Recent advances in cell-free systems have opened up new capabilities in synthetic biology from rapid prototyping of genetic circuits and metabolic pathways to portable diagnostics and biomanufacturing. A current bottleneck in cell-free systems, especially those employing non-E. coli bacterial species, is the required use of plasmid DNA, which can be laborious to construct, clone, and verify. Linear DNA templates offer a faster and more direct route for many cell-free applications, but they are often rapidly degraded in cell-free reactions. In this study, we evaluated GamS from λ-phage, DNA fragments containing Chi-sites, and Ku from Mycobacterium tuberculosis for their ability to protect linear DNA templates in diverse bacterial cell-free systems. We show that these nuclease inhibitors exhibit differential protective activities against endogenous exonucleases in five different cell-free lysates, highlighting their utility for diverse bacterial species. We expect these linear DNA protection strategies will accelerate high-throughput approaches in cell-free synthetic biology.

中文翻译：

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保护无细胞表达系统中的线性 DNA 模板免受各种细菌的侵害。

无细胞系统的最新进展为合成生物学开辟了新的能力，从遗传电路和代谢途径的快速原型设计到便携式诊断和生物制造。当前无细胞系统的瓶颈，尤其是那些使用非大肠杆菌细菌物种的系统，是需要使用质粒 DNA，而质粒 DNA 的构建、克隆和验证可能很费力。线性 DNA 模板为许多无细胞应用提供了更快、更直接的途径，但它们通常在无细胞反应中迅速降解。在这项研究中，我们评估了来自 λ 噬菌体的 GamS、含有 Chi 位点的 DNA 片段和来自结核分枝杆菌的Ku因为它们能够保护不同细菌无细胞系统中的线性 DNA 模板。我们表明，这些核酸酶抑制剂在五种不同的无细胞裂解物中对内源性外切核酸酶表现出不同的保护活性，突出了它们对不同细菌物种的效用。我们预计这些线性 DNA 保护策略将加速无细胞合成生物学中的高通量方法。