New England Biolabs
Hosts: Drs. Ruben Gonzalez and John Hunt
Title: Recombinant Protein Synthesis and Synthetic Systems
Abstract: I will talk about two lines of interest in my lab. The first is our attempts to experimentally test how well evolutionarily conserved factors involved in protein translation are functionally compatible between different bacterial species. Our approach is in vitro reconstitution of minimal protein translation machineries from gram-negative E. coli and Thermus thermophilus, and gram-positive Mycobacterium. By establishing in vitro protein synthesis, we examine functional contributions and compatibilities of individually purified and recombinant components. This reconstitution approach could help to study bacteria intractable to conventional genetic means. The second line of interest is to design and build cell-like synthetic systems to enable high-throughput characterization of protein sequence-function relationships. One aspect of this bottom-up approach establishes a general assay platform for protein interactions by recapitulating bacterial transcription regulation and gene expression. I will describe an in vitro two-hybrid system (IVT2H) that mimics the cell-based system and allows gene-to-function in just a few hours. I will demonstrate the use of our in vitro systems for high throughput deep mutational characterization of proteins in μ-liter-scale 96-well microplates and picoliter-scale droplet-based microfluidics.
(1) Engineering bacterial transcription regulation to create a synthetic in vitro two-hybrid system for protein interaction assays. (2014) J Am Chem Soc. 136(40):14031-8.
(2) A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders. (2016) Nature Sci Rep. 6:22575.
(3) Reconstitution of Protein Translation of Mycobacterium Reveals Functional Conservation and Divergence with The Gram-Negative Bacterium Escherichia coli. (2016) PLoS One. 11(8):e0162020.