 and 4 (when I mixed the test tubes before putting 400ul in the cuvettes for measurement). Maybe using larger volumes would be helpful?