Abstract
The goal of this project is to clone four selected genes from the marine microbe, Roseobacter denitrificans, which plays a significant role in the dynamic and biogeochemical interactions of oceaninc ecosystems. Two of these genes, rodA and cheY, which have known functions involved in structure, development, motility, and chemotaxis, will be used as controls to elucidate the functions of two hypothetical genes, "N-AcGlu" and rhaI, which are believed to be involved in sugar metabolism. R. denitrificans genomic DNA was isolated via the QIAgen DNA Purification Kit, and Restriction Fragment Length Polymorphism of 16S ribosomal DNA with the restriction endonucleases, HindIII, RsaI, and MseI, was used to verify the species. Polymerase Chain Reaction (PCR) will be used to amplify the four target genes. Then, carefully constructed plasmid vectors, antibiotic derivatives of pBBRI-MCS, will be used to clone each gene. The plasmids will be transformed into Escherichia coli JM109 competent cells, so that they can constantly regenerate and be extracted. A future aim of this project is to conduct site-directed mutagenesis of the cloned genes, which causes a decrease in gene expression and altered gene products with compromised functions. The mutated plasmids can be transformed into R. denitrificans to study the effects of the genetic mutations and ultimately the functions of the target genes. These techniques can be applied to many cultivable microbes to provide insight into the annotation of novel genes of interest, particularly those involved in secondary metabolism of pharmaceuticals, antimicrobial agents, and anti-tumor agents used in cancer treatment and prevention.
Faculty Sponsors
Dr. Julie Torruellas Garcia, Dr. Jose Lopez
Project Type
Event
Location
Alvin Sherman Library
Start Date
4-4-2014 1:00 PM
End Date
4-4-2014 5:30 PM
Cloning of Functional and Regulatory Hypothetical Genes in Marine Microbe Roseobacter denitrificans OCh 114
Alvin Sherman Library
The goal of this project is to clone four selected genes from the marine microbe, Roseobacter denitrificans, which plays a significant role in the dynamic and biogeochemical interactions of oceaninc ecosystems. Two of these genes, rodA and cheY, which have known functions involved in structure, development, motility, and chemotaxis, will be used as controls to elucidate the functions of two hypothetical genes, "N-AcGlu" and rhaI, which are believed to be involved in sugar metabolism. R. denitrificans genomic DNA was isolated via the QIAgen DNA Purification Kit, and Restriction Fragment Length Polymorphism of 16S ribosomal DNA with the restriction endonucleases, HindIII, RsaI, and MseI, was used to verify the species. Polymerase Chain Reaction (PCR) will be used to amplify the four target genes. Then, carefully constructed plasmid vectors, antibiotic derivatives of pBBRI-MCS, will be used to clone each gene. The plasmids will be transformed into Escherichia coli JM109 competent cells, so that they can constantly regenerate and be extracted. A future aim of this project is to conduct site-directed mutagenesis of the cloned genes, which causes a decrease in gene expression and altered gene products with compromised functions. The mutated plasmids can be transformed into R. denitrificans to study the effects of the genetic mutations and ultimately the functions of the target genes. These techniques can be applied to many cultivable microbes to provide insight into the annotation of novel genes of interest, particularly those involved in secondary metabolism of pharmaceuticals, antimicrobial agents, and anti-tumor agents used in cancer treatment and prevention.
