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Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium.
Sakai, Andrei; Jonker, Aafke J; Nelissen, Frank H T; Kalb, Evan M; van Sluijs, Bob; Heus, Hans A; Adamala, Katarzyna P; Glass, John I; Huck, Wilhelm T S.
Afiliación
  • Sakai A; Institute for Molecules and Materials, Radboud University, Nijmegen 6525AJ, The Netherlands.
  • Jonker AJ; Institute for Molecules and Materials, Radboud University, Nijmegen 6525AJ, The Netherlands.
  • Nelissen FHT; Institute for Molecules and Materials, Radboud University, Nijmegen 6525AJ, The Netherlands.
  • Kalb EM; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455, United States.
  • van Sluijs B; Institute for Molecules and Materials, Radboud University, Nijmegen 6525AJ, The Netherlands.
  • Heus HA; Institute for Molecules and Materials, Radboud University, Nijmegen 6525AJ, The Netherlands.
  • Adamala KP; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota 55455, United States.
  • Glass JI; Synthetic Biology & Bioenergy, J. Craig Venter Institute, La Jolla, California 92037, United States.
  • Huck WTS; Institute for Molecules and Materials, Radboud University, Nijmegen 6525AJ, The Netherlands.
ACS Synth Biol ; 12(6): 1616-1623, 2023 06 16.
Article en En | MEDLINE | ID: mdl-37278603
Cell-free expression (CFE) systems are fundamental to reconstituting metabolic pathways in vitro toward the construction of a synthetic cell. Although an Escherichia coli-based CFE system is well-established, simpler model organisms are necessary to understand the principles behind life-like behavior. Here, we report the successful creation of a CFE system derived from JCVI-syn3A (Syn3A), the minimal synthetic bacterium. Previously, high ribonuclease activity in Syn3A lysates impeded the establishment of functional CFE systems. Now, we describe how an unusual cell lysis method (nitrogen decompression) yielded Syn3A lysates with reduced ribonuclease activity that supported in vitro expression. To improve the protein yields in the Syn3A CFE system, we optimized the Syn3A CFE reaction mixture using an active machine learning tool. The optimized reaction mixture improved the CFE 3.2-fold compared to the preoptimized condition. This is the first report of a functional CFE system derived from a minimal synthetic bacterium, enabling further advances in bottom-up synthetic biology.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bacterias / Sistema Libre de Células Idioma: En Revista: ACS Synth Biol Año: 2023 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Estados Unidos
Texto completo
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Imprimir
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Bacterias / Sistema Libre de Células Idioma: En Revista: ACS Synth Biol Año: 2023 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Estados Unidos