Cloning the flp-l and flp-2 genes of Actinobacillus pleuropnemoniae into plasmid vector, pKBll; Significance to Biofilm Formation
Szombati, Dana M.
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Actinobacillus pleuropneumoniae (A. pleuropneumoniae), a severe swine pathogen, has significant impact directly on the health and lives of the entire swine population and indirectly on human beings who utilize these swine for a variety of purposes. Despite the serious threat this bacterium poses, little research has been conducted on A. pleuropneumoniae and its methods of virulence. The goal of this research was to clone the flp-tad operon in A. pleuropneumoniae starting first with the flp-I and flp-2 genes of the operon and the promoter that expresses the operon into plasmid vectors. Once the genes were cloned into these plasmid vectors, further studies would analyze both the expression and regulation of these genes. Utilizing luciferase assays, the regulation of the promoter under different environmental conditions such as those that promote biofilm formation would also be explored. Due to the limited outside research conducted on A. pleuropneumoniae, this research project was shaped by previous research conducted on a genetically similar bacterium, Actinobacillus actinomycetemcomitan (A. actinomycetemcomitans). The cloning of the flp-I and flp-2 genes was executed utilizing a variety of techniques including PCR, polymerase chain reaction, digestion with restriction enzymes, gel extraction, electrical and chemical transformation, ligation, and colony growth. By obtaining successful clones of the flp-I and flp-2 genes into the plasmid vector, pKB II, it was established that A. pleuropneumonia could be copied and manipulated in a laboratory setting. This result was necessary in order to guide further research which will analyze the role of the flp genes of A. pleuropneumoniae in the formation of biofilms and propagation in the host organisms.