Week 8-Semester 3

Hello, and welcome back.

Spring break is upon us, so unfortunately this means that no lab time will be logged this week. Yet, a blog is still due...so this week, in lieu of any experiments you will find here the abstract I submitted to the Estrella Mountain Student Conference. Cheers!

16s ribosomal gene sequencing is a standard technique for identification of bacterial species in the laboratory (Janda & Abbot 2007). Preparation of a bacterial sample for genomic sequencing is initiated by extracting DNA from the target species and conducting a polymerase-chain reaction (PCR) to amplify the DNA into a quantity sufficient for the sequencing process (Mao et. al. 2012). Nucleotide primers currently in widespread use by laboratories are targeted at specific variations of the 16s ribosomal gene found conserved among different bacterial species and are used during the PCR process to amplify the DNA sample. However, certain primers known as Universal Rice Primers (URPs) have been found to have universal application during the PCR across multiple bacterial species (Eun, et. al. 2002). This study analyzed URPs 2F, 4R, and 9F for universal application against eight bacterial species commonly found in laboratories. The bacterial species utilized were Salmonella enterica, Shigella sonnei, Proteus vulgaris, Proteus mirabilis, Klebsiella oxytoca, Providencia stuartii, Serratia marcescens, and Escherichia coli. The study also identified six previously unknown primers for analysis of universal application across the eight bacterial species. The identified primers were PCAT-1f-2015, PCAT-1r-2015, PCAT-2f-2015, PCAT-2r1-2015, PCAT-2r2-2015, PCAT-3f-2015, PCAT-3r-2015, PCAT-4f-2015, and PCAT-4r-2015. The study determined viability of the designated primer groups for use in universal application during the PCR process to enable subsequent identification of the target organisms.

BTW, here is an interesting link to a lay article about cloning the extinct mammoth.

 http://www.cbc.ca/news/technology/woolly-mammoth-cloning-attempt-revives-ethical-debate-1.2867654

And an image from the same article @ www.cbc.ca:

Week 7-Semester 3

Hello!

This week, I do not have an exciting development or even reams of data to report regarding my primers project. Due to a scheduling conflict with the BIO 181 labs, the thermocycler is unavailable for usage this week. Therefore, the next phase of my project is on hold until after the return from break.

This is particularly unfortunate, as I have been quite interested in testing my primers against the genetic sequences of my target organisms. As you may recall, I have already analyzed certain Universal Rice Primers (URPs) and found them to be not truly universal in application against the selected species under the controls I selected; the next phase is to examine whether or not the primers of my own design fare any better using those same controls. The essence of my project is the search for primers that can be used universally to amplify common bacterial species during a polymerase-chain reaction. This has gotten me a bit excited, as you may imagine, so having to wait another two weeks for results is stoking an excited anticipation that is almost untenable.

Instead of conducting experiments, I have been focused this week on expanding my project to include additional bacterial species. Welcome, Pseudomonas aeruginosa, to the fold. Also, in lieu of thermocycler time, I cultured fresh stock of the species I am analyzing and conducted DNA extractions on them. I banked them for later analysis. This, in a nutshell, was the progress I made this week.

Tomorrow brings the first field trip of the semester for our S-STEM group. We will be heading to the Phoenix Zoo for a little rest and relaxation, along with (hopefully) a little learning about the zoo's conservation efforts.

Until the semester resumes, be good to yourself.

PS: Here are some links to information about the black-footed ferret population recovery program, of which the Phoenix Zoo is a participating member. This only native North American ferret species was thought to be extinct until a lone colony was discovered in Wyoming in 1981. Since then, hundreds of kits have been bred and successfully introduced into the wild, including here in Arizona.

http://www.blackfootedferret.org/captive-breeding

http://www.azgfd.gov/w_c/blackfooted_ferret.shtml

Week 6-Semester 3

Hello! Welcome back to my blog.

As mentioned last week, I have been spending some time perusing the 16s gene sequences of my target organisms and was able to design a number of primers for use in PCR. The primers have been ordered, and that portion of my research is in a holding pattern until the order arrives.

In the meantime, I have been testing Universal Rice Primers (URPs) for universality against the eight bacterial species I am studying. URPs are called universal arbitrary primers because, rather than targeting specific gene sequences without variation, they adhere to and amplify random nucleotide sequences during the PCR process. Theoretically, this allows them to be used with a greater variety of organisms than the sequence-specific primers I am testing. However, in my first attempt to amplify DNA using these primers and a protocol previously used by a former S-STEM student, my PCR was unsuccessful. I surmised that this was likely due to the lack of necessary data from the previous student's experiment, as all I had to go on when attempting replication of the experiment was the poster that had been created with a synopsis of the experiment. Detailed, relevant information, such as the concentration of the primer dilution used, was missing.

In order to surmount this obstacle, I devised an experiment that utilized repetition and manipulation of variables to potentially identify the reason(s) for the failure of the first experiment. Using the same DNA samples that were utilized in the first test, I changed the annealing temperature of the PCR protocol to a lower temperature than previously used; I also repeated the original primer dilution but added another dilution factor as a variable to test concentration effect on PCR results. Given the variables being tested, I had a total of forty-eight samples being tested in one experiment.

The results? DNA amplification successful! Data analysis indicated that primer concentration did affect PCR results, as there were variances in banding frequency and size when the electrophoresis gels were reviewed post-PCR. However, I determined that temperature was the primary factor for the differences in results between experiments #1 and #2; given that I repeated all of the original controls, changed only the annealing temperature, and left the original primer concentration intact for the second experiment, this indicated that temperature was the culprit in the first experiment.

The addition of a primer concentration variable to the experiment did provide valuable data. Gel analysis revealed that DNA banding was less prevalent at the lower concentration for all but one of the primers, which instead showed an increase in banding clarity. This result will help me in designing future experiments using these URPs, as it revealed the optimal dilution concentration formula for primers tested at the annealing temperature selected.

Next week, I will be repeating the experiment using primers of my own design. Until then, in the words of S'chn T'gai Spock, "Live long and prosper."

And speaking of rice, here is an interesting video on GMO'd golden rice.

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.