Lab rotation project description
For the mini project we will design a 'bite-sized' program addressing a simple question that can be addressed in the limited time available. The laboratory continually generates candidate recombinases with improved activities. Several of these will be made available to the student to investigate. Various approaches may be taken, ranging from biochemical analysis of the purified proteins, to testing their gene delivery and recombinational potential in cultured human cells. Ideally, biochemical analysis, using our established assays, would be correlated with the efficiency of transgenesis in cultured mammalian cells. This would provide the student with a broad-based training in the routine techniques of molecular and cell biology, as well as an exposure to advanced recombineering techniques.
A specific example would be to use the crystal structure of the protein to identify residues that could be mutated to increase or decrease the strength of DNA binding. Intuitively, tighter interactions might be expected to increase activity. However, our recent discovery that double occupancy of the transposon ends actually decreases activity, suggests otherwise. Site-directed-mutagenesis will be used to make candidate mutations, which will be assayed for gene-delivery activity in human cell culture models.
LR2 and LR3
Nature has furnished us with a large number of enzymes that provide exquisitely precise tools for the manipulation of DNA. These tools have now penetrated all branches of biology and spawned the biotechnology industry. Generation of new tools is a BBSRC strategic priority because they feed forward into the bio-based knowledge economy, including industrial biotechnology, bioenergy, bioprocessing, novel materials and biosensors.
DNA is an interesting material because it is flexible across large distances, but very stiff over short distances. To deal with this situation, nucleic acid enzymes often assemble in to higher-order complexes with several components and numerous dynamic interactions. Because of the stiffness of DNA the complexes experience the types of physical strains we are familiar with in the macro-world. They therefore represent nano-machines, which we can harness for our own genome engineering purposes. The host laboratory is focused on the use of transposons and site-specific recombinases. We recently made a break-through in understanding the autoregulation of a eukaryotic transposon. We discovered that regulation was an emergent property of mechanism responsible for assembling the higher-order complexes. This insight suggested several general approaches to engineer more efficient transposon tools, which we are pursuing in several eukaryotic and prokaryotic transposons. The current project is focused on the multimeric state of the transposase proteins and how this dictates the efficiency of the reactions that they catalyse. For example, if a transposase self associates to form the active multimeric form in the absence of DNA, it automatically generates an autoregulatory feedback circuit. Thus, an increase in transposase concentration beyond a certain point decreases the rate of transposition. However, if the transposase multimerizes after transposon end binding, an increase in transposase concentration will always provide an increase in the rate of the reaction. The project is investigating the pathways used for assembling the higher-order complexes and seeking ways in which these can be exploited to control the rate of the reaction. Another aim of the project is to acquire structural information to facilitate a knowledge-driven approach to generating hyperactive proteins.
Recent publications from the lab 1 Liu, D. & Chalmers, R. Hyperactive mariner transposons are created by mutations that disrupt allosterism and increase the rate of transposon end synapsis. Nucleic Acids Res 42, 2637-2645, doi:10.1093/nar/gkt1218 (2014). 2 Claeys Bouuaert, C., Walker, N., Liu, D. & Chalmers, R. Crosstalk between transposase subunits during cleavage of the mariner transposon. Nucleic Acids Res 42, 5799-5808, doi:10.1093/nar/gku172 (2014). 3 Loh, E. et al. Temperature triggers immune evasion by Neisseria meningitidis. Nature 502, 237-240, doi:10.1038/nature12616 (2013). 4 Claeys Bouuaert, C., Lipkow, K., Andrews, S. S., Liu, D. & Chalmers, R. The autoregulation of a eukaryotic DNA transposon. elife 2, e00668, doi:10.7554/eLife.00668 (2013). 5 Claeys Bouuaert, C. & Chalmers, R. Hsmar1 transposition is sensitive to the topology of the transposon donor and the target. PLoS One 8, e53690, doi:10.1371/journal.pone.0053690 (2013). 6 Siddique, A., Buisine, N. & Chalmers, R. The transposon-like Correia elements encode numerous strong promoters and provide a potential new mechanism for phase variation in the meningococcus. PLoS Genet 7, e1001277, doi:10.1371/journal.pgen.1001277 (2011).