Non-canonical genetic code implementation with an optimized, easily scaled cell free lysate system

Naturally occurring protein polymers are limited by the chemistry of the canonical genetic code. By incorporating non-canonical amino acids (ncAA) monomers into proteins, the catalytic, sensing, and physical properties of proteins can be engineered to generate novel reagents and materials. To address this synthetic biology need, Fabrico Technology and Professor Andrew Ellington at the University of Texas at Austin (UTA) propose to develop an innovative One Pot cell free lysate system for the scalable in vitro transcription and translation (IVTT) of proteins/polymers incorporating natural and ncAA. The proposed One Pot system should be extremely cost-effective and only requires a single purification step to obtain active protein factors for IVTT. The Ellington lab has utilized this technology to previously achieve site-specific incorporation of ncAA via orthogonal aminoacyl-tRNA synthetase/ tRNA pairs. In Phase I, Fabrico and the Ellington Lab will demonstrate the incorporation of 2 ncAAs and scale this capability to a 2-liter bioreactor. This demonstration will provide Fabrico with in-house reporters that can be adapted to diagnostic assays. In Phase II, we intend to incorporate 3 or more ncAAs, and scale the production capacity to a 10-liter bioreactor resulting in optimization of 10 or greater factor production from current systems.