Presentation Type
Poster
Location
Portland State University, Portland, Oregon
Start Date
5-12-2015 11:00 AM
End Date
5-12-2015 1:00 PM
Subjects
DNA topoisomerases, Molecular biology, Archaea
Abstract
Reverse gyrase is the only known topoisomerase enzyme with positive supercoiling activity on covalently-closed DNA. This positive supercoiling is required to prevent DNA from denaturation at high temperatures. The gene that codes for this protein is present in all hyperthermophiles and absent from all mesophilic and thermophilic genomes, suggesting that this enzyme is the only hyperthermophile-specific protein. To investigate if this protein is vital for the cells, we knocked out its gene from the genome of living organism Pyrococcus furiosus. Pyrococcus furiosus is a hyperthermophilic archaeon that grows between 70°C to 103°C with an optimum growth temperature of 100°C. The knocking out process was done as follows. Recombinant DNA was made using Gibson Assembly technique. This DNA, which included a selection marker for ability to synthesize uracil, was used to transform Pyrococcus furiosus strain ‘COM1’. Uracil prototrophs were selected on growth medium lacking uracil, and the deletion of the target gene was confirmed using PCR. Mutants then were grown at different temperatures ranging from 75°C to 105°C and growth curves were compared to that of COM1. Although mutants do not survive at temperatures over 93°C, no significant difference was observed between the growth rate and density of COM1 and mutants in temperatures below that. We demonstrated that although reverse gyrase is not essential for cell survival, it shifts the maximum living temperature from 103°C to 93°C.
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Persistent Identifier
http://archives.pdx.edu/ds/psu/19823
Reverse Gyrase is Not Necessary for Survival of Hyperthermophilic Archaeon Pyrococcus Furiosus
Portland State University, Portland, Oregon
Reverse gyrase is the only known topoisomerase enzyme with positive supercoiling activity on covalently-closed DNA. This positive supercoiling is required to prevent DNA from denaturation at high temperatures. The gene that codes for this protein is present in all hyperthermophiles and absent from all mesophilic and thermophilic genomes, suggesting that this enzyme is the only hyperthermophile-specific protein. To investigate if this protein is vital for the cells, we knocked out its gene from the genome of living organism Pyrococcus furiosus. Pyrococcus furiosus is a hyperthermophilic archaeon that grows between 70°C to 103°C with an optimum growth temperature of 100°C. The knocking out process was done as follows. Recombinant DNA was made using Gibson Assembly technique. This DNA, which included a selection marker for ability to synthesize uracil, was used to transform Pyrococcus furiosus strain ‘COM1’. Uracil prototrophs were selected on growth medium lacking uracil, and the deletion of the target gene was confirmed using PCR. Mutants then were grown at different temperatures ranging from 75°C to 105°C and growth curves were compared to that of COM1. Although mutants do not survive at temperatures over 93°C, no significant difference was observed between the growth rate and density of COM1 and mutants in temperatures below that. We demonstrated that although reverse gyrase is not essential for cell survival, it shifts the maximum living temperature from 103°C to 93°C.