I previously completed an Msci in Biochemistry at Newcastle University, working in three different labs on a range or projects while there. Firstly, I worked in the lab of Professor Jeremy Lakey on the Caf1 protein from Yersinia pestis, better known as the plague bacterium. This was where I got my first real lab experience and developed my passion for protein biochemistry and cell biology. After this I worked on the autophosphorylating PrkC kinase from Listeria monocytogenes in Professor Rick Lewis’ lab. This was where I first probed protein dynamics and and protein-protein interactions. Finally, I worked in Dr Dave Bolam’s lab on novel glycoside hydrolases from the microbiota. Putting my protein research experience in the contexts of a commensual bacterial environment. It also allowed me to experience X-ray crystallography techniques. This project with Professor Barilla and Professor Leake intrigued me because of its focus on protein dynamics in areas I not dived into before. Protein-DNA interactions have always interested me in both prokaryotic and eukaryotic fields so It seemed a great idea for a PhD. The project also allows me to become versed in high resolution microscopy, a technique I have wanted to master since a first glance of electron microscopy in my first lab. It also allows me to bring forth the experience I gained in protein biochemistry from all the labs I’ve been lucky enough to part of.
My work surrounds the plasmid segregation system for the low copy plasmid TP228. This plasmid confers resistance to kanamycin, streptomycin and mercuric ions. Conferring multiple selection advantages to E. coli. The plasmid segregation system is composed of a series of imperfect tetrameric repeats called parH, the centromere binding protein ParG and an ATPase ParF. Together these elements help guide the plasmid to the poles of the cell where they are effectively retained by daughter cells during replication. Although the proteins have been well characterised there is much to discover about how the system effectively segregates plasmids to opposite cell poles. In this project I hope to ellucidate the method by which it is done by viewing the system with super high resolution microscopy techniques. For example using 3D SIM. This will give valuable in vivo data to help us refine the existing Venus fly-trap model of plasmid partition. I also wish to combine these techniques with characterised mutants to help probe their effects in vivo. Finally I also hope to investigate ParF’s interactions with DNA using biochemical techniques and atomic force microscopy.