Research – Temporal Dynamics of Viral Communities

Hi Everyone,

Today is July 17, the final day of my seven weeks here this summer. I cannot believe it has gone by this quickly. It has been a very busy, productive and gratifying summer. I am honored that I had this opportunity. It has confirmed for me that research is the field that I want to work in the rest of my life. But, before I head back up to Alexandria, I want to leave you with some final thoughts about my research and plans for the fall.

As I mentioned in the first post, this project was begun in April and will continue, at minimum, until next April. The water processing, PCR, tRFLP, microscopy and DNA extractions will continue. And, I could not be more excited. Dustin, a colleague of mine, and I have decided that we should never have to take another class again. We would rather spend our days in Dr. Williamson’s lab. This summer was simply that good.

Of course, I wish we had not had to face the various frustrations we did along the way. Troubleshooting PCR and DNA extraction from soils took time and energy that we would rather have devoted to continuing our projects. Added to that, manufacturer error at Millipore has completely halted our viral enumeration by fluorescence microscopy.  Despite these setbacks and frustrations, we have learned a great deal.

 We have hammered out methodologies, working out the kinks in our various protocols in order to optimize our data collection efficiency. We have learned how to think critically and problem solve. We have learned to deal with frustration and work around problems. We have even gained some experience in dealing with manufacturers and other laboratories. All in all, we have gained invaluable experience that will shape us as scientists.

As I prepare to leave, I look back at the work I have done and I am astounded by the sheer volume of data I have gathered and that we have gathered as a lab. The next step is data analysis. I have no idea where this data will lead us, but I am excited to forge ahead. You may feel as though you gained no new insight into the microbial world of Lake Matoaka and it’s watershed. And maybe that’s true, not yet at least. But, look for a paper one day with Dr. Williamson’s name, my name and the names of each of my peers. We have plenty to share and there is plenty left to learn.

 Thanks so much for reading this summer! I know I had a great time and learned a lot. I hope you learned something, too!

Dana

Hello Everyone!

You may be wondering what the point is of all of the work in the previous posts. Where does this research all lead? What is she trying to determine? Well, it all leads right here, to Terminal Restriction Fragment Length Polymorphism or tRFLP.

In brief, tRFLP is a means of creating a community profile of the various assemblages of bacteria within Lake Matoaka, the Spillway, the Inlet and the soil around the lake. By digesting the terminally labelled PCR products of the 16s ribosomal rNA gene (a highly conserved gene present in all bacteria) with the same enzymes, the varying sizes of each fragment can be analyzed using capillary electrophoresis. Very simply, different fragment sizes indicate different groups of bacteria. Identical fragment sizes indicate identical or very similar groups of bacteria.

However, before capillary electrophoresis is carried out, the PCR amplicons must be purified, prepared and digested. Purification is done by means of a Qiagen kit (which I have heard referred to by one very wise professor as the “Miracle in the Blue Box”). The kit removes all of the Taq Polymerase, extra dNTPs and such from the amplicons.  These particles would inhibit the process of capillary electrophoresis. After PCR product purification, the amplicons are digested with Mung Bean nuclease. This is an enzyme that cleaves any single-stranded overhangs on the PCR products. Blunted, double-stranded DNA is ideal for a restriction digest. With the blunting complete, the DNA is digested with two enzymes HinfI and MspI. These two restriction enzymes recognize palindromic sequences of five and four base pairs respectively. Anywhere the enzyme recognizes this specific sequence of DNA is cleaves the DNA apart, creating smaller double-stranded fragments. These fragments are then used in capillary electrophoresis.

Capillary electrophoresis is carried out in the ABI3130 Genetic Analyzer. Essentially, the termini of these DNA fragments were labelled with the forward primer, 27f-HEX. This primer fluoresces green when hit by a laser. The capillary electrophoresis machine separates the fragments by size and funnels the fragments individually through the tubing. When each fragment passes the laser, the electrons in the fluorescent primer are excited and send a signal to the computer.  This signal creates an electropherogram that looks something like this:

Each peak represents a fragment size. The height of the peak indicates the number of fragments of that size. Any peaks that are the same for every sample indicate an overlap in the bacterial community.

Thus far, I have generated tRFLP electropherograms for the May, June and July soil and water samples. Detailed analysis has yet to be performed. However, preliminary analysis indicates that while there are bacteria groups present in all samples, there is a fair amount of divergence amongst the sample sites. It will be interesting to see who exactly is out there!

 Dana

Hello All,

 As you may have guessed from the title of this post, sometimes research does not go as planned or as expected. We have recently begun Polymerase Chain Reaction (PCR) work as a part of our analysis of the community profile of the bacterial population in Lake Matoaka and it’s watershed. Here our research hit a snag or two. PCR was not working as expected and our research was stalled. Our focus shifted from community profiling to optimizing PCR.

Briefly, PCR is a means of amplifying small quantities of DNA. Using an enzyme called Taq Polymerase, the three step process amplifies DNA over a period of several hours. The first step denatures the double-stranded DNA, making it single-stranded. Without single stranded DNA, amplification is not possible. The next step is the annealing step. The primer needed for elongation is annealed to the single-stranded DNA. The third and final step is the elongation phase. Taq Polymerase adds complimentary nucleotides making a new, semi-conserved strand of double-stranded DNA. This entire process takes a couple of hours, the results of which are assessed by gel electrophoresis. For more detailed information about PCR please see, http://www.dnalc.org/ddnalc/resources/pcr.html.

This is a fairly straightforward procedure that can come with a plethora of problems. After running several gels of different sets of PCR products, we had nothing to show for it, but pictures of empty gel lanes. It was very frustrating. Troubleshooting PCR began with literature searches and contacting the manufacturers of the various elements of the process. One solution kept popping up, Bovine Serum Albumin (BSA). BSA is used to prevent non-specific binding of the Taq Polymerase to plastics. As the tubes we use are plastic, the BSA coats the sides of the tubes and prevents the Taq from biding to the sides of the tubes. The Taq is now free to bind to the DNA in the tubes.

We have not had perfect success with PCR every single time. We joke that there is a certain amount of voodoo involved in getting PCR to work. Aside from taking extra precautions in using aseptic technique, there is little else we can do.

All in all, we have each learned a valuable lesson. Research is about solving problems and sometimes those problems arise in the methodology. Frustration and failure are innate facets of research. It is better that I understand that now  in dealing with something as simple as PCR. My chosen career will not be easy. Like I said in the title, problems continue in research and that is why they call it research!

 Dana

Hello All,

 Again, sorry for the delay in updating my blog, but research has kept me very busy. Anyway, as the designated “water girl,” the first week of research was spent collecting water samples and processing them by different methods of filtration. The overarching goal of the filtration process is to concentrate viruses from eight liters of raw water to two milliliters of viral concentrates. It’s an astounding feat that more often than not leaves me excitedly stammering, “Cool.”

The first step of this process involves collecting eight liters of water from the Inlet, Keck Pier and Spillway sites, as denoted on the previous graphic. These samples are then run through dead-end filtration. A series of filters measuring 10um, 5um, 1um and 0.22um in pore size are connected by piping. A peristaltic pump (It is called a peristaltic pump because it functions using peristalsis, a series of contractions that force the water through the piping. This same process is what pushes food through the digestive tract of the human body.) Each filter registers a decrease in pore size. This essentially screens out smaller and smaller particles that may be in the raw water. For instance, the bacteria in the water, which tend to be bigger than 0.22um in diameter, get stuck on the 0.22um filter. This filter can then be saved and bacterial DNA can be extraced from it. Viruses, however, are much smaller than 0.22um and pass freely through the filter. The water collected after this step in the filtration process has only viruses in it – along with proteins, heavy metals, etc.

The virus water gathered in the first step of the water filtration protocol is then further filtered in a Vivaflow Tangential Flow Filter. Tangential flow filtration involves the passing of water through a filter block. The application of an “out” tube that is smaller in diameter than the “in” tube causes back-pressure to build in the filter block. This forces the water through a 30 kilo-Dalton filter (30 kilo-Daltons is the diameter of most proteins). Viruses get stuck on the filter and virus-free water exits the filter block as waste. After all eight liters of water has passed through the block (taking about four hours), 50mL of virus-free water is flushed backwards through the filter. This frees the viruses from the filter and allows for 50mL of virus water to be collected.

This 50mL is then filtered through a Sterovex filter of 0.22um in diameter. Essentially, a filter is attached to the end of a syringe and the 50mL of water are forced through the filter. This filtrate is then placed in a Centricon filter. This final step of filtration occurs in the centrifuges. The Centricon filter is another 30 kilo-Dalton filter that is designed to trap viruses in the filter when centrifugal force is applied. After centrifugation, the filters can be flipped over and that same force pulls the viruses off the filters. The viruses are concentrated in two milliliters of water. This concentrate can be used for abundance enumeration, DNA extraction and much more. It’s a day long process per sample taken, but it is well worth it!

 Dana

Hi All,

 I realize this post is a little late, but I would like to give everyone an overview of the project I’ll be working on this summer. I will be continuing my research in Dr. Williamson’s Phage Ecology Lab. This summer I will continue working to determine the viral and bacterial abundance and diversity in Lake Matoaka and it’s surrounding watershed. Six sample sites have been determined (three water and three soil sites) and our samples will be drawn from these sites for processing this summer and in the coming year.  Please see the attached graphic for a visual representation of these six sites.

From these samples, the abundance of the viral and bacterial communities will be determined via flourescence microscopy. Slides will be made of the samples at various concentrations, stained with SYBR Gold and the viruses and bacteria present on the slides  will be enumerated under the microscope. The SYBR Gold intercolates with the nucleic acid is the individual viruses and bacteria and causes the specimen to flouresce and become visible for counting.

To determine the diversity of the bacterial communities in Lake Matoaka and the surrounding soil, a process called Terminal Restriction Fragment Lenght Polymorphism (tRFLP) will be used. Please see this link for a good summary of this process: http://en.wikipedia.org/wiki/Terminal_restriction_fragment_length_polymorphism. To determine the diversity of the viral communities in Lake Matoaka and the surrounding soil, a process called Pulsed Field Gel Electrophoresis (PFGE) will be used. Please see this link for a good summary of this process:  http://en.wikipedia.org/wiki/Pulsed_field_gel_electrophoresis. Some DNA sequencing will be used as well.

In addition to determining the abundance and diversity of the viral and bacterial communities, we will work to determine the level of overlap between the water and soil environments through statistical anaylsis. We will also examine such metadata as pH, salinity (water), Cholorphyll a levels, water temperature, air temperature, rainfall events, moisture content (soil), etc., looking at what possible effects, if any, these factors have on the soil and water bacterial and viral communities.

This project is a very unique one in the field of virology and I can’t wait to get started. I’ll write again soon!

 Dana

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