Development of prokaryotic cell-free systems for synthetic biology

Chiao, Abel C.; Murray, Richard M.; Sun, Zachary Z.

doi:10.1101/048710

Cited by 4 publications

(3 citation statements)

References 101 publications

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“…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.…”

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confidence: 99%

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

2021

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Cell-free protein synthesis (CFPS) is a platform biotechnology that has enabled the on-demand synthesis of proteins for a variety of applications. Numerous advances have improved the productivity of the CFPS platform to result in highyielding reactions; however, many applications remain limited due to long reaction times. To overcome this limitation, we first established the benchmarks reaction times for CFPS across inhouse E. coli extracts and commercial kits. We then set out to finetune our in-house extract systems to improve reaction times. Through the optimization of reaction composition and titration of low-cost additives, we have identified formulations that reduce reaction times by 30−50% to obtain high protein titers for biomanufacturing applications, and reduce times by more than 50% to reach the sfGFP detection limit for applications in education and diagnostics. Under optimum conditions, we report the visible observation of sfGFP signal in less than 10 min. Altogether, these advances enhance the utility of CFPS as a rapid, user-defined platform.

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confidence: 99%

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

2021

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“…Several varieties of CFPS systems have been described for different target applications. 18 Most of these reactions utilize an Escherichia coli cell extract combined with exogenous DNA, substrates for energy consumption and regeneration, and often T7 RNA polymerase. The choice of cell strain, lysis method, and energy mixture composition 20 varies between laboratories.…”

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confidence: 99%

Quantification of Interlaboratory Cell-Free Protein Synthesis Variability

et al. 2019

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Cell-free protein synthesis (CFPS) platforms, once primarily a research tool to produce difficult to express proteins, are increasingly being pursued by the synthetic biology community for applications including biomanufacturing, rapid screening systems, and field-ready sensors. While consistency within individual studies is apparent in the literature, challenges with reproducing results between laboratories, or even between individuals within a laboratory, are discussed openly by practitioners. As the field continues to grow and move toward applications, a quantitative understanding of expected variability for CFPS and the relative contribution of underlying sources will become increasingly important. Here we offer the first quantitative assessment of interlaboratory variability in CFPS. Three laboratories implemented a single CFPS protocol and performed a series of exchanges, both of material and personnel, designed to quantify relative contributions to variability associated with the site, operator, cell extract preparation, and supplemental reagent preparation. We found that materials prepared at each laboratory, exchanged pairwise, and tested at each site resulted in 40.3% coefficient of variation compared to 7.64% for a single operator across days using a single set of materials. Reagent preparations contributed significantly to observed variability; extract preparations, however, surprisingly did not explain any of the observed variability, even when prepared in different laboratories by different operators. Subsequent exchanges showed that both the site and the operator each contributed to observed interlaboratory variability. In addition to providing the first quantitative assessment of interlaboratory variability in CFPS, these results establish a baseline for individual operator variability across days that can be used as an initial benchmark for community-driven standardization efforts. We anticipate that our results will narrow future avenues of investigation to develop best practices that will ultimately drive down interlaboratory variability, accelerating research progress and informing the suitability of CFPS for real-world applications.

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“…Here we present an overview of different types of lysate preparation steps ( Figure 1A), and their effects on lysate properties. The history of the field, recent advances, as well as the development, optimization, and applications of TX-TL systems are covered in recent reviews (Chiao et al, 2016;Silverman et al, 2019a).…”

Section: E Coli Lysatesmentioning

confidence: 99%

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

Laohakunakorn

Grasemann

Lavickova

et al. 2020

Front. Bioeng. Biotechnol.

103

101

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Cell-free systems offer a promising approach to engineer biology since their open nature allows for well-controlled and characterized reaction conditions. In this review, we discuss the history and recent developments in engineering recombinant and crude extract systems, as well as breakthroughs in enabling technologies, that have facilitated increased throughput, compartmentalization, and spatial control of cell-free protein synthesis reactions. Combined with a deeper understanding of the cell-free systems themselves, these advances improve our ability to address a range of scientific questions. By mastering control of the cell-free platform, we will be in a position to construct increasingly complex biomolecular systems, and approach natural biological complexity in a bottom-up manner.

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Development of prokaryotic cell-free systems for synthetic biology

Cited by 4 publications

References 101 publications

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

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

Quantification of Interlaboratory Cell-Free Protein Synthesis Variability

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

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