The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system.

中文翻译：

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无细胞蛋白质合成作为哺乳动物合成生物学的原型平台。

哺乳动物合成生物学领域正在迅速扩展，用于工程化大型合成基因电路的技术也越来越容易获得。然而，对于哺乳动物细胞工程，传统的组织培养方法缓慢且麻烦，并且不适合用于高通量表征测量。在这里，我们利用HeLa细胞提取物和液体处理自动化技术来进行哺乳动物无细胞蛋白质合成（CFPS）分析，以替代组织培养和基于流式细胞仪的测量。我们的CFPS分析需要几个小时，并且我们已经使用自动声学液体处理技术建立了针对小批量反应的优化方案。作为概念验证，我们描述了CFPS中多种类型的遗传调控，包括T7组成型启动子变体，内部核糖体进入位点（IRES）组成型翻译起始序列变体，CRISPR / dCas9介导的转录抑制和L7Ae介导的翻译抑制。我们的数据表明，用于哺乳动物细胞的简单调控元件可以在CFPS模型系统中快速原型化。