Perturbing and Sensing Changes to Complex Pathways
The YRC is developing technology to make complex changes to cells and report on the effects of some of these changes. We are developing novel genome engineering approaches, selection systems, methods to disrupt protein-protein interactions, and biosensors. These efforts will allow us to make both large-scale and targeted changes to cells, to identify among the changed cells those with useful properties, and to detect the level of expression of either a ligand or a protein in cells that have been engineered.
Yeast Genome Engineering
Ability of tetO variants to drive expression of genes for the production of lycopene in S. cerevisiae correlates with their affinity for the Tet repressor protein. See: Cuperus et al. 2015.
There continues to be a need for improved genome engineering strategies in yeast. Even with the advent of facile genome engineering in any organism through the use of CRISPR/Cas9 technology, yeast can play a major role in demonstrating the power of large-scale mutagenesis approaches in a eukaryote. We have previously developed a toolkit of reagents and methods in yeast to generate strains with a defined set of elements to control gene expression. This toolkit employs variants of the tet operator (tetO) sequence to bind to a Tet repressor–VP16 activator with differential affinity. We are working to increase the ease, efficiency, and application of this method.
We are working to develop more sensitive library selection systems using continuous culture. The YRC has previously developed technology to characterize libraries via assaying the competitive fitness of each variant. However, typical competition experiments in batch culture present several shortcomings, including confounding selection variables and compressed dynamic range. We are building upon the chemostat and turbidostat platforms to develop new continuous culture systems to improve the selectivity of library competition experiments.
Disrupters of Protein-Protein Interactions
Protein-protein interactions play critical roles in biology both inside and outside cells. However, it is currently difficult to probe the importance of specific intracellular protein-protein interactions at a particular developmental stage and location or in a particular cell type. Few protein-protein interfaces can be disrupted specifically with small molecules, and even when these molecules are available, it is difficult to deliver them to a specific tissue. Dr. David Baker in the YRC is building upon his existing protein design process to develop technology to disrupt specific protein-protein interactions in order to examine mechanisms and regulation of cellular processes.
Biosensors that are capable of sensing and responding to ligands in vivo have wide-ranging applications, including their use in regulating metabolic pathways, optimizing biosynthetic pathways, measuring metabolite concentration, imaging, detecting toxins and triggering therapeutic responses. While biosensors have a long history of development and use, there have been no generally applicable strategies to generate a biosensor for a new molecule of interest. The YRC, in collaboration with George Church’s lab at Harvard, have developed two approaches that can lead to biosensors for potentially almost any molecule or protein of interest. The YRC is continuing to adapt and refine this technology for making biosensors to detect small molecules or proteins.