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Journal Articles ACS Synthetic Biology Year : 2023

Clonal Amplification-Enhanced Gene Expression in Synthetic Vesicles


In cell-free gene expression, low input DNA concentration severely limits the phenotypic output, which may impair in vitro protein evolution efforts. We address this challenge by developing CADGE, a strategy that is based on clonal isothermal amplification of a linear gene-encoding dsDNA template by the minimal Φ29 replication machinery and in situ transcriptiontranslation. We demonstrate the utility of CADGE in bulk and in clonal liposome microcompartments to boost up the phenotypic output of soluble and membrane-associated proteins, as well as to facilitate the recovery of encapsulated DNA. Moreover, we report that CADGE enables the enrichment of a DNA variant from a mock gene library via either a positive feedback loop-based selection or highthroughput screening. This new biological tool can be implemented for cell-free protein engineering and the construction of a synthetic cell.

Dates and versions

hal-04098688 , version 1 (16-05-2023)



Zhanar Abil, Ana Maria Restrepo Sierra, Christophe Danelon. Clonal Amplification-Enhanced Gene Expression in Synthetic Vesicles. ACS Synthetic Biology, 2023, 12, pp.1187 - 1203. ⟨10.1021/acssynbio.2c00668⟩. ⟨hal-04098688⟩
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