# Preparing for E. coli in DDW, DI, and D2O

Sorry for the lack of notebooking today. I had to send a bunch of emails this morning, catch up on teaching work, and then had to write an informal CV this afternoon. Wanna see it?

Anyways, I’m doing the prep work for the E. coli studies and showing Alex how to use an autoclave. I also am getting into sterile technique and Alex will have something up later about all of this to which I will link.

Tomorrow we are going to mess around (and get triple doubles) in a safe environment and grow some e.coli and see what happens. I also hope to get my hands on some yeast. There are a few barriers before we can get any serious data. First I have to successfully grow microscopic creatures in small containers. I have to get acclimated to using the nanodrop as an OD measuring device. Then I have to do all this with different water types.

# Project Outline for DDW Effects on Microorgansims

Another page for project outlining that will change over time.

UPDATE: I changed the embed code so the map has live update capabilities.

# Today I learned how to synchronize yeast cells

I spoke with Kelly Trujillo about cell synchronizing after Koch had mentioned to me that we may need this for ddw yeast studies. He mentioned that he was going to be synchronizing for a study that he was setting up and that I could come learn the process. So I took him up on his offer.

I have to say, the protocol is surprisingly simple. Kelly grew a colony, diluted it to about 0.1 OD and then let it divide up to 0.4 OD. We looked at the asynchronous growth and then he added some mating pheromone (details to come) to some of the culture to halt the cells in G1 phase of cell division. From here, he could synchronize them to enter S phase by using hydroxyurea (HU) after washing out the mating pheromone. The remaining culture was arrested in G2/M phase with the drug Nocodazole.

We paused during each step to look at each culture (asynch, G1, S, and G2) but I have no pictures. I do have the knowledge to do it myself and hope to incorporate this in the DDW experiments shortly. A preplanning thread will come soon.

# Effect of Deuterium Depeleted Water on Life

Hydrogen has several isotopes and one of them, deuterium, exists quite naturally in water to form $D_{2}O$. In previous experiments and several papers by Gilbert Lewis, it has been found that life is hindered in the presence of $D_{2}O$. While this may be true, my PI Steve Koch wondered if life had found a use for it because naturally occurring water has about a 17mM (millimolar) concentration of deuterium.

To put that number into perspective, when I do a typical polymerase chain reaction of DNA I add 10mM of each base of DNA (which is less than the amount of naturally occurring deuterium) to create millions of copies of a DNA template from an amount that is 1000x less then what the reaction yields. In fact most chemicals in most of my buffers on the order of the amount of naturally occurring deuterium.

So you can see it isn’t a stretch to think that nature has found a use for $D_{2}O$ since it is quite abundant and life has been constantly evolving for billions of years. I want to test this hypothesis in a variety of different organisms:

1. Tobacco Seeds – to act as a foil to Lewis’ experiments in which he grew tobacco seeds in pure $D_{2}O$.
2. Mustard Seeds – from what I’m told mustard seeds are the powerhouse of the botanical genetics world much like Drosophila and S. cerevisiae are in their respective genetic fields.
3. Escherichia coli – another molecular biological powerhouse that is very easy to grow and may be easy to see results with. We just got the facilities to be able to grow E. coli and damn it I want to use them!
4. Saccharomyces cerevisiae (Yeast) – I know a guy who grows yeast for his experiments and I’m sure it wouldn’t be a stretch to get him to do so in deuterium depeleted water.

So the idea would be to try to grow these in regular water and in deuterium depleted water (no $D_{2}O$), and in the case of E. coli and yeast, perhaps in pure $D_{2}O$ because I don’t think those experiments have been carried out yet. Hopefully I will be able to conclusively state whether or not life has developed a need/use for $D_{2}O$ which would be a very interesting discovery indeed!