Rebecca Downs' bursary placement
Rebecca Downs from Lancaster Girls Grammar School took a four week bursary placement in the Department of Biological Sciences at Lancaster University. Her project was called 'Site Directed Mutagenesis of Polymerase Beta'.
The aims of the project
This project concentrated on understanding how cells carry out DNA repair. This involves making human DNA repair enzymes in bacteria, purifying these enzymes and then seeing how they repair artificially damaged DNA.
How the project was carried out
Before my project commenced, my supervisor had made some recombinant plasmids. These contained Kanamycin resistance genes (antibiotic resistance) that were expressed enabling the hosts that had taken up the plasmids to be identified, since they were grown on a growth medium containing Kanamycin and Chloramphenicol (this selects for the strain of bacteria required).
A swab of the bacterial cells was taken and treated so that they were ready for protein purification. The protein (enzyme) was then purified and an SDS-PAGE gel electrophoresis was run on it, to check the purity of the protein. The protein was very pure in the wild type polymerase beta and slightly less pure in the mutated version of the enzyme.
Then activity assays were carried out on the enzyme to see that it was functioning correctly. This showed how the enzyme worked (because it was active), and was less effective at lower concentrations. It also shows that the some of the mutated versions of the polymerase beta were less active than other mutated versions of the enzyme.
The conclusions reached at the end of the project
My work showed that these mutations of the polymerase beta were still active and could be put into mammalian cells. The results of this investigation will be sent to Grigory L. Dianov at MRC Harwell who will then put these mutations into mammalian cells to investigate how polymerase beta levels are regulated in cells. The purified enzymes will be used for other experiments to help understand the results from the mammalian cells.
The most valuable thing about the bursary experience
I found working on a project funded by the North West Cancer Research that I knew could potentially benefit future cancer sufferers a very rewarding experience. By undertaking this research I have learnt how to use pieces of scientific equipment I had only heard about in school. I have gained an understanding of how science is being used in the world and how important scientific research is. This is vastly different to A-Level coursework, which changes little over the years.