Hydrogen has several isotopes and one of them, deuterium, exists quite naturally in water to form . In previous experiments and severalpapers by Gilbert Lewis, it has been found that life is hindered in the presence of . 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 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:
Tobacco Seeds – to act as a foil to Lewis’ experiments in which he grew tobacco seeds in pure .
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.
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!
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 ), and in the case of E. coli and yeast, perhaps in pure 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 which would be a very interesting discovery indeed!
From now on, I’m going to refer to this experiment as DDW3, so I don’t have to type all that out when I’m updating pics. I’m also still not sure how frequently I’m going to update (no less than every 3 days though). The reason is because I don’t need to track growth rates (although maybe that would work), but instead I need to look for phenotypical changes to the plants (like the root hairs in trial 1).
I have four samples per organism and I have two organisms. Now I’m doing both tobacco seeds and arabidopsis. Arabidopsis is one of the model organisms for genetics and hopefully will be great in this series of experiments. Let’s discuss the setup:
Initial preparation began with preparation of water types. DI, DDW, and tap water were placed in 15ml centrifuge tubes to be added to samples later. 0.5ml of D2O were placed in a PCR tube for mixing to be done later.
5 seeds per sample were counted and added to 8 prelabeled macro cuvettes (from VWR, see Experiments’ Products Page). Seeds were handled with tweezers and dropped into the cuvettes (this proved very difficult with the arabidopsis seeds because they are at least 25% the size of the tobacco seeds).
I used Columbia arabidopsis and Virginia Gold #1 tobacco seeds (also on the Product Page). I chose Virginia Gold #1 because of its inclusion in the prior experiments, and I chose Columbia because it was listed as a participant in the genome sequencing project of the species.
3ml of each water type were then added to the cuvettes (1 type per cuvette) with the exception of the D2O. For the fourth sample, I added 2999.5ul (or as close to that as I could possible get) of DDW and added .42ul of D2O. This sample is what I’m calling simulated water and is based on the Vienna Standard Mean Ocean Water. I used the proportion of D to H from that standard to determine how much D2O to add to DDW to create that same proportion. (It should be noted that this volume here is about 10x smaller than what I initially calculated last week and that post will be amended to reflect these values).
Cuvettes were then closed and placed in my photography rack, custom made by me from leftover Thor Labs cage system parts.
In previous experiments, I have done a few samples of presoaked seeds. In this trial I’m postponing those samples until these experiments are complete because there is a lack of space on my rack. I need to buy more cage system parts (the rest are being used in the previously unmentioned Optical Tweezers our lab has constructed) and then I can build a better photo rack.
In the past I’ve complained about the static repulsion the tobacco seeds have from the tweezers I use. Well with the arabidopsis seeds, the repulsion plays a much larger effect. The seeds are way smaller than the tobacco seeds and thus have less mass. So when they feel an electric force, they are propelled farther. I’ll go into the physics in the next post because it just dawned on me that I should have a picture showing the size difference. But once I figured out a decent technique of grabbing the seeds (it involved lots of jukes, saying “look over there,” and convincing the seeds that I had something great to show them) the setup didn’t take too long, and was considerably easier once I moved on to the tobacco seeds.
So now that I’ve had a whole post to discuss the setup and thus time to think it over, I think I’ll take pictures daily until I’ve determined that this data is useful or irrelevant. I’ll take a picture of my setup as-is now and post that so you can see what I’m working with.
Here is the setup I have for taking pictures of the seeds. As you can see, it is on the same assembly that I use for the Repeating Crumley Experiment.
The camera is on a rail that is kept parallel to the seed rack that I created. I use rubber bands to keep the two cage rods as tight as possible on the cuvettes. This prevents them from falling out of the assembly, and provides a nice divider between organisms. The arabidopsis seeds are on the left and the tobacco seeds are on the right.
Despite the appearance, the structure is actually quite stable, as long as there is no direct impact with the cuvettes. With that said, I have some minor modifications in mind once I buy some more equipment.
Today is much better from a setup standpoint. The seeds have soaked adequately and have sunk to the bottom. I tilted the cuvettes for a little so that all the seeds would be as close to the same focal plane as possible (at the front of the cuvette). Pictures taken indicate this.