Transposon epigenetics and applications for genome engineering, synthetic biology and gene therapy applications.

The student will initially receive training in the human cell culture and the techniques of molecular biology required to build recombinant plasmid vectors. This represents the foundation of a broad-based approach to molecular and cell biology that will develop towards an advanced understanding of cutting-edge recombineering techniques. During the rotation the student will have the opportunity to address a 'bite-sized' question that they have a good chance of answering in the limited time available. Specifically, they will develop a genetic screen to identify hyper-active variants of the human Hsmar1 transposase protein. The laboratory has established reporter assays that allows the facile screening of large mutant libraries. The student will generate the library of mutations using error prone PCR. The aim will be to fine-tune the reaction conditions to produce between two and three mutations per kb of coding sequence. This "ideal" density will aid screening because it minimizes the number of clones that either lack mutations or those that have multiple mutations, which confounds subsequent analysis. Hyper-active clones will be sequenced to identify the individual mutations. The biochemical mechanism underpinning the hyper-activity will be investigated by mapping the position of the mutations onto the crystal structure of the protein.