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.