CDT Lab Projects
bioc3160
The model organism in the lab is Staphylococcus aureus, which has a reputation as a human pathogen resistant to the majority of antibiotics available. Consequently we're investigating several enzymes that are involved either in the replication of plasmids that confer resistance to these antibiotics, the spread (through conjugative transfer) of genes that confer this resistance, as well as essential enzymes required by the Staphylococci for maintenance of their DNA. Ultimately these essential processes may become the targets for novel antibiotics directed against the organism.
Several of the enzymes we study have "DNA relaxase" (or, more correctly, transesterase) activity. They are capable of breaking a phosphodiester bond in the backbone of the DNA, and form a covalent protein-DNA phosphotyrosine intermediate. Much of our work is based on sequence alignment, site directed mutagenesis and enzyme assay, combining bioinformatics and protein engineering to deduce the roles of domains or even individual amino acids in the function of these enzymes.
In all cases we are aiming to determine the three-dimensional structure of these enzymes, which can be combined with the biochemical evidence to understand how they work at the molecular level.
Which project? You are interested in the subject matter itself, or would like to have the opportunity to gain first-hand experience in one of the techniques we use in the lab. Either way, please visit me to discuss project titles.
useful references
There are several good reviews on plasmid biology, DNA replication, and DNA topoisomerases - just a few of these are listed below. Use these as a starting point to familiarise yourself with the topic. If you are accessing this page from campus, you should find the full text for most of these is available online by following the links:
- "The Biology of Plasmids" David K. Summers (Blackwell Science, 1996) Library
- "The Horizontal Gene Pool" ed. Christopher M. Thomas (Harwood Academic, 2000) Library
- Replication of Plasmids in Gram-Negative Bacteria Kues, U. & Stahl, U. (1989) Microbiol. Rev. 53(4):491-516 PubMed
- Staphylococcal Plasmids and their Replication Novick, R.P. (1989) Annu. Rev. Microbiol. 43:537-565 PubMed
- Replication and Control of Circular Bacterial Plasmids del Solar, G., Giraldo, R., Ruiz-Echevarria, M.J., Espinosa, M. & Diaz-Orejas, R. (1998) Microbiol. Mol. Biol. Rev. 62(2):434-464 PubMed
- Plasmid copy number control: an ever growing story del Solar, G. & Espinosa, M. (2000) Mol. Microbiol. 37(3):492-500 PubMed
- Nicking by transesterification: the reaction catalysed by a relaxase Byrd, D.R. & Matson, S.W. (1997) Mol. Microbiol. 25(6):1011-1022 PubMed
- Enzymology of DNA transfer by conjugative mechanisms Pansegrau, W & Lanka, E. (1996) Prog. Nucleic Acid Res. Mol. Biol. 54:197-251 PubMed
- DNA processing reactions in bacterial conjugation Lanka, E. & Wilkins, B.M. (1995) Annu. Rev. Biochem. 64:141-169 PubMed
postgraduate
Many different projects suitable for Wellcome Trust students on rotation are available. Two projects ready and waiting right now include:
- NMR study of a novel DNA binding binding protein
- SELEX-based investigation of structure-based DNA recognition
We also carry out a lot of gene cloning, site directed mutagenesis and protein engineering in the lab, and use a wide variety of techniques to investigate protein:DNA interactions. If you would like to join in to acquire these skills as part of your rotation, please get in touch!