Clonal Amplification-Enhanced Gene Expression in Synthetic Vesicles
Zhanar Abil (TU Delft - BN/Christophe Danelon Lab, TU Delft - BN/Bionanoscience, Kavli institute of nanoscience Delft)
Ana Maria Restrepo Sierra (Kavli institute of nanoscience Delft, TU Delft - BN/Gijsje Koenderink Lab, TU Delft - BN/Bionanoscience)
Christophe Danelon (Kavli institute of nanoscience Delft, INSA Toulouse, TU Delft - BN/Bionanoscience)
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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 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.
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