Tag Archives: intro

Introducing Shotgun DNA Mapping

Before I got immersed in the world of deuterium-less water, I was a rabid DNA-man. The project was Shotgun DNA Mapping which was a term we invented to describe a quick protocol for mapping a DNA sequence.

In theory, the technique was awesome: unzip a DNA sequence with optical tweezers and compare the data to a library of simulated data for a given genome to figure out where you are in the genome. This would lead to a bigger project called Shotgun Chromatin Mapping which was the similar except you could map protein locations on DNA fragments using the same technique.

For this to work, you need three components:

  1. Optical Tweezers – to unzip DNA and record data
  2. DNA – to unzip
  3. A computer simulation – to simulate DNA unzipping and match recorded data to simulated data

I dedicated a few blog posts in my other blog to discussing the basic principles of the project in case you need to get caught up to speed. But in case you don’t have time for all that, here is the whirlwind summary:

Optical tweezers are an optical system that requires a laser, a microscope objective, a condensor, some steering components, and a detector (in our case a quadrant photo diode) among other things. The laser is focused by the objective and this focus can exert forces on tiny dielectric particles. Our particles are microspheres. (The blog posts linked explain the physics of this in great detail.)

Using some principles of biochemistry I can attach a microsphere to a DNA fragment that is specially designed to: (1) tether to a glass slide using antibody-antigen interactions, (2) contain a weak point in the DNA backbone to begin unzipping, and (3) be versatile enough to use a variety of different DNA sequences.

I can then tether the DNA to slides and place them in the path of the laser. The focus will attract the beads, and if the tethering process works properly then the beads will be attached to DNA. This is how we are able to exert forces on the DNA. Our detector is used to track the laser movement, and those signals get converted into force data. The forces recorded are on the order of pN, which is insanely small but enough to distinguish from background noise.

Once we have unzipping data we can use a computer program to compare this information to a library of simulated unzipping data for a genome. In our proof of principle study we used the yeast genome, so we simulated unzipping fragments for the entire genome and then used actual yeast genomic DNA to unzip.

Unfortunately I hit an impassable road block in the experiment. The DNA I created wouldn’t unzip. I tried everything I could think of, reworked the entire process and tried to come up with alternate methods for creating the DNA fragments. Ultimately I had to switch to the project I’m working on now…

…But that doesn’t mean that the project was a complete failure. I’m sure the protocols and techniques I employed can be useful to someone, somewhere, someday and so I’m going to highlight posts from my old notebook here as a way to kind of direct attention to the protocols that summarize my project well and were most useful for me.

In this way, one wouldn’t need to sift through mounds of information just to find one thing. And it would provide visitors here a little more information about my background and something I keep alluding to. All in the name of open science!

Kinesin Stability in D2O

A main focus of the lab is to examine the effects of D2O on the kinesin motor protein. Andy Maloney (now Dr. Maloney) spent quite a bit of time perfecting an experiment known as the gliding motility assay which allows an experimenter to study kinesin processivity by analyzing microtubule movement. Microtubules are relatively long protein filaments that kinesin has the ability to “walk” on which it does to carry cargo to various locations of a cell.

Below is a video of the gliding motility assay. Since kinesin molecules are too small to be resolved we actually depend on seeing the microtubules (which are in turn only visualized because of fluorescent dye proteins. What we are seeing below is that the microtubules (squiggles) are being propelled by the kinesin.

The reason that I mention this is because Andy used these experiments to determine if D2O affected kinesin movement at all. Initial results looked promising because it appeared kinesin would push the microtubules slower than identical experiments in regular DI water. It turns out that the decrease in speed could be related to the increased viscosity of deuterium oxide.

Despite this, Dr. Koch hypothesizes (based on introductory reports by Gilbert Lewis, a very popular name on this blog, and others regarding life in general) that D2O can stabalize kinesin. What do I mean by this? Well proteins in general don’t retain their shapes forever. There is some probability that a protein can denature (unfold) and this is expedited by certain cellular conditions (temperature, time, function, pH, charge, etc.). So it is believed that the inclusion of D2O can affect these conditions to ensure the “survival” of the protein. Currently kinesin suspended in buffer has a relatively short shelf life (but I’m not familiar with the lifetime). This would be very useful for use in the lab when chemicals, proteins, etc are stored for long term use.

We have proposed a couple of experiments that may help determine if deuterium oxide does indeed affect the storage life of kinesin (and perhaps other proteins/enzymes). The first is to detect aggregation which happens when proteins unfold and stick together to form large clumps of amino acids. The second experiment would involve detecting decreasing kinesin activity over time possibly through ATP hydrolysis (ATP turning into ADP+P).

Over the next few weeks (months?) I will be exploring these avenues. I have a slight head start on experiment 1 because an REU student named Kenji Doering spent the summer in our lab and explored the possibility of D2O affects on stability with ovalbumin which is a protein from egg whites. This will of course be the topic of my next post (or two) and I will be posting some rather interesting data from those experiments and some others.

Mindmapping Proposed Projects (Updated)

Mindmeister and WordPress apparently do not work well together. I spent a good hour trying to figure out how iframes work in WordPress and it turns out they don’t so I had to install a plugin. I used Embed Iframe and it works, but it turns out that (well at least for me) Mindmeister wants to stick a big panel in front of the mindmap blocking at least 50% of the map. Booo! There is a mindmeister plugin, but I don’t know if I want to play with it right now. Maybe later, and then I’ll tell you all about it.

Back on track, if the above mindmap embed doesn’t work for you then the link is here to see it in full page glory.

Update: I found out that there was a plugin for Mindmeister and replaced the previous iframe coding with the new plugin. The process of discovery can be found here.

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!