rapid-characterization-of-genetic-parts-with-cell-free-systems

Rapid Characterization of Genetic Parts with Cell-Free Systems

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the ...

US Army Combat Capabilities Development Command Chemical Biological Center

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.

Quantification of Interlaboratory Cell-Free Protein Synthesis Variability.

Cell-free systems that mimic essential cell functions, such as gene expression, have dramatically expanded in recent years, both in terms of applications and widespread adoption. Here we provide a review of cell-extract methods, with a specific focus on prokaryotic systems. Firstly, we describe the diversity of  genetic strains available and their corresponding utility. We then trace the history of cell-extract methodology over the past 20 years, showing key improvements that lower the entry level for new researchers. Next, we survey the rise of new prokaryotic cell-free systems, with associated methods, and the opportunities provided. Finally, we use this historical perspective to comment on the role of methodology improvements and highlight where further improvements may be possible.

Stephanie D. Cole

US Army Combat Capabilities Development Command Chemical Biological Center

Rapid Characterization of Genetic Parts with Cell-Free Systems