Document Type
Thesis - University Access Only
Award Date
2005
Degree Name
Master of Science (MS)
Department / School
Biology
Abstract
Microbiology is a vast field that, until recently, has been constrained in its ability to analyze microbial communities due to the fact that most microbes cannot be grown in standard laboratory culture. Thus any research which depends on the bacteria to be grown in culture will not be able to give an accurate picture of the total microbial community. New laboratory techniques, such as Polymerase Chain Reaction (PCR) using 16S ribosomal DNA (rDNA) and Denaturing Gradient Gel Electrophoresis (DGGE), only require that the bacterial DNA be extracted from the soil, and are not dependent on culturing the bacteria. The focus of this research was to isolate nucleic acid sequences from soil bacteria in a Prairie Pothole Wetland in order to analyze the cyanobacterial community. Soil core samples were taken from a wetland site near Madison, SD. These cores were separated into three depths to be analyzed (0-5 cm, 5-10 cm, and 10-15 cm]. Then PCR using primers specific for the 16S rDNA of cyanobacteria were employed. The amplified DNA sequences were then separated from one another using DGGE. The individual bands on DGGE were excised, DNA was eluted from the gel, and the DNA samples were reamplified using PCR. The re-amplified products were run on a DGGE gel in order to confirm purity of the sample and migration distance. An initial attempt to directly sequence the purified PCR products yielded no useable results, thus cloning vectors were later used in order to obtain samples with single cyanobacterial l 6S rDNA segment insertions. Isolated and purified PCR samples were then inserted into a cloning plasmid vector and cloned into chemically competent E. coli. The transformed E. coli cells were then stored at -80° C in glycerol. Revived stocks were used to obtain plasmids that were purified and sent off for sequencing. Sequences obtained were compared against known cyanobacterial sequences using the BLAST database. The sequences obtained from this research showed similarity to G/oeobacter violaceus, Anabaena virabi/is, Synechococcus sp., and Proch/orococcus marinus. Sequences were aligned using the ClustalW program on Biology Workbench. From these aligned sequences a PHYLIP unrooted phylogenetic tree was created. Sequences from Gloeobacter violaceus, Synechococcus sp., and Prochlorococcus marinus were included in the unrooted phylogenetic tree in order to compare the relationship between the sequences isolated by this laboratory against known cyanobacterial sequences. Sequences were also analyzed for chimeric sequences using Bellerophon chimera checker and Chimera Check programs. Results from the Bellerophon chimera check and Chimera Check program indicated that no chimeric sequences were found in the sequences obtained from the samples.
Library of Congress Subject Headings
Cyanobacteria -- Prairie Pothole Region
Soil microbiology -- Prairie Pothole Region
Wetlands -- Prairie Pothole Region
Gel electrophoresis
Format
application/pdf
Number of Pages
84
Publisher
South Dakota State University
Recommended Citation
Kruse, Jill, "Partial Analysis of the Cyanobacterial Community in a Prairie Pothole Wetland Using Denaturing Gradient Gel Electrophoresis" (2005). Electronic Theses and Dissertations. 1198.
https://openprairie.sdstate.edu/etd2/1198