Well then...

Well, there may be some issues with my extraction technique. As the protocol I used will be undergoing some drastic changes, and could possibly be scrapped completely for another method, I will give just an overview of what is going on.

The above sample of leaf material that I collected was ground to a slurry in a lysis buffer solution that served to extract the DNA from other plant cell material. DNA is a negatively charged molecule and there are positively charged "beads" in the solution that adhere to them. The entire solution was then centrifuged to separate the beads and DNA from the heavier plant material. After filtration and a wash with isopropanol, I should have had a viable sample of DNA to run gel electrophoresis.

In gel electrophoresis, DNA is loaded into an agarose gel and current is applied (in this case 120 volts for 30 minutes). As I mentioned before DNA holds a negative charge and by placing it in the gel near the negative probe, it is drawn through the gel towards the positive probe. As DNA travels through the gel segments with more base pairs (Adenine with Thymine, and Guanine with Cytosine) will travel much slower than those with fewer base pairs, and the bands can be then observed and recorded.

This is the gel box I used. Apologies for the poor quality picture, I believe I deleted the wrong picture.

In my case, this did not go so well. The DNA sample did travel, but it went the wrong way! THe sample traveled to the negative end of the gel. This indicates to me that the positively charged beads used in the extraction actually made it through the filtration and wash process and were still bound to the DNA and pulled it to the negative pole. After discussion with Anil, and Matt, we have a couple different ways to change our extraction protocol. We think that the protocol we used was designed more for animal cells than plants, and the filtration wasn't up to par with the plant material.

I am not sure when I will continue with the extractions as the weight of the end of the semester is upon us. My research paper on the bacterial identification draft needs some polishing into a final draft, and I still need to develop an oral presentation of it as well (not to mention papers due in my other classes and impending finals). This extended Thanksgiving weekend will not be quite as relaxing as those passed...

HAPPY THANKSGIVING ALL!

In my last post, I left a question hanging: where is the line between the separate clone entities and one connected organism. Well, after some digging I have found another species that clones in much the same fashion: Populus tremuloides, the Quaking Aspen. Not only has it exhibited the same cloning adaptation, an example of it also happens to be THE WORLD'S LARGEST LIVING ORGANISM! According to the US Forest Service, Pando (as the grove is known), covers roughly 106 acres, weighs in at an estimated 13 million pounds and consists of over 40,000 individual trees that are, you guessed it, CLONES! These clones sprout form the root system in much the same manner as my little old Palo Verde.

"Pando" courtesy of USFS

This will definitely help me in laying out the proof of cloning (along with good DNA testing results, of course). I am currently trying to track down a couple of peer reviewed journal entries that relate experimental data with the aspen in much the same fashion as I am beginning to do with the Palo Verde. Unfortunately I have only been able to read through the abstracts as of yet and i want the data. A little time and effort and I should be able to find similar research. 

As far as my experimentation goes, today I conducted my first DNA extraction from one of the many samples I have collected. Next week I should have some gel results that will show wether or not I was successful at extraction. I will leave explaining the method of extraction for next week's post where I will fully explain the entire process from extraction to running the gels.

I think I'm a clone now...

Well, the ball is finally rolling on my research project. I will be looking at plant species that are known to produce sexually and attempting to show the fact that they do reproduce through cloning as well. The first species I will be looking at is Palo Verde.

When attempting to decide what topic I would cover for my project, i came to a decision that I would like to learn more of the nuts and bolts of genetic analysis. After extensive conversations with Matt and Josh, I decided to pursue an avenue of research that Matt suggested. He felt pretty confident through his  personal observations that the Palo Verde may actually do some reproduction from through cloning. The Palo Verde is a tree that has adapted well to the desert environment. It's dispersal appears to be pretty broad but it is often concentrated in washes. It specifically has adapted to growing in caliche, hardened deposits of calcium carbonate that acts like a cement holding gravel, sand, clay, and silts together in a solid mass. The Palo Verde is able to utilize voids in the caliche to develop it's root system. While very hard, this caliche layer is subject to forces of erosion. This erosion is where the fun begins with the Palo Verde. The suspicion is that when the Palo's root become exposed due to erosion that they will actually begin to grow another tree.

 This is a prime example of what I have been looking for. Notice the main tree left of center in the photo with a large root mass trailing to the right. On the right side is another tree that has matured, and between the two, new "shoots" can be seen. I think that this specimen will make a positive control.

In this picture you can see the large root heading from the main mass of trees on the right to the smaller one on the left. Is this a clone?

The same specimen from a different angle, showing that there are three separate tree systems here.

I have begun collecting samples from multiple Palo Verdes at Squaw Peak. I am collecting samples from trees I suspect to be cloning and trees that show no indications of it, all of which are in relatively close proximity to each other (map to come). Next week I will begin extracting DNA and soon hope to be running gels for analysis.

The question that looms in my head is where is the division line between one organism and another? For example in the first photo, there are three obvious tree growths connected to the same root system. Are they the same organism? Will I be able to find a situation where there are genetically identical trees that are no longer connected by the same root system? This may be difficult to do, and is definitely difficult to research. I have not been able to find literature on this subject as yet.

The digging continues...

All's fair in love and bacteria

The remaining differential test results are in and my unknown bacterium is, Staphylococcus epidermidis... or, well, maybe not...

In my last post I mentioned that there were two more tests that I was conducting. The first of these was a test referred to as the glucose O/F medium. This test would indicate wether the bacteria was capable of metabolizing carbohydrates (glucose) through oxidation or fermentation. Two test tubes of the glucose media and bromthymol blue (a pH indicator) were inoculated with samples of the bacteria. To one of these tubes a layer of mineral oil was added to create an anaerobic environment. The theory here is that a bacteria that ferments will exhibit growth in the anaerobic tube with the mineral oil, and the oxidizer will grow in the open or aerobic tube. The metabolic processes will result in the production of an acid that will change the pH indicator from dark blue to yellow. If yellow is seen in the open tube, the bacteria is an oxidizer. If the yellow is seen in the closed tube it is a fermentor.

These are the tubes after inoculation, and before incubation. Notice the layer of mineral oil on top of the media in the red capped tube.

This picture is after the incubation period as seen with backlighting. There was no perceivable difference under normal lighting. The aerobic tube on the left showed the faintest bit of a haze in the center of the tube. The anaerobic tube on the right does show what appears to be a colony growth and a yellow color change in the center of the tube. I have conducted this test in a previous experiment with a different bacteria that was a fermentor and the results were much more conclusive: the tube showed a large amount of color change. I interpreted the current result as being a weak positive for fermentation due to the apparent colony growth in the anaerobic tube. My thoughts were that maybe it was a slow grower and thus the minimal results.

I then analyzed the results of a mannitol fermentation broth. This test determines wether the fermentation of glucose results in the production of an aqueous acid or a gas. It was inconclusive on both counts. This stumped me as I believed the glucose O/F test indicated that this was a fermentor. It should have produced in the mannitol test as well, and would have been Staphylococcus epidermidis. 

I then consulted with Josh. He informed me that the glucose O/F test has been problematic in the past. My unknown should have produced in the aerobic tube and not the anaerobic tube. He did not elaborate on the reasons why the test had shown problems in the past. I suspect that the reasons are as follows. In the aerobic tube, the inoculation method required a "stab" with an inoculation needle through the gel like media. This may have created a situation that actually made it anaerobic. When the inoculation needle is withdrawn from this gel media it is plausible that the media could have "resealed" itself (the media may have been more fluid than it should have been) and not allowed oxygen to the sample once inoculated so it did not have the air needed for oxidation to occur. In the anaerobic tube, it is conceivable that the opposite occurred. Movement of the needle during inoculation could have created a void that filled with air and then was trapped in by the mineral oil. This small amount of air could have allowed some oxidation to occur and show the mild results I saw. At this point this is all speculation on my part and I need to dig deeper.

So, had the glucose O/F test showed the results it should have I would have correctly identified my unknown bacteria as Micrococcus luteus.