Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium

Andrei Sakai, Aafke J. Jonker, Frank H.T. Nelissen, Evan M. Kalb, Bob van Sluijs, Hans A. Heus, Katarzyna P. Adamala, John I. Glass, Wilhelm T.S. Huck

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1616-1623
Number of pages8
JournalACS Synthetic Biology
Issue number6
StatePublished - Jun 16 2023

Bibliographical note

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.


  • JCVI-syn3A
  • Mycoplasma
  • active machine learning
  • cell-free expression system


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