The all-E. coliTXTL toolbox 3.0: New capabilities of a cell-free synthetic biology platform

David Garenne, Seth Thompson, Amaury Brisson, Aset Khakimzhan, Vincent Noireaux

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


The new generation of cell-free gene expression systems enables the prototyping and engineering of biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription-Translation (TXTL) platforms remains a major goal to either execute larger DNA programs or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all-E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch-mode reactions, the synthesis of the fluorescent reporter protein eGFP (enhanced green fluorescent protein) reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced at concentrations of >8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kb and ∼60 genes, is produced at a concentration of 1013 PFU/ml (plaque forming unit/ml). This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for testing regulatory elements and circuits, biomanufacturing biologics or building synthetic cells.

Original languageEnglish (US)
Article numberysab017
JournalSynthetic Biology
Issue number1
StatePublished - 2021

Bibliographical note

Publisher Copyright:
© 2021 The Author(s) 2021. Published by Oxford University Press.


  • bacteriophages
  • cell-free transcription-Translation
  • gene circuits
  • synthetic cell


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