Location

Portland State University, Portland, Oregon

Start Date

12-5-2015 11:00 AM

End Date

12-5-2015 1:00 PM

Subjects

DNA topoisomerases, Molecular biology, Archaea

Description

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.

Persistent Identifier

http://archives.pdx.edu/ds/psu/19823

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May 12th, 11:00 AM May 12th, 1:00 PM

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.