Activation of Energy Metabolism through Growth Media Reformulation Enables a 24-Hour Workflow for Cell-Free Expression

Abstract

Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, point-ofcare detection, therapeutics, and education using affordable laboratory equipment and reagents. The Escherichia coli cell extract-based platform, being one of the most affordable and versatile CFPS platforms, has been broadly adopted. In spite of the promise of simplicity, the cell-free platform remains technically nuanced, posing challenges to reproducible implementation and broad adoption. Additionally, while the CFPS reaction itself can be implemented on-demand, the upstream processing of cells to generate crude cell lysate remains time-intensive, representing one of the largest sources of cost associated with the biotechnology. To circumvent the lengthy and tedious upstream workflow, we have redesigned the processes by developing a longlasting autoinduction media formulation for cell-free that obviates human intervention between inoculation and harvest. CelI-free autoinduction (CFAI) media supports these advantages through the production of highly robust cell extracts from high cell density cultures nearing stationary phase of growth. Growth of cells to high density and autoinduction of T7 RNAP expression can be achieved by incubation overnight, eliminating the need for user intervention for the entirety of the process. The total mass of cells obtained is substantially increased, which directly results in a 400% increase in total extract volume obtained compared to past workflows. Based on these advances, we outline a new upstream processing workflow that allows researchers to go from cells on a streak plate to completing CFPS reactions within 24 hours while maintaining robust reaction yields of sfGFP (>1 mg/ml). We hope this advance will improve the time and cost-efficiency for existing CFPS researchers, increase the simplicity and reproducibility, and reduce the barrier-to-entry for new researchers interested in implementing CFPS.

Characterizing and Improving Reaction Times for E. coli-Based Cell-Free Protein Synthesis

“…E. coli -based cell extracts are a well-established, low-cost, high yielding, and broadly adopted extract type that supports numerous CFPS applications. , For this reason, we sought to examine CFPS reaction time courses using three separate in-house E. coli -based extracts. Among these are extracts from cells grown in 2xYTPG (yeast, tryptone, PEP, glycerol) media to OD 600 2.5 (referred to as “2xYTPG”), CFAI (cell-free autoinduction) media grown to OD 600 10 (referred to as “CFAI 10”), and CFAI media grown to OD 600 2.5 (referred to as “CFAI 2.5”), each of which have been previously used to achieve high protein titers >1000 μg/mL sfGFP . We had previously demonstrated that each cell-growth method results in a unique metabolism within the cell extracts; here we evaluated whether these differences affect CFPS reaction rates (Figure A–G).…”

“…Here we report our optimization results using extract from cells grown in 2xYTPG (Yeast, Tryptone, Phosphate, Glucose) to OD 600 2.5. 2xYTPG media is a traditional E. coli extract preparation method capable of supporting high protein titers in CFPS and has become the standard growth medium for many applications of E. coli -based CFPS . For this reason, we wanted to establish a benchmark reaction time course for CFPS with 2xYTPG extract using the PANOx-SP energy system.…”

“…Cells grown in CFAI media to OD 600 10 (CFAI 10) is a recently developed E. coli -based extract preparation method. CFAI provides an “Instant Pot”-type utility for the growth of cells suitable for extract preparation, in which the higher OD 600 harvest results in a greater than 400% increase in extract production without sacrificing CFPS productivity . The reduced production costs and improved reproducibility that CFAI provides make the method well suited for supporting education and diagnostics applications, especially in resource-limited environments, while also being compatible with biomanufacturing applications.…”

“…Cell-free protein synthesis (CFPS) is a biotechnology platform that provides a robust user-defined strategy for the on-demand production of proteins to support a variety of applications. − Innovations of the CFPS platform have led to a large array of compatible extract types, energy systems, and DNA templates capable of producing high protein titers. − Despite these advancements, slow reaction rates remain a bottleneck for design–build–test–learn cycles, and also limit the effectiveness of CFPS in applications such as diagnostics and education in which rapid readouts are desired.…”

“…Toward our first goal we chose to evaluate E. coli -based cell extracts due to their low-cost, reproducibility, and general prevalence as a broadly utilized extract type. ,, However, even among E. coli extracts, the preparation method and energy system can impact transcriptional, translational, protein expression, and folding rates, as well as overall protein yield . Therefore, we quantified CFPS reaction times using three different in-house E. coli extracts prepared from cells grown in 2xYTPG media to OD 600 2.5 (2xYTPG), CFAI (cell-free autoinduction) media grown to OD 600 10 (CFAI 10), and CFAI media grown to OD 600 2.5 (CFAI 2.5) with CFPS reactions run using the PANOx-SP energy system.…”