Clonal Amplification-Enhanced Gene Expression in Synthetic Vesicles.
ACS Synth Biol
; 12(4): 1187-1203, 2023 04 21.
Article
in En
| MEDLINE
| ID: mdl-37014369
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 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 via either 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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA
/
DNA-Directed DNA Polymerase
Language:
En
Journal:
ACS Synth Biol
Year:
2023
Document type:
Article
Affiliation country:
Netherlands