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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.

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Figure 1: Basic mechanisms that control protein co-expression and co-purification from a single bacterial consortium.
Figure 2: Design and optimization of the synthetic bacterial consortia.
Figure 3: Reducing the number of bacterial strains in the synthetic consortia.
Figure 4: Applications of the translation-mix one shot (TraMOS) in cell-free synthetic biology.

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Acknowledgements

We appreciate the discussion of the manuscript with members of Tan lab. This work is supported by Society-in-Science, Branco–Weiss Fellowship to C.T. L.E.C.-L. is supported through a UC MEXUS-CONACYT Doctoral Fellowship.

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Contributions

F.V. and C.T. designed the study. M.C. and C.T. wrote Matlab codes and performed computer simulations. F.V. performed all experiments and data analysis. L.E.C.-L. assisted with protein purification and quantification of protein yields. C.T. gave technical and conceptual advice. F.V. and C.T. wrote the paper. Y.D constructed the Ngo plasmids. J.F. and T.P. assisted with the study of the Ngo plasmids.

Corresponding author

Correspondence to Cheemeng Tan.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–10, Supplementary Figures 1–24 and Supplementary Notes 1–3 (PDF 14714 kb)

Life Sciences Reporting Summary (PDF 69 kb)

Supplementary Dataset 1

Identified proteins and quantified counts from 34-strain TraMOS. (XLSX 19 kb)

Supplementary Dataset 2

Identified proteins and quantified counts of TraMOS I with low activity. (XLSX 12 kb)

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Villarreal, F., Contreras-Llano, L., Chavez, M. et al. Synthetic microbial consortia enable rapid assembly of pure translation machinery. Nat Chem Biol 14, 29–35 (2018). https://doi.org/10.1038/nchembio.2514

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