Category Archives: E. Coli

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).

WT E. coli colony (on D2O LB agar) morphology

Yesterday I posted some pictures of E. coli colony morphologies. This was one of the colonies, but it wasn’t as developed, so today I’m adding the extra day’s growth image.

WT E. coli grown on DI LB agar
WT E. coli grown on DI LB agar

Looks great! It’s interesting to note that the colonies grown on D2O agar grow out. Instead of getting thick like it normally does, it grows in an outward direction. I guess I would attribute that to the stress induced by being in D2O.

Comparing the results from today to WT E. coli grown onĀ  DI media and D2O adapted E. coli grown on D2O media, it seems there is an interesting mix of morphological behavior. The adapted E. coli is very “brainy” and obviously the normal WT is “smooth,” but today’s specimen is in between smooth and brainy. Unfortunately I can’t make out the topographical features because the E. coli (as I mentioned above) is very flat. But the contour is very feature rich.

E. coli cells in D2O

I’m not going to make many comments about these cells. It seems that in D2O, E. coli is more likely to say fused but it’s not as obvious as it is with yeast. I make no other observations.

E. Coli Colony Morphology in D2O

Here are the results of yesterday’s setup. Here I’m comparing 4 samples:

  1. Wild type (WT) E. coli grown on DI LB agar
  2. WT E. coli grown on D2O LB agar
  3. D2O adapted E. coli grown on DI LB agar
  4. D2O adapted E. coli grown on D2O LB agar

All 4 samples were incubated for the same amount of time, and taken from starter cultures of similar absorbance. The absorbances of the starter cultures are as follows:

  1. Wild type (WT) E. coli grown in DI LB – 0.641
  2. WT E. coli grown in D2O LB – 0.325
  3. D2O adapted E. coli grown in DI LB – 0.489
  4. D2O adapted E. coli grown in D2O LB – 0.112

The D2O adapted E. coli took over the DI LB plate! I’ve re-inoculated those cells from that plate to get a picture that would show a typical colony and compare that morphology to the rest. I’ve also allowed the two D2O media samples to incubate for another 24 hours.

It should also be said that the D2O adapted colonies grown on D2O media look distressed compared to the WT colonies on DI media. But compare the two D2O adapted colonies and it’s tough to discern. Whatever mechanism gives the colonies a distressed appearance on D2O media, seems to be completely uninhibited on DI media. It’s tough to tell if the cells are distressed or just out of control.

Still these results seem pretty comparable to the last time I did these experiments (except using YPD). I’ll update again tomorrow, and insert these results into my dissertation.

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…

D2O Adapted E. coli Experimental Replication

Yesterday while writing I realized that the images of the D2O adapted E. coli that I’ve taken were grown on D2O YPD. In an effort to figure out if the morphologies are due to the YPD or the D2O, I’ve decided to redo the experiments on LB agar.

Today I made some D2O and DI LB broth:

  • 1.84g LB in 92ml of D2O
  • 1g LB in 50ml of DI water
  • Filtered broth for sterilization

Then from there I made some LB agar

  • 40ml of liquid D2O LB with 0.8g of agar (2% agar)

I already have solid LB plates with normal water (commercial).

I then incubated E. coli at 37C in liquid media so that I can streak the plates and analyze them. I used 2 different E. coli:

  1. Normal competent cells
  2. Cells from Day 33.
  3. I also had made a separate culture from an unlabeled glycerol stock that I’m pretty sure I made when I finished the experiment. I’ll check it out tomorrow after the sample has developed.