PT - JOURNAL ARTICLE AU - Wiegand, Daniel J. AU - Lee, Henry H. AU - Ostrov, Nili AU - Church, George M. TI - Establishing a cell-free <em>Vibrio natriegens</em> expression system AID - 10.1101/331645 DP - 2018 Jan 01 TA - bioRxiv PG - 331645 4099 - http://biorxiv.org/content/early/2018/05/26/331645.short 4100 - http://biorxiv.org/content/early/2018/05/26/331645.full AB - The fast growing bacterium Vibrio natriegens is an emerging microbial host for biotechnology. Harnessing its productive cellular components may offer a compelling platform for rapid protein production and prototyping of metabolic pathways or genetic circuits. Here, we report the development of a V. natriegens cell-free expression system. We devised a simplified crude extract preparation protocol and achieved &gt;260µg/mL of super-folder GFP in a small-scale batch reaction after three hours. Culturing conditions, including growth media and cell density, significantly affect translation kinetics and extract protein yield. We observed maximal protein yield at incubation temperatures of 26°C or 30°C, and show improved yield by tuning ions crucial for ribosomal stability. This work establishes an initial V. natriegens cell-free expression system, enables probing of V. natriegens biology, and will serve as a platform to accelerate metabolic engineering and synthetic biology applications.