Cell-free protein expression systems (CFPS) can be used as tools for Synthetic Biology with several key advantages over in vivo systems, including the ability to rapidly screen for the functionality of novel genetic components. We are developing and optimizing CFPS from E. coli Nissle 1917, a protective probiotic strain of E. coli, as a tool to rapidly test the functionality of sensors and constructs built for this strain. While preparing E. coli Nissle 1917 extracts we varied several parameters in a protocol established for E. coli BL21* DES, a strain optimized for high protein production and commonly used for generating CFPS. For example, during cultivation of the E. coli Nissle 1917 we selected cells from different growth phases (mid-exponential and late-exponential). Additionally, we optimized the total sonication energy input during lysis, the magnesium concentration of the extract buffer, the cell-free reaction conditions, as well as other parameters. Initial cell-free reactions using the Nissle 1917 extracts and a vector with GFP expressed under the T7 promoter produced modest, but detectable amounts of GFP over background. Optimizing different parameters improved the functionality of the Nissle 1917 system and current work focuses on high throughput testing of relevant components. For example, we plan to engineer E. coli Nissle 1917 to detect stress in the human gut and subsequently produce a response to remediate this condition. A functional Nissle 1917 CFPS provides us with the capability to rapidly test components for this application and others.

Optimization of probiotic E. coli Nissle 1917 cell-free protein expression system for rapid testing of novel components

255th National Meeting (2018)

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