Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
Center for Synthetic Microbiology (SYNMIKRO), Philipps-Universität Marburg, Marburg, Germany.
Methods Mol Biol. 2025;2850:197-217. doi: 10.1007/978-1-0716-4220-7_11.
Cell-free transcription and translation (TXTL) systems have emerged as a powerful tool for testing genetic regulatory elements and circuits. Cell-free prototyping can dramatically accelerate the design-build-test-learn cycle of new functions in synthetic biology, in particular when quick-to-assemble linear DNA templates are used. Here, we describe a Golden-Gate-assisted, cloning-free workflow to rapidly produce linear DNA templates for TXTL reactions by assembling transcription units from basic genetic parts of a modular cloning toolbox. Functional DNA templates composed of multiple parts such as promoter, ribosomal binding site (RBS), coding sequence, and terminator are produced in vitro in a one-pot Golden Gate assembly reaction followed by polymerase chain reaction (PCR) amplification. We demonstrate assembly, cell-free testing of promoter and RBS combinations, as well as characterization of a repressor-promoter pair. By eliminating time-consuming transformation and cloning steps in cells and by taking advantage of modular cloning toolboxes, our cell-free prototyping workflow can produce data for large numbers of new assembled constructs within a single day.
无细胞转录翻译（TXTL）系统已成为测试遗传调控元件和回路的有力工具。无细胞原型制作可以极大地加速合成生物学中新功能的设计-构建-测试-学习循环，特别是在使用易于组装的线性 DNA 模板时。在这里，我们描述了一种 Golden-Gate 辅助的无克隆工作流程，通过从模块化克隆工具盒的基本遗传部件组装转录单元，快速生成用于 TXTL 反应的线性 DNA 模板。由启动子、核糖体结合位点 (RBS)、编码序列和终止子等多个部分组成的功能性 DNA 模板在一锅 Golden Gate 组装反应中体外生成，然后进行聚合酶链反应 (PCR) 扩增。我们展示了启动子和 RBS 组合的组装和无细胞测试，以及对抑制剂启动子对的表征。通过消除细胞中耗时的转化和克隆步骤，并利用模块化克隆工具盒，我们的无细胞原型制作工作流程可以在一天内为大量新组装的构建体生成数据。
