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Advanced ape that understands logic, creativity, emotion, and expression. Displays great cognitive skills with somewhat limited communication skills. I'm some kind of new smart ape, artist extraordinaire, lover and fighter, and damned fine scientist.
Kinesin Stability in D2O

Kinesin Stability in D2O

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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.

DDW1, Water Type Effects on Organism Growth

Updated Preliminary Tobacco Seed Growth “Results”

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Those are images of the first batch of samples (Dark Virginia seeds, not pre-soaked), I’ll take pictures of everything and post it next week. But here are the preliminary reports of what different water types do to tobacco seeds:

  • Every seed submerged in deuterium depleted water (DDW) sprouted little hairs on the initial root (the radicle). The interesting thing is this happened almost immediately after emerging from the seed coat.
  • Typically the seeds submerged in deionized water (DI water) germinated the slowest. More will come on this when I replicate the Crumley experiment.
  • Little hairs sprouted inconsistently on the seeds in tap and DI water but are more prevalent on the DI water seedlings. They are not as abundant on these seedlings as they are on the ddw seedlings. If I had to give an analogy (and I do) then I would say the ddw seedlings can grow a nice ‘fro, while the other seeds exhibit male pattern baldness.
  • The little hairs remain localized on the end of the root and aren’t distrubted along the hypocotyl (early stem, and I almost said axon, lol!) so I’m inclined to believe that this is an early root system that is developing because of a lack of nutrients in the water (seen on ddw, most DI, and almost no tap water seeds). But I’m no botanist so I’m just guessing. The fact that the hairs are really prominent in the ddw seeds might suggest the plant recognizes the lack of deuterium, but I’m not willing to make that leap yet.

I setup a new photography system for these seeds. Dr. Koch lent me his Nikon D40 dSLR camera and I purchased some magnification lenses for it. I have the camera setup on an optical post and use a cylindrical lens holder to mount the seed samples (in cuvettes). The picture quality is much better now, with a much higher resolution. I’ll be looking into microscope images soon. Soon I’ll be developing a reliable way to measure the germination, but let’s not jump the shark now. All will be revealed in due time.

Also I was going to measure the pH of the samples at this stage of their development to gain some insight into whether the germination event drastically alters the pH, but the probe in the lab is too big and I don’t have enough sample volume. So I’m thinking that next week I combine the volumes of the water (of each type) to do one “average” measurement. There are four samples of each water type, two for each seed species, and each is filled about 2ml which would give me 8mL of combined volume for each water type. I’m just waiting for the pre-soaked samples to reach full germination (ie shed the seed coat). Now I’m not saying this will work, and it may not be reliable, but hopefully it is a decent approximation for expectations for now until I learn a little bit more.

RC1, RC2, RC3, RC4, Water Type Effects on Organism Growth

Repeating Crumley et al (1950)

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Koch asked me in a comment to replicate an experiment done in 1950 by Helen A Crumley et al demonstrating Tobacco seed growth in deuterium oxide (D2O). The experiment is rather simple (and the figure from the paper is shown below) as Crumley placed 100 seeds in differing amounts of D2O (double distilled water, 33%, 66%, and 99.8% D2O) and analyzed the growth.

Influence of ordinary water, 33%, 66%, and 99% deuterium oxide on tobacco seed germination. Counts made at daily intervals for 39 days. (From Crumley, Fig 3)

So here I am planning the experiment. I will change some things from their experiment. First they placed the seeds on wet cloths (paper towels?), and I will submerge the seeds in the water amounts they used. They also used a variety of plant species (tobacco, clover, radish, Kentucky bluegrass), where I will just use tobacco seeds (but I will try two different species). Finally they talk about their results in terms of percent germination, but it isn’t clear from the paper if they mean number of plants that have exhibited germination, or if they are referring to some amount of growth exhibited by each plant. I will look for both possibilities and report the results as I find them.

In a preliminary experiment I will submerge the plants in water in petri dishes and seal it with parafilm. I will be looking into a more airtight solution as time goes on. I also won’t do 100 seeds but probably on the order of 33 seeds per sample. And in the future I will look into figuring out a way to measure the seed growth.

Original Crumley paper can be found here.

This post is open to read and review on The Winnower.

Open Notebook

OpenPCR: Understanding the Final Hold Issues

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I’ve been writing about the OpenPCR platform for a couple weeks now and together we’ve had our ups and downs. The most prevalent issue that I’ve had in my tests is a failure to hold. By that I mean, after completing a program, a PCR reaction is usually set to hold at a certain temperature in case the user isn’t around to claim his prize (a complete reaction). I usually set the hold at 4C but discovered OpenPCR can’t handle below ~12C, but I’ve also discovered that in my case (maybe other cases) the machine doesn’t hold at all. It just ends the program and returns to room temp.

So today I investigated…

The short results: I could not consistently get the machine to hold after trying multiple programs. Nor could I get the machine to run the designated program consistently. When the machine did perform a hold in two cases there was a glitch in the LCD, but otherwise reports are ok.

Now for the long answer:

I ran 5 experiments and then gave up. I don’t like admitting that, but after a couple hours of frustrations I think it is warranted noting. Here are all my notes:

  1. I ran the “Simple Experiment” that is pre-programmed into the software. On my first attempt (this is after completing the PCR experiment I posted earlier), the experiment crashed. The software displayed “Done!”, while the machine sat idly. (Get used to that because this will happen a lot in this discussion.) I tried to restart and this time the machine did nothing while the software displayed the time left screen. I turned the machine off and on and tried again. This time the program ran and the machine cooled to hold after completion. While in the hold phase there was a glitch on the LCD that said “Final H” and next to the H was a rectangle and three stacked horizontal lines. (I should have taken a picture of that screen 🙁 )
  2. I reran the “Simple Experiment.” On my first attempt the machine did nothing while the software displayed the time left screen. I turned off and on the machine. Tried a rerun and this time, the machine held at 20C. It again displayed that weird screen on the LCD.
  3. I created a new program: 1 cycle – 12C (for 30s), 20C (30s), 30C (30s), hold at 12C. On my first attempt, the lid heated and the software said done. Then the program started and the software started the countdown (after saying Done!). The machine got to about 15C and stayed that way for a while (time unknown) and so I stopped it. I tried to run the program again, but changed the lid heating to 0C hoping it would just not activate the lid. This time the program never started and the machine did nothing. I then changed the lid temp to 75C because I noticed it was cooling and thought maybe it needs to reach some temp before the program could start. The machine never started running.
  4. Another new program: 1 cycle – 37C for 30s, hold at 20C. On the first try (yay!) the machine ran, completed, and held. The LCD actually read “Final Hold” too. On a second attempt, the software said “Done!” but the experiment hadn’t started yet. It then ran and completed.
  5. In my last experiment, I did 1 cycle – 16C for 30s, hold at 16C. I tried twice, and both times the machine never started the program.

By that point I had given up and decided to do this write up. If I had to guess I would say there is a glitch whenever the software asks the machine to hold at anything less than 20C. There is definitely a glitch if you want to do two experiments back to back, because a lot of those times is when the machine would not do anything after completing the first of the back-to-back experiments. Other than those observations I have to give a big shoulder shrug and hope that the OpenPCR guys can give me some troubleshooting ideas. Anyone else with the system having any problems?

Open Notebook

OpenPCR Reaction Try 2

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1% Gel Stained with Sybr Safe

Today I ran the same reaction that I ran on Friday, this time I did two sets of reactions. One set, I put in the OpenPCR machine and the other set I placed in our Thermo thermal cycler. The image on the left shows the results. Lanes 1-5 contain the samples from the OpenPCR machine (Lane 5 also contains a 1kb ladder), and lanes 6-10 contain the samples from the Thermo machine. The bands are brighter towards the top than towards the bottom because I stained prior to running the gel (and the stain runs in the opposite direction from the DNA). I tried to post-stain for about 15 min to improve image quality.

Notice that the bands are all the same length and the same brightness, which was not the case in the reaction from last week. The spreadsheet of the reaction can be found below.

Open Notebook

OpenPCR Reaction Results

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Here are the results of the reaction that I posted earlier today. In lane 1 is a 1kb DNA ladder. The second visible band from the bottom is 1kb in length. The next band is 1.5kb. Lanes 2-6 are each 10ul (mixed with 2ul of 6x loading buffer) from their respective PCR reaction tubes (all the same reaction). Lane 2 clearly worked the best, while lane 3 apparently contained a failed reaction. The other lanes (4-6) worked well enough in this test run. And as you can see, since the band travel distance is right between the 1kb and 1.5kb mark, that puts these fragments at right around 1.1kb (primers annealed at ~980 and 2008 on the template strand). More tests will need to be conducted in the future, but the preliminary results appear quite promising.

Note: It should be noted that this gel does not display a PCR reaction after cleanup. I went straight from the thermal cycler to the gel. The faint bands at the bottom of the image are the primers used in the PCR reaction.

Open Notebook

OpenPCR Reaction

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This reaction is based on the reaction for making the 1.1kb anchor used in the unzipping construct, which I haven’t discussed in this space, will in the future, and some background can be found on OpenWetWare.

This particular reaction makes a 1.1kb dsDNA product that is labeled on one end with a digoxygenin molecule and has no label on the other end. The reaction can be found here.

DDW1, Water Type Effects on Organism Growth

Unsoaked tobacco seed growth

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In most of these samples there is one seedling that shed the seed coat, and in few that seedling is floating at the water surface. It also seems that the branching in the second DD water sample (the Virginia Gold species) has been shed on one of the seedlings (perhaps the one that shed the seed coat). There is a stringy thing floating in the background, which I’m not sure is visible in the image but definitely there in the sample.

Anyone notice anything else?

DDW1, Water Type Effects on Organism Growth

Pre-soaked tobacco seeds early stage growth

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Note: There is some wordpress gallery error that won’t display the second and third picture captions. They are: 2. Dark V in Tap Water. 3. Dark V in DI Water

These images are from the presoaked tobacco seeds. I soaked them in their respective buffers (deuterium depleted water (DD water), tap water, and deionized water (DI water)), and then stored them in the fridge (4C) for 5 days in case there is any drastic chemical exchange between the seed and the water (which would change the buffer). It appears that the deuterium depleted water has started sprouting first in both cases, which is speculative but noteworthy. Also the branching that I noted in the last post appears again here, but only in the DD water samples, which is interesting. I wonder what that could be.

 

Open Notebook

Mindmeister Plugin Demo

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I found a WordPress plugin for Mindmeister that is really easy to implement and decided to write this page that demonstrates it.

The syntax is:

[mindmeister url="MINDMEISTER-URL"]

You can find the plugin here.

And as an aside I had to find a way to display syntax in the blog. First I tried Syntax Highlighter Compress, but that didn’t work and so then I learned that WordPress plugins are called using shortcode syntax which led me to Show Shortcode. Using this plugin is just the shortcode:

[showshortcode][/showshortcode]

And you place your shortcode in between the tags.

Note: The use of shortcodes is very much like coding in HTML except in some cases (like the Mindmeister plugin) no closing tag is needed.