Didovyk Andriy, Tonooka Taishi, Tsimring Lev, Hasty Jeff
BioCircuits Institute, University of California San Diego , La Jolla, California 92093, United States.
San Diego Center for Systems Biology, University of California San Diego , La Jolla, California 92093, United States.
ACS Synth Biol. 2017 Dec 15;6(12):2198-2208. doi: 10.1021/acssynbio.7b00253. Epub 2017 Aug 21.
Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.
无细胞基因表达系统正成为一个重要平台，用于各种合成生物学和生物技术应用，包括生产坚固耐用、可直接用于实际环境的生物传感器。在此，我们将程序性细胞自溶与冻融或冻干循环相结合，创造出一种实用、可重复且经济高效的方法，用于快速生产用于无细胞基因表达的细菌裂解物。使用这种方法，无需专门设备即可在广泛的规模上生产出强大且高活性的细菌细胞裂解物，使无细胞基因表达易于实现且广泛可用。此外，活的自溶菌株可以直接冻干，随后在复水时裂解以产生活性裂解物。我们通过调节蛋白质的产生和降解、实施群体感应以及展示GamS蛋白对线性DNA模板的定量保护，证明了自溶产物在合成生物学中的实用性。为了实现基于多功能且灵敏的β-半乳糖苷酶（LacZ）的读数，我们生产出无可检测背景LacZ活性的自溶产物，并使用它们来生产具有LacZ介导的比色和荧光输出的灵敏汞（II）生物传感器。自溶方法可以促进无细胞技术在无细胞基因表达以及其他合成生物学和生物技术应用（如代谢工程、天然产物生物合成或蛋白质组学）中的更广泛应用。
