Systematic genetic code reprogramming / Centre for chemical and synthetic biology
Our lab has pioneered the development and application of methods for reprogramming the genetic code of living organisms. These approaches allow the site specific incorporation of designer unnatural amino acids, beyond the canonical 20, into proteins in diverse cells and organisms.
A fundamental challenge in reprogramming the genetic code of cells is to direct the incorporation of unnatural amino acids into proteins. We have shown that it is possible to create an orthogonal translation pathway in the cell in which a new ribosome, the orthogonal ribosome, is directed to a new message. Since the orthogonal ribosome, unlike the natural ribosome, is not essential it is possible to evolve the orthogonal ribosome to read new genetic codes on the orthogonal message. We have created a ribosome that efficiently reads quadruplet codons and amber codons, that are inefficiently read on the natural ribosome. By directing tRNAs aminoacylated with unnatural amino acids to the orthogonal ribosome we have created a parallel genetic code in the cell. This parallel translation pathway provides a series of blank codons that may be assigned to new amino acids.
We have pioneered the use of pyrrolysyl -tRNA synthetase/tRNA pairs for genetic code expansion. Since we demonstrated that this pair could be evolved for unnatural amino acid incorporation this system has been widely adopted. We continue to investigate and expand the scope of this system for unnatural amino acid incorporation.
We have developed approaches to make proteins bearing post-translational modifications that were previously inaccessible. By investigating the modified recombinant proteins we have created – using structural biology, enzymology and single molecule approaches – we have provided new insight into the role of post-translational modifications, including lysine acetylation, methylation and ubiqutination, in regulating protein structure and biological function.
We have developed approaches that allow us to rapidly control enzymatic activity and protein transport in cells with a millisecond pulse of light. In one series of experiments we demonstrated that we can photo-control protein kinase activity in vivo. Using this approach we have begun to provide insight into the kinetics of elementary steps in signal transduction and feedback regulation. In previous work we have also developed methods for defining protein interfaces involved in protein interactions by genetically encoded photocrosslinkers.
We are developing approaches that allow any site in a protein of interest to be rapidly, specifically and efficiently labelled with a probe of interest in vivo. We have recently described approaches for site specifically labelling proteins in and on cells with a range of small molecule fluorophores for live cell imaging.
While initial efforts at genetic code expansion focused on E. coli we have shown that it is possible to incorporate a range of unnatural amino acids in yeast and mammalian cells. We have recently been able to expand the genetic code of two established multicellular model organisms, C. elegans and D. melanogaster. By applying the approaches we have created in cells for controlling and imaging protein function in whole animals we hope to provide new insight into processes, including embryonic development, tissue morphogenesis, tumor biology and neuronal plasticity, that can only be studied at the organism level.