Category Archives: Kinesin Stability in D2O

The Biophysical Effects of Heavy Water – My Defense Presentation

Defense Outline

Just over a week away now…

  1. Introduction
    1. What is D2O?
    2. The history of D2O
      1. Gilbert Lewis:
        1. purification
        2. biological effects
        3. The hypothesis
      2. Joseph Katz
        1. various experiments
    3. Uses of D2O
      1. NMR, mass spec
      2. The need for a D2O adapted organism
    4. Experiments in DDW
      1. use for space travel
      2. cure for cancer?
  2. The effects on life
    1. Tobacco Seeds
      1. The Crumley experiment and repeating the experiment
      2. Tobacco seed germination rate
      3. tobacco seed growth rate in low deuterium concentration
    2. Arabidopsis
      1. arabidopsis growth rate
      2. arabidopsis morphology
    3. E. coli
      1. growth rates
      2. adaptation and adapted growth
      3. morphology
    4. Yeast
      1. growth rates
      2. adaptation – can’t adapt
      3. morphology
        1. stall during cell division
        2. microtubule stabilization in D2O
  3. Molecular effects
    1. Stabilization of biomacromolecules
      1. DLS experiments
        1. Catalase
        2. Ovalbumin
      2. YPD longevity
    2. Investigation of HD exchange
      1. mechanism and exploitation for protein struture studies
      2. FT-IR analysis
      3. Cavity ring-down analysis
        1. low cost measurement of local atmosphere isotopic composition
    3. Effect on DNA
      1. The pursuit of shotgun DNA mapping
      2. optical tweezers
      3. methods
      4. overstretching data
  4. Future Work
    1. Arabidopsis
      1. adaptation
      2. seed growth in low deuterium
    2. Tobacco growth in low D2O
    3. Yeast morphology in taxol
    4. E coli protein expression in D2O and protein structure analysis
    5. DNA
      1. overstretching in D2O with intercalators

Well there is my idea of how to present my dissertation. I’m not sure if/where I should put my discussion on open notebook science. Also there are a couple things that I could see going elsewhere. I could describe the yeast and e. coli stuff in parallel instead of one after another. Also the HD exchange stuff could easily go right after the yeast, e. coli, or even the tobacco seed stuff. What to do…

Otherwise I think the story is pretty compelling: history of D2O and the unanswered question by Lewis. Investigations into D2O effects and trying to understand low D2O concentration effects, effects on macromolecules, and the understanding of large volume/long-term HD exchange.

Any feedback you may have would be GREATLY appreciated. I’ll send you a figshare t-shirt, or if you are XL, I’ll send you a hoodie (but I only have one).

Protein Aggregation Graphs

I just finished some beautiful graphs by combining the aggregation data sets from the protein aggregation studies (Ovalbumin and Catalase). Check them out:

ovalbumin visual aggregation: (left) non-aggregated sample, (right) after aggregation
ovalbumin visual aggregation: (left) non-aggregated sample, (right) after aggregation
plot of ovalbumin aggregation
plot of ovalbumin aggregation
Plot of catalase aggregation
Plot of catalase aggregation

Preliminary Results of YPD deterioration

Absorbance of DI YPD (pink), D2O YPD (green), and blank (red)
Absorbance of DI YPD (pink), D2O YPD (green), and blank (red)

These are the results of the experiment I stated a couple weeks ago. I have been tracking the deterioration (previously called aggregation, but I’m not entirely sure aggregation is the correct terminology) of YPD in both solvents. Today they looked pretty well degraded so I thought I’d share the results. Between the two, the DI YPD is more absorbent than the D2O YPD at nearly every wavelength measure (major uncertainty below 350nm).
I’m associating degradation with absorbance since the blank (which is also DI YPD) has an absorbance of zero at all the same frequencies.

D2O YPD also records 0 for absorbance at 600nm, which is the wavelength used for cell count studies, so there would be no interference from the solution. Whether or not the media is still usable by cells is undetermined.

I’m beginning a second experiment that would track the absorbance every few days via the same mechanism. If you recall, I began this experiment taking pictures and eventually moved toward using the nanodrop. This probe seems to do a good job so its continued use is reasonable.

Man I’ve been writing my dissertation for too long…

YPD aggregation update

Remember this experiment?

Checking in on it, the YPD has already begun it’s aggregation in both cases, but it is more prevalent in the DI YPD case. Which should be expected. At this point I can see a concentration gradient in both solutions, but the full-on aggregation hasn’t happened yet. As is, it’s not worth photographing because there isn’t much difference.

But thinking back to here, I remembered we used a Dynamic Light Scattering Machine (the DynaPro Titan TC by Wyatt), which just uses scattered light to calculate particle size. In the aggregation experiments we didn’t care so much about specific particle size, just that we wanted to see the particles grow bigger.

If in fact the YPD is aggregating, the solution would become cloudier by eye. So since our Nanodrop measures absorbance, we could potentially see the same effect. The catch is that the DLS machine mentioned above measures at 830nm, and my nanodrop measures a broad spectrum from 250nm-700nm. In that range I’m not sure where it is most accurate except at 260nm, 280nm, and 600nm (which is where it measures DNA, proteins, and cell counts).

I’ll do a more thorough study on Monday, but here is the quick analysis:

red - blank sample (normal YPD) orange  - aggregated DI YPD green - aggregated D2O YPD
red – blank sample (normal YPD)
orange – aggregated DI YPD
green – aggregated D2O YPD

YPD aggregation study

Two summers ago Kenji Doering (an REU student that summer) and I did some protein aggregation studies comparing D2O and DI water. The results were pretty consistent in that proteins don’t aggregate under the same conditions in both samples. What that meant was the D2O is almost certainly better for longevity of chemicals.

A few months ago I noticed that my ypd stocks go noticeably bad after a couple of weeks. But they seemed to last longer in D2O, I just never quantified that. Well now I’m going to.

I have two cuvettes sitting on my bench. One is filled with 1ml of DI YPD and the other is 1ml of D2O YPD. They are sitting next to the benchtop cooler to supply a bit of heat to speed up the aggregation reaction. I will take daily pictures of the solutions to compare the aggregation times of each media. Here is the day=0 time point (on the left is the DI YPD sample, and the right is the 99% D2O sample):

20130219-105637.jpg

Dissertation Plan

Here is my dissertation and defense plan. My research is composed of three areas:

  1. Shotgun DNA Mapping
  2. D2O Effects
  3. Open notebook science

All of it will be tied together because of ONS and D2O. Let’s get to it!!!


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