Post 5: Final Thoughts about the Summer

Hello again!

Well, the summer is officially over. I’ve put in 10 long weeks of work and I’ve accomplished quite a bit. While this series of blogs has been focused on my honors thesis project, I’ve also been working on the tRFLP project described in last summer’s blog series. With that project, I have gotten fragment data for every month from Mat 2009 to August 2010! The next step is the analysis of the fragments and creation of dendrograms to compare similarity in the bacterial community in each month over the course of the year. I’ve got my work cut out for me there too!

Moving into the fall semester, I’m really excited to get back into the lab! I have 92 sequences to get and analyze, in silica digestion of the sequences to perform, similarity matrices to create and a thesis to write! It’s going to be a busy year, but I am confident I can get it all done!

Despite the setbacks (and because of the setbacks) in my research this summer, I have learned so much! I am more in love with research than ever before and I know for sure this is what I want to do with my life! I am so grateful to Mr. Chappell, my thesis funding donors, the Charles Center and Dr. Williamson for giving me this opportunity! I truly wouldn’t have wanted to be anywhere other than our freezing cold, windowless, absolutely wonderful lab for my summer!

And to you, readers, thanks for coming with me on this journey, following bacterial DNA on it’s roller coaster ride to being sequenced! I hope you enjoyed this as much as I did!

For the last time,

Dana

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Post 4: Mini Prep Mayhem

Hi All,

To recap – I began the summer by extracting bacterial DNA from 0.22 micron filters that held bacteria from Lake Matoaka. I then amplified the 16s rRNA gene by PCR. These amplicons were ligated into plasmid vectors, transformed into E. coli cells and hundreds of copies of each amplicon were made. What’s next? Since the end goal is to sequence the bacterial 16s gene, I need to get the plasmid back out of the E. coli cell. Enter: Mini-Prep kits. These are brilliant little kits that have made the process of removing plasmid DNA much easier.

To begin, I picked individual colonies into LB broth with kanamycin and grew them up overnight. Once that was completed, I pelleted the bacterial cells out of the broth by centrifugation. (I also archived 500 ul of the culture in 60% glycerol and froze the sample at -20C). Using the reagents in the kit and a benchtop microcentrifuge, I proceded to lyse open the cells and purify out everything but the plasmid DNA. The plasmid DNA was eluted and the plasmid concentration determined by NanoDrop. To date: I have performed 124 mini-preps on the June 2009 Keck Pier samples and 50 mini-preps on the December 2009 Keck Pier samples. Whew!! Click on the link to learn even more about Mini-Preps: http://www.qiagen.com/Products/Plasmid/QIAprepMiniprepSystem/QIAprepSpinMiniprepKit.aspx#Tabs=t1

However, this is research. Something had to go wrong! The concentration of plasmid DNA was startlingly low. I was expecting about 200 ng/ul of DNA. I got about 50 ng/ul. I knew there were no contamination issues because my spectrophotometer curves were correct. What could it be? Back to the drawing board. I tried everything I could think of. I increased the incubation time of the broth cultures. I changed the amount of broth I used for the broth cultures. I changed the incubation time of the elution buffer step. I tried to concentrate the DNA in smaller volumes of buffer. I tried different volumes of culture to make the initial bacterial pellet. My friend Zac even did Mini-Preps on two of the samples with his kit. Nothing seemed to work.

However, every cloud has a silver lining. Despite the low plasmid concentration, I still had enough for sequencing reactions. I tentatively handed four of my samples off the Lidia with M13 forward primer. The next day she emailed with great news! The sequences were perfect!! I was able to identify my first four bacterial phyla! It was so cool!!

Now to finish sequencing 44 more June samples and 48 more…

Dana

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Post 3: Transformation Nightmare!!

Hi All,

You’re probably dying to know the results of the transformation reactions that I set up last post! Sorry it’s taken me so long to get back to you all!

As you can probably tell from the title, they didn’t go so well. When I very excitedly removed my samples from the incubator the following morning, I was immediately sobered. The plates were clear. There were no little white colonies. None – as in nada, zip, zero! I was flabbergasted! These TOPO-TA kits were supposed to be fool proof! And so began the tedious task of figuring out what was going wrong and why.

I set up a series of tests to try and answer this disturbing question. They are as follows:

1. A control plasmid, pUC19, was transformed into the DHa E. colicells provided in the kit on an LB plate with ampicillin.

2. Amplicons from my PCR reactions were ligated into TOPO 2.1 plasmid vectors from a TOPO kit provided by Dr. Forsyth and plated on LB+ kanamycin plates and allowed to grow overnight. This was done to determine whether or not my TOPO-TA kit was a “bad” one.

3. Amplicons from from my PCR reactions were ligated in TOPO Blunt plasmid vectors from a TOPO kit provided by Dr. Forsyth and plated on LB+ kanamycin plates and allowed to grow overnight. This was done to test the ligation of my amplicons into the plasmid vector. If the Taq Polymerase I was using for PCR was high fidelity there would be no adenine overhangs needed for ligation in my TOPO-TA kit.

4. E. coli cells from my kit were plated on LB plates with no antibiotics. This was done to test the viability of the cells in my kit.

The results were in the next morning. The cells with no plasmids grew up beautifully. Thus, my cells were viable. The  plate with the blunt TOPO vectors showed no colonies. My PCR amplicons were not being blunted by the Taq Polymerase I was using. The control plate also showed no colonies. This was disturbing. This could mean my TOPO-TA kit came from a bad lot. This hypothesis was confirmed by the presence of more than 100 little white colonies on the TOPO-2.1 plate! MY transformations were working! My kit was bad.

Needless to say, we ordered a new TOPO-TA kit. My transformations have been working beautifully since! Stay tuned to follow the bacterial DNA on to the next step: Mini-Prep Mayhem!

Dana

P.S. Much thanks to Dr. Forsyth for helping me uncover what was going wrong with my transformations!

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Post 2: Ligation and Transformation

Hello All!!

After several weeks of DNA extractions, I finally moved on to the next steps in my thesis research!!

The end goal of my thesis project is to create a detailed profile of the bacterial communities in Lake Matoaka based on bacterial gene sequences. I began with a giant mix of environmental DNA from almost a year’s worth of sample collections. The next step is to get that DNA into a specific, sequenceable form and get a lot of it.  Using PCR amplification of the 16s rRNA gene (a gene that is highly conserved in all bacterial species), I can get a mix of specific and sequenceable amplicons. However,  I only have the ability and means to sequence a few genotypes out of the vast array of 16s amplicons. How can I narrow down the field, if you will? The solution: TOPO-TA Cloning Kits for Sequencing (aka my new bffs). The TOPO-TA Kit allows me to perform ligation and transformation reactions to narrow the vast amount of DNA to just a few purified sequences. The kit works in two distinct steps.

The first step is the ligation step. A mixture of the 16s amplicons (in my case from the samples collected in June and December of 2009) is added to a vial containing plasmid vectors. These vectors are prepared in such a way as to allow for insertion of the 16s amplicons at A to T overhangs into the plasmid. Imagine you are building a circular toy train track. You’ve almost completed your circle and are missing one piece. Each piece fits together by overhanging pieces.   Click the missing train piece into place and you’ve created a perfect, circular train track. In this case, the plasmid is the almost completed train track and the 16s amplicon (insert) is the missing piece.

Once ligation is completed, transformation is next. Chemically competent E. coli cells – cells that have been treated so as to make their membranes more permeable – are now mixed with the insert-containing plasmids. A combination of incubations on ice and heat shocks is used to force the competent bacteria to take up the plasmid vectors. The transformed E. coli are then spread onto selective media plates and incubated overnight.

Did the transformation work? How do I get those inserts back out of the E. coli? How do I sequence the inserts? These questions and more will be answered in my upcoming posts! Stay tuned!

Dana

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Post 1: DNA Extraction

Hi All,

I realize that June is almost over and I haven’t posted any updates about my research. June has been a very busy month for me.  But, the bulk of my work has centered around the extraction of bacterial DNA from the 0.22 micron filters that are used to collect the bacteria from Lake Matoaka on a monthly basis. So far this month, I’ve extracted nine months (and counting) worth of DNA from filters that have been stored at -80 C for almost a year now. 

The extraction protocol is a time consuming one that usually takes me two days to complete per sample. It combines both physical and chemical techniques. The initial steps of the extraction process deal with breaking open the bacterial cells and releasing the cellular contents (lipids, proteins and nucleic acids). Three specific enzymes are utilized to this end. Lysozyme is an enzyme found in egg whites (and human tears) that degrades peptidoglycans, which are important molecules that compose the bacterial cell membrane. Sodium dodecyl sulfate (SDS) is a detergent that degrades lipids and Proteinase K is an enzyme that digests proteins. These three chemicals/enzymes in combination with vigorous shaking and a freeze-thaw process that stresses the cellular membrane effectively lyse open the bacterial cells captured on the sample filters.

Once the contents of the bacterial cells have been released into the extraction buffer, a series of chemicals in combination with centrifugation is used to purify the nucleic acids from the rest of the cellular contents. Phenol removes proteins and chloroform removes lipids and excess phenol from the mix of bacterial cellular contents. Finally, isopropanol is used to precipitate the nucleic acids out of the extraction buffer. Once re-suspended in stabilizing TE Buffer, the bacterial nucleic acids are ready for further use.

My next few posts will follow the progress of the extracted nucleic acids through PCR, enzyme restriction, the creation of clone libraries and sequencing. Stay tuned!

Dana

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Summer 2010 Introduction

Hello Everyone!!

My name is Dana Hardbower. I’m a junior and a biology major here at William and Mary. This is my fifth semester of undergraduate research.

Welcome to the summer of 2010. I began this blog last summer to keep anyone interested informed about my research in Dr. Williamson’s Environmental Virology lab. Lasy summer, much of my discussion centered around the concentration and maintainence of viral concentrates from Lake Matoaka and the analysis of the bacterial community by terminal restriction fragment lenght polymorphism (tFRLP). This summer’s project is both a continuence of last summer’s project and a new project that adds another facet to the scope of the overall study of the temporal dynamics of viral and bacterial assemblages in Lake Matoaka right here on William and Mary’s campus.

The specific subject of my summer research project is the temporal dynamics of the bacterial assemblages in Lake Matoaka. I will be using two techniques – Terminal Restriction Fragment Length Polymorphism (tRFLP) and clone libraries to create and accurate and detailed profile of the bacterial assemblages in Lake Matoaka. tRFLP is a powerful technique that will allow me to catalogue the changes in the bacterial assemblages over time in Lake Matoaka. Much of the data for this protocol was collected over the past year and I am now moving into the analysis phase of this portion of the project. I will use GeneMapper software for the statistical analysis of the data. The creation of a clone library of the major bacterial species in the lake will be accomplished by TOPO-TA kits and will provide me with sequence specific data about the major species in the lake. The clone libraries will allow me to add detail to the general profile I am creating with tRFLP. I am perhaps the most excited about the latter part of this project because it will constitute my honors thesis research. Moreover, this work remains unique and novel. I sincerely hope to greatly contribute to the field of freshwater microbial ecology with this work.

I hope you are as excited about following my research this summer as I am about sharing it with all of you! Look for more new posts starting June 1.

Dana

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Post 5: Final Thoughts about the Summer

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

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Post 4: tRFLP

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

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Post 3: PCR (Problems Continue in Research…)

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

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Post 2: Water Sampling

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

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