Lubock, Nathan B. (2014 - 2017)
Nathan is in the Department of Chemistry and Biochemistry. He joined the training program in 2014. He received a B.S. degree in 2013 from Duke University.
Mentor: Dr. Sriram Kosuri
A Next-generation Two-hybrid for Genomic-scale Screens
Protein-protein interactions (PPIs) coordinate nearly all cellular processes, and it is estimated that nearly 600,000 PPIs exist in humans. Thus, determining which proteins interact in vivo is critical for a mechanistic understanding of the molecular machines that control cells, as well as for our ability to intervene when these complexes are disrupted as in many diseases. However, attempts to extend current methods, such as yeast two-hybrid screens, to investigate PPIs at a genome-wide scale have been stymied by a lack of throughput and sensitivity. We will overcome these limitations by developing a next-generation two-hybrid screen that leverages two existing technologies: protein fragment complementation assays and next-generation sequencing. Briefly, upon a successful PPI, two adenylate cyclase domains will refold, resulting in the production of cAMP. cAMP production then drives expression of an mRNA barcode that can be read by RNA-sequencing (RNA-seq). The RNA-seq readout will provide a quantitation of the full range of PPIs, including transient or weak interactions. Finally, we will harness recently developed array-based oligo synthesis methods to create protein libraries of over an order of magnitude larger than any previous reported and screen them in multiplex.
