Cell-free Protein Expression Using the Rapidly Growing Bacterium <em>Vibrio natriegens</em>

The marine bacterium Vibrio natriegens has garnered considerable attention as an emerging microbial host for biotechnology due to its fast growth rate. A general protocol is described for the preparation of V. natriegens crude cell extracts using common laboratory equipment. This high yielding protocol has been specifically optimized for user accessibility and reduced cost. Cell-free protein synthesis (CFPS) can be carried out in small scale 10 μL batch reactions in either a 96- or 384-well format and reproducibly yields concentrations of > 260 μg/mL super folder GFP (sfGFP) within 3 h. Overall, crude cell extract preparation and CFPS can be achieved in 1-2 full days by a single user. This protocol can be easily integrated into existing protein synthesis pipelines to facilitate advances in bio-production and synthetic biology applications.