RT Journal Article
SR Electronic
T1 Clonal amplification-enhanced gene expression for cell-free directed evolution
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.11.17.516858
DO 10.1101/2022.11.17.516858
A1 Abil, Zhanar
A1 Restrepo Sierra, Ana Maria
A1 Danelon, Christophe
YR 2022
UL http://biorxiv.org/content/early/2022/11/17/2022.11.17.516858.abstract
AB 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 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.Competing Interest StatementZ.A. and C.D. have filed patent 22315279.4 entitled Micro-compartmentalized ultra-high throughput screening from single copy gene libraries.