Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, 2629HZ Delft, The Netherlands.
Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRAE, INSA, 31077 Toulouse, France.
ACS Synth Biol. 2023 Apr 21;12(4):1187-1203. doi: 10.1021/acssynbio.2c00668. Epub 2023 Apr 4.
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.
在无细胞基因表达中，低输入 DNA 浓度严重限制了表型输出，这可能会影响体外蛋白质进化的效果。我们通过开发 CADGE 策略来解决这一挑战，该策略基于最小 Φ29 复制机制对线性基因编码 dsDNA 模板进行克隆等温扩增，以及原位转录-翻译。我们在批量和克隆脂质体微隔间中证明了 CADGE 的实用性，以提高可溶性和膜相关蛋白质的表型输出，并促进封装 DNA 的回收。此外，我们报告称，CADGE 可通过基于正反馈回路的选择或高通量筛选从模拟基因文库中富集 DNA 变体。这种新的生物工具可用于无细胞蛋白质工程和合成细胞的构建。
