Unlocking Applications of Cell-Free Biotechnology through Enhanced Shelf Life and Productivity of E. coli Extracts

Abstract

Cell-free protein synthesis (CFPS) is a platform biotechnology that enables a breadth of applications. However, field applications remain limited due to the poor shelf-stability of aqueous cell extracts required for CFPS. Lyophilization of E. coli extracts improves shelf life but remains insufficient for extended storage at room temperature. To address this limitation, we mapped the chemical space of ten low-cost additives with four distinct mechanisms of action in a combinatorial manner to identify formulations capable of stabilizing lyophilized cell extract. We report three key findings: (1) unique additive formulations that maintain full productivity of cell extracts stored at 4 °C and 23 °C; (2) additive formulations that enhance extract productivity by nearly 2-fold; (3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. These findings provide a simple and low-cost advance toward making CFPS field-ready and cost-competitive for biomanufacturing.

The Genetic Code Kit: An Open-Source Cell-Free Platform for Biochemical and Biotechnology Education

“…Transitioning the CFPS platform from a research-focused technology to one that is broadly accessible to high school and university classrooms required extensive simplification, reduced costs, and improved reagent stabilization. Our work to date has taken incremental steps toward these milestones by reducing the number of pipetting steps in CFPS setup (Levine et al, 2019a), creating a less-labor intensive cell extract preparation workflow (Levine et al, 2019b), and identifying a low-cost formulation of additives that enables storage and transport of cell-free extract at room temperature (Gregorio et al, 2020). These advances are part of a concerted effort by the research community to make CFPS accessible to classrooms around the world (Huang et al, 2018;Stark et al, 2018Stark et al, , 2019Collias et al, 2019).…”

Cell-free protein synthesis andin situimmobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion

“…13 Moreover, it is known throughout literature that reproducible lysate production from batch to batch is a crucial task, and much effort has been undertaken to develop protocols that solve this issue. 12,13,35,36 The prepared lysates were supplemented with 1 mM of Chi6 DNA to prevent degradation of the functional, linear PCR fragments used for implementing genetic information into microgels that served as reaction environment for CFPS. 37,38 Experiments indicate that addition of Chi6 is not benecial in every experiment (Fig.…”

“…2 The expected effect of increasing activity of the CFPS by increasing the crowding agent was not observed. 18,34,36,38,39 While the cytosol of E. coli cells is a very crowded environment of approx. 300-400 mg mL À1 of macromolecules, 40 the reason for our results may lay in a PEG-induced precipitation of mRNA, for instance.…”

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

“…A body of work dedicated to optimization of extract preparation and reaction conditions has simplified, expedited, and improved the cost and performance of E. coli CFE systems (22,28,29). Optimized E. coli-based CFE reactions: (i) quickly synthesize grams of protein per liter in batch reactions (30)(31)(32), (ii) are scalable from the nL to 100 L scale (33,34), and (iii) can be freeze-dried for months of shelf-stability and distribution to the point of care (6,22,29,(35)(36)(37)(38)(39)(40). Freeze-dried CFE systems are poised to make disruptive impacts in biotechnology, having already been leveraged for point-of-use biosensing (41)(42)(43)(44)(45)(46), therapeutic and vaccine production (37,38,47), and educational kits (22,(48)(49)(50).…”