Development of an in Vitro Protein Production Platform from Tobacco Chloroplasts to Facilitate Plant Engineering

Plants represent an emerging frontier for synthetic biology with opportunities in discovering and producing pharmaceutically relevant compounds as well as deploying biosensors for monitoring the environment. Unfortunately, engineering plants is limited by their complex genetic makeup, slow growth, and transformation idiosyncrasies. To address these limitations, we present a cell-free system from Nicotiana tabacum chloroplasts as a way of accelerating biological design. Our cell-free platform enables rapid, high-throughput (up to 10⁴ construct per week) screens for precise characterization of genetic parts. To build our cell-free platform, we developed a combined transcription and translation system from N. tabacum chloroplast extract that works with plasmid or linear DNA. Specifically, we optimized lysis and processing of the extract as well as the reaction environment to enable yields of luciferase of up to 100 µg/mL. Towards using the cell-free system as a prototyping platform, we tested libraries of genetic parts. We anticipate that our system will increase the pace of building genetic programs in plants and offer a fast track to the discovery and synthesis of cheap, pharmaceutically, or agriculturally relevant products.