The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

中文翻译：

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差异优化的无细胞缓冲液能够从外切核酸酶缺陷提取物中未受保护的线性 DNA 中实现稳健表达

在无细胞系统中使用线性 DNA 模板有望加速合成基因回路的原型设计和工程。一个关键的挑战是线性模板会被细胞提取物中存在的核酸外切酶快速降解。目前的方法通过添加核酸外切酶抑制剂和 DNA 结合蛋白来保护线性 DNA 来解决这个问题，这需要额外的时间和资源密集型步骤。在这里，我们从大肠杆菌中删除了recBCD核酸外切酶基因簇BL21 基因组。我们展示了所得的无细胞系统，带有专门针对线性 DNA 优化的缓冲液，可以在线性 DNA 模板中实现接近质粒水平的 σ70 启动子的表达，而无需采用额外的保护策略。在缓冲液校准步骤中使用线性或质粒 DNA 模板时，使用线性 DNA 获得的最佳谷氨酸钾浓度始终低于使用质粒 DNA 获得的相同提取物的浓度。我们证明了核酸外切酶缺陷提取物在七个不同批次和两个不同实验室的各种实验条件下的稳健性。最后，我们说明了ΔrecBCD的使用两种应用的提取物：立足点开关表征和酶筛选。我们的工作为在无细胞系统中使用线性 DNA 模板提供了一种简单、高效且具有成本效益的解决方案，并强调了为最终实验设置专门定制缓冲液组成的重要性。我们的数据还表明，类似的外切核酸酶删除策略可以应用于适用于无细胞合成生物学的其他物种。