Synthetic microbial consortia enable rapid assembly of pure translation machinery

Abstract

Assembly of recombinant multiprotein systems requires multiple culturing and purification steps that scale linearly with the number of constituent proteins. This problem is particularly pronounced in the preparation of the 34 proteins involved in transcription and translation systems, which are fundamental biochemistry tools for reconstitution of cellular pathways ex vivo. Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure. The expression of these proteins is controlled by synthetic genetic modules to produce the proteins at the correct ratios. We show that the pure multiprotein system is functional and reproducible, and has low protein contaminants. We also demonstrate its application in the screening of synthetic promoters and protease inhibitors. Our work establishes a novel strategy for producing pure translation machinery, which may be extended to the production of other multiprotein systems.

A simple, robust, and low-cost method to produce the PURE cell - free system

“…This observation is in agreement with results obtained for TraMOS (17 ). Based on these results the OnePot PURE system achieved similar levels of purity as PURE produced in-house using the standard method.…”

Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems

“…Cell‐free expression systems have long been established as a simplified in vitro approach to overcome these challenges by maintaining cellular processes in the absence of an intact cell membrane (Swartz, ). Cell‐free systems, made up of either individually reconstituted cellular components (Shimizu et al , ; Wang et al , ; Villarreal et al , ) or cell lysates (Jewett et al , ; Garamella et al , ), can support a variety of catalytic reactions in vitro when supplied with energy sources, cofactors, and ions (Calhoun & Swartz, , ; Jewett et al , ). These cell‐free approaches have facilitated the development of therapeutically useful natural products (Dudley et al , ; Maini et al , ) and biologics with non‐standard amino acids (Martin et al , ) or chemical moieties otherwise challenging to synthesize (Jaroentomeechai et al , ).…”

Elucidating the potential of crude cell extracts for producing pyruvate from glucose

“…This methodology has been successfully demonstrated for hydrogen production and protein synthesis among others (6,7). While recent efforts in copurification of full reaction cascades have reduced costs, any process utilizing bulk purified proteins remains expensive (8,9). To date, the use of purified components for CFME has resulted in long running systems capable of catalyzing reactions for several days, but with the drawback of slow catalysis rates.…”