Week 5-Semester 3

Hello! Welcome back.

Since my last post, I have been spending a great deal of time doing research on the genetic sequences of my target species and attempting to identify primer sequences that will be successful at DNA amplification across my chosen organism set. It is a bit of a painstaking process, as I am manually looking for nucleotide sequence similarities among the 16s ribosomal genes of Salmonella enterica, Klebsiella oxytoca, and Serratia marcescens instead of using a sequencing program. This means I take a piece of code that looks like this: (actual S. enterica 16s sequence)

cagagatggatttgtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcgattaggtcgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggcgcatacagaagtcggaatcgctagtaatcgtggatcagaatgccacggtgaatacgttcccgggc

and compare it to something like this: (actual K. oxytoca)

atgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtatgaagaaggccttcgggttgtaaagtactttcagcggggaggaagggagtgaggttaataaccttattcattgacgttacccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggctggagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggat

and this: (actual S. marcescens)

gaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcgaggaggaaggtggtgagcttaatacgttcatcaattgacgttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgggctcaacctgggaactgcatttgaaactggcaagctagagtctcgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcgggcccctggacgaagactgacgctcaggtgccaaagcgtggggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaaatcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctactcttgacatccagagaactttccagagatggattggtgccttcgggaactctgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggttcggccgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaa

and attempt to find a sequence between 20-25 base pairs long that matches within each organism. It is not difficult, but it is certainly time consuming. Luckily, I love a good puzzle, this being no exception; therefore, I am glad to report that I am thoroughly enjoying the research portion of this project. I was also able to identify multiple sequence similarities, so I have placed an order for the appropriate primers and will be testing them for universality across all eight of my species as soon as the post arrives.

Whether or not my chosen target sequences will successfully amplify during the PCR process is an unknown at this point. Amplification should be successful with the three noted species, as the 16s gene of each strain carried in this lab has been mapped, and I used those maps (pictured above) to develop the primers. The remaining five species, however, are of particular strains that have not yet been mapped, and I cannot predict what the results will be for those strains. I will post the results when the experiment has concluded.

Until then, please enjoy the following Bio-Rad commercial. Cheers!

Week 4-Semester 3

Hello! Welcome back.

Since the last blog post and experiment, I have been focused on doing background research on how to design my own primers for use in amplifying DNA during the polymerase-chain reaction (PCR) process. Essentially, I am having to improve my knowledge of the structure of DNA and genetic sequences, so that I can determine which code sequences share similarities among the organisms I have selected for this study. Once I have identified a sequence that I find promising, I will have the primers made and proceed with that portion of the experiment.

In the meantime, I took the DNA samples which I have previously tested with my original study primers, and ran them through a PCR using Universal Rice primers (URPs). These kinds of primers are non-specific to any one gene sequence, and showed promise with bacterial DNA amplification in a study conducted by a prior S-STEM student.

The PCR I ran using the URPs was negative for strong DNA banding. Some DNA was present, but amplification appeared to be weak.This may be to one or a number of factors, as the variables in my experiment differed from the prior experiment in several ways. The DNA extraction itself was performed using a different protocol; the prior study used a commercially available kit, while mine used a simple SDS/alcohol method. PCR primer and DNA sample volumes also differed between the two tests. Furthermore, a major unknown when I attempted to compare the two studies was the primer concentration used by the other tester; that data has been lost to time, so I could not repeat those elements. Finally, I used an annealing temperature of 62.4°C during my PCR, as opposed to the 54°C used previously.

Next week, I will continue the background research portion of this project while attempting to replicate the previous student's experiment as closely as possible. Until then, happy science, and please enjoy this image from NASA of the Earth (taken from space.)

 Photo credit: NASA, Cmdr. Chris Hadfield.

Week 3- Semester 3: PCR Success

Hello! Welcome back!

This week, I decided that in order to really move forward with my project, I had to be certain that the techniques I have been using are viable. Of course, one of the best ways to verify results is to simply repeat them using different variables. So I decided to test my extraction and electrophoresis gel techniques by applying them to additional gram-negative bacterial species. You may recall that to-date I have been working solely with E. coli; this week, I expanded my research to include Salmonella enterica, Shigella sonnei, Proteus vulgaris, Proteus mirabilis, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens. 

I conducted extractions on all eight species, including E. coli, using the isopropanol method I have posted on here previously. I then ran two electrophoresis gels on the eight extraction samples, using the gel production method that fellow scholar Matt Hill and I developed last semester. Both techniques had demonstrated positive results in previous experiments. The results this time were gratifying; all eight samples showed positive results for DNA banding. (picture below)

This was extremely encouraging news, so I decided to push my luck and also test my polymerase-chain reaction (PCR) protocol and the primers from the first phase of my study. Despite my best efforts, I had been unable in phase 1 to achieve DNA amplification during a PCR.

The result? Good news! Three of my eight species were positive for banding post-PCR. This is a desired result, because it proved that my basic techniques were viable through that point. And because the results were not positive for all tested species,  they also indicated that the primers selected were not universal. In other words, I can move forward with the next phase of my research: identifying a sequence of genes, in each bacterial species, that is similar; and developing a set of primers that will successfully target that gene sequence.

As we say around the lab, "There are no wrong answers in science, only new directions." Now, with the results of this experiment, I know what direction I need to go in next.

Until next week, enjoy!

 

Gel, pre-PCR, eight species sample. Source: Paul C.

Week 2-Semester 3: An Overview of My Research Project

Hello! Welcome back to my blog.

This week, I was ready to begin my project in earnest and move quickly into the next phase, which will deal primarily with polymerase-chain reaction (PCR) primer design for application to multiple bacterial species. What the heck is that, you say?

Well, for you new readers, the gist of my project is as follows: When a lab has a sample of unknown DNA, it can begin the identification of that DNA by running it through a thermo-cycler process known as a PCR. This process subjects the sample to repeated healing and cooling, which breaks down the DNA contained in the sample. A substance called a primer is added to the DNA sample before the PCR is run, and during the PCR cycle that primer binds to the DNA fragments and assists in replicating the DNA until there is a larger quantity of it. The resulting larger sample is then sequenced to determine what organism it originated from. Why is this important? Imagine, if you will, that a violent crime has been committed and a miniscule DNA sample, left behind by an unknown suspect, has been found at the crime scene. This sample is too small to be sequenced (checked for its unique series of genes, contained in every organism's DNA), so it is useless for identifying who the suspect is. But by adding the tiny DNA sample to the proper primer and subjecting it to the PCR process, we can create more of the DNA, sequence it, and potentially identify the culprit.


There are many different primers used in PCRs; in fact, each primer is specific to a particular organism because primers target and adhere to a certain section of genes, unique to that organism only, during the PCR. The means that the need to use the correct targeting primer during a PCR, without knowing the origin of the DNA sample you wish to add it to, can be a little like a roll of the dice in certain circumstances. If a primer could be developed that was universal, or able to be applied to multiple unknown organisms, it would greatly shorten the amount of time needed to identify an organism.

That's where my project comes in. I am looking for primers that can be universally applied to bacterial species. Because E. coli has had its entire genomic sequence mapped, and a particular section of genes that was found in E. coli has also been identified in other bacterial species, I hypothesized that there must be a primer that would work during a PCR for all bacteria. In order to prove my hypothesis, however, I first had to get a crash-course in molecular biology techniques.

As a result, previous semesters have been focused on learning, developing, and implementing those techniques. I had to not only learn the vocabulary of a new field, I had to learn DNA extraction, verification, and amplification procedures. And since a major goal of my project is to also identify protocols that could be adapted into an easy method that the average student could potentially do in a Microbiology class, I had to also simplify those procedures as much as possible while still retrieving a viable, undamaged DNA sample.

That's what I have been doing, in a nutshell. The entire project has kept me fairly busy, but like I said this week I was ready to move forward. I did, in fact, manage to significantly expand my project beyond E. coli to include seven additional bacterial species, and I have begun the process of developing a primer that will target the specific genetic sequence contained in the DNA of those bacteria. Exact details of those developments to follow in a later blog.

Until then, have a most excellent week. Enjoy this pic of some human/chimpanzee gene sequences. Notice the similarities?

www.panspermia.org
"Multiple sequence alignment of the gene sequence of the human gene CLLU1 and similar nucleotide sequences from the syntenic location in chimp and macaque.

Week 1-Semester 3

Hello! Welcome back to my blog!

Today was my first day working back in the lab, and I have to say that I hadn't realized how sorely the lab crew and environment was missed until I was amongst it again. I felt like I had come home the moment I opened the micro fridge and pulled out bacteria to start my first cultures of the year.I am looking forward to spending time in the lab again, with my colleagues and friends.

I am expecting this semester to be the most challenging for me yet, both inside and outside the lab. Professionally, I have continued to pick up extra work in education, thanks to the stewardship of PC's resident Developmental Education guru, Robin Ozz. She continuously pushes me to set the bar higher for myself, and under her tutelage I was recently selected for a DevEd position working with high school students. I will be prepping a group of seniors (four times a week) for the college placement exams over the next twelve weeks. Add that to the English 091 and 101 courses I am already tutoring, my ongoing job with the Phoenix Zoo, my class schedule, and the S-STEM scholarship, and I will be a very busy man indeed through mid-May. I am not complaining, however; I am very lucky to be able to do what I do. I remain always cognizant of the fact that I am able to reach for my dreams.Not everyone has that opportunity.

I am also very lucky to have the S-STEM faculty and students supporting me. Thank you in advance for helping me succeed this year! It is much appreciated.

Academically, my S-STEM project is the bright spot this semester, though I am also extremely excited to be enrolled in Biology 181+lab with Dr. Anna Marti-Subirana. I also have Calculus 1, but please excuse me if I don't jump for joy over that one. Luckily, I realized my mathematical shortcomings when I booked my schedule, so I rounded out my credit load with a 100 level class to yin the yang of Calculus.

Those of you who read my blog regularly know that I rarely get personal on here. It is, however, the first week of lab work, with little in the way of science to report. But don't get used to me being a frail human! The progress reports begin in earnest next week. :)

Until then, please enjoy the following. It is a short video on the polymerase-chain reaction (PCR), an essential component of my research. Note that the video mentions primers, a substance which allows DNA replication during the PCR; finding PCR primers that work with DNA samples from multiple bacterial species is the core of my research project.

Week 14, Semester 2-Wrap Up.

Hello! This final week of the semester has been consumed by data collection and its requisite formatting into both the required research paper and oral presentation, but I did manage to find time in the lab to complete one last experiment. As mentioned in previous Semester 2 blogs, the primary focus this term has been on perfecting extraction protocols and electrophoresis gel production techniques. This focus was rewarded by repeated successful DNA extractions from the initial bacteria, E. coli, and the project was also extended to included two additional gram-negative species, P. aeruginosa and P. vulgaris, as well as one gram-positive organism, S. aureus.

As a final test before the next phase of my project, I took my successfully extracted samples and ran a PCR on them using my existing primers. I then tested the resultant samples for DNA using the gel technique I developed this semester. The result? Negative for DNA. However, this is not discouraging news. In fact, the results simply confirmed my suspicion that the primers I selected based on my original review of results published in Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16s rRNA Genes (Frank, et. al., 2008) would not work for the species in this study. This leads me directly to the next step of this project, designing my own primers, which will resume in January, 2015. Until then, cheers!

 A scanning electron micrograph image of Pseudomonas aeruginosa cultured onboard shuttle mission STS-115, courtesy www.nasa.gov .

Week 13, Semester 2-Success!

This week my DNA research project yielded new results, which upon discovery nearly had me leaping into the air from excitement. However, I refrained from doing so lest I give lie to the calm and studious façade I have cultivated this semester, and instead allowed myself only a silent "Hallelujah" and a brief victory trot through the lab to share my results with any available listeners.

A slight change in electrophoresis gel creation methodology yielded the clearest DNA banding I have yet received during this project. I have tested different gel recipes to-date, with varying results, and recently I began testing the optimal conditions for adding the DNA stain SybrGreen directly to my uncongealed gel solution before pouring and setting the gels. Previously, I simply added the DNA stain into each well as I added the DNA sample for testing; however, these gels showed limited or zero presence of DNA banding. To test the hypothesis that a direct addition would result in improved gel performance, I conducted a comparison experiment of the two gel types. I prepared four DNA samples using an identical protocol. I then created two gels from the same buffer/agarose base. I poured one gel, added DNA stain directly to the remaining mixture, then poured the second and allowed both to set. Finally, I loaded the samples. After thirty-five minutes running @200 volts, gel one showed zero DNA present, yet gel two showed clear banding. While this result was expected, it was exciting nonetheless.

Gel protocol:

The gels utilized for this test were created using a 0.9 g agarose/100 mL buffer mixture heated for 1:45 minutes on high in the microwave. Immediately after heating, gel #1 was poured. The remaining 75 mL mixture was allowed to cool to exactly 60°C, and then 3µl of 10,000x concentration SybrGreen was added and diffused into solution using a five-second swirling action. Gel #2 was poured, then both gels were allowed to set for 20 minutes in darkness (to prevent degradation of the SybrGreen due to UV light). The buffer mixture contained 980 mL/20mL de-ionized water/50X TAE buffer stock.

I was also able to expand the project to include additional bacterial species, which was another turn towards progress as my original hypothesis theorized the existence of universal primers that can be applied to multiple bacterial species. To-date, all experiments have utilized only E. coli. With this experiment, Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus were also subjected to extraction and gel testing. All four species yielded positive results after electrophoresis, with the gram-positive S. aureus providing the most limited results. These were the results I both projected would occur and hoped for. After some additional testing to replicate this week's results with the added bacterial species, this project should now be able to move forward to the next phase, which is intended to focus on primer design.

Altogether, it was a successful week. Please enjoy this photo of my gel results until the next blog. Cheers!

Left to right: E. coli, P. aeruginosa, P. vulgaris, S. aureus