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bioRxiv · 10.1101/2022.11.17.516858

Clonal amplification-enhanced gene expression for cell-free directed evolution

Danelon, C.·Restrepo Sierra, A. M.·Abil, Z.
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Abstract

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 {Phi}29 replication machinery and in situ transcription-translation. 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 either via a positive feedback loop-based selection or high-throughput screening. This new biological tool can be implemented for cell-free protein engineering and the construction of a synthetic cell.

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Danelon, C., Restrepo Sierra, A. M., Abil, Z.. 2022-11-17. Clonal amplification-enhanced gene expression for cell-free directed evolution. https://doi.org/10.1101/2022.11.17.516858

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