Date of Award

Spring 2015

Document Type

Thesis

Department

Biology

First Advisor

Dr. Ruth Plymale

Second Advisor

Dr. Joe Jeffers

Third Advisor

Dr. Terry DeWitt

Abstract

My research consisted of two separate parts, both involving the PCR methods. In part one I was trying to identify bacteria isolated from a chicken coop. In part two I was trying to identify bacteria isolated from the bluff. In both parts, I used a technique called colony PCR. This concept uses a single colony of bacteria to complete the PCR reaction. One pipet tip from a single colony is selected from the plate and mixed into a tube containing ultrapure water. A small amount of this bacterial mixture is then added into the PCR reaction. In the first part of my research, in the spring of 2014, I used bacteria isolated by the Fall 2013 microbiology class. The original samples were taken from a local chicken coop, and from there, each of the six groups isolated a single bacterial species. Many metabolic tests were run on each bacterium. The results of the metabolic tests were used to identify the isolated species by using a guide book. Time did not permit for the bacteria to be sent off for sequencing to see if each group had predicted the correct species of bacterium. Plates streaked with each group’s bacterium were stored in the refrigerator. The goal of my part of the project was to take each group’s bacteria, amplify the 16S rRNA gene using PCR, run the product through a gel to check for the presence of DNA, and send the samples off to be sequenced.

Because PCR had not previously been done on these bacteria, I had to pick two primer sets to be used. I found two articles that both used the 16S rRNA amplification for bacterial identification and decided to try the primers from both papers. The first article discussed a project that used DNA extraction procedures and PCR conditions for DGGE (denaturing gradient gel electrophoresis) analysis of bacterial populations in freshwater samples (Yamaguchi, 2013). Their goal was to study microbial changes in the water caused by industrialization and urbanization. They used 16S rRNA forward primer 933F (5′-GCA CAAGCGGTGGAGCATGTGG-3′) and reverse primer 1387R (5′-GCCCGGGAACGTATTCACCG-3′) (Yamaguchi, 2013). The second set of primers I used came from an experiment that was identifying bacteria found in the gut of broiler chickens (Torok, 2008). The data were then used to correlate the composition of the microbial communities in the gut to the amount of energy that is able to be metabolized. The primers used by these scientists were universal 16S primers, forward primer 27F and reverse primer 907R (Torok, 2008). The sequences of these primers are 5’-AGAGTTTGATCMTGGCTCAG-3’ and 5’-CCGTCAATTCMTTTRAGTTT-3’ (“16S Ribosomal DNA”).

In the second part of my research, in the spring of 2015, I used bacteria isolated by the Fall 2014 microbiology class from soil samples collected from the bluff near Ouachita’s campus. Each group isolated several species of bacteria and tested them for the production of antibiotics. If the group had a bacterium that produced antibiotics, further metabolic testing was done in an attempt to provisionally identify the species. PCR, using the 8F, 1492R primer set, was then done to amplify the 16S rRNA gene for confirmatory bacterial identification. If the reaction was successful, the DNA samples were purified and sent off for sequencing. Each group’s bacteria were stored in the refrigerator until needed. Each group was able to use PCR to obtain the sequence to identify the species of their isolated bacteria, except for two groups–Hall and Feather. The Hall bacterium is very waxy, which could have been the reason it did not work. The goal for my part of this project was to try several method adaptations to see if the PCR will provide enough DNA for the bacterial species can be identified. Unlike the first part of my research, in this part I used a pre-made PCR bead. The bead contained all the ingredients necessary for the reaction except for the primers and source DNA. The primers for the second part are­­­ universal 16S forward and reverse primers—forward primer 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and reverse primer 1492R short (5’-ACGGCTACCTTGTTACGACTT-3’) (“16S Ribosomal DNA”s). This was the same primer mix used in the Fall 2014 microbiology class.

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