OnePot PURE Cell-Free System

The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for cell-free synthetic ...

École Polytechnique Fédérale de Lausanne

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription-translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System.

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.

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

Self-regeneration is a fundamental function of all living systems. Here we demonstrate partial molecular self-regeneration in a synthetic cell. By implementing a minimal transcription-translation system within microfluidic reactors, the system is able to regenerate essential protein components from DNA templates and sustain synthesis activity for over a day. By quantitating genotype-phenotype relationships combined with computational modeling we find that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieve simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.

A partially self-regenerating synthetic cell.

Cell-free synthetic biology offers an approach to building and testing gene circuits in a simplified environment free from the complexity of a living cell. Recent advances in microfluidic devices allowed cell-free reactions to run under nonequilibrium, steady-state conditions enabling the implementation of dynamic gene regulatory circuits in vitro. In this chapter, we present a detailed protocol to fabricate a microfluidic chemostat device which enables such an operation, detailing essential steps in photolithography, soft lithography, and hardware setup.

Article	Year
A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System. ACS synthetic biology\| PubMed ID: 30632751	2019
Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology. Frontiers in bioengineering and biotechnology\| PubMed ID: 32266240	2020
A partially self-regenerating synthetic cell. Nature communications\| PubMed ID: 33311509	2020
Steady-State Cell-Free Gene Expression with Microfluidic Chemostats. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 33405223	2021

Barbora Lavickova

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