Tuning Cell-Free Composition Controls the Time Delay, Dynamics, and Productivity of TX-TL Expression,ACS Synthetic Biology

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Tuning Cell-Free Composition Controls the Time Delay, Dynamics, and Productivity of TX-TL Expression
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-09-09 , DOI: 10.1021/acssynbio.1c00136
Grace E Vezeau ₁ , Howard M Salis _{1,

2}

Affiliation

The composition of cell-free expression systems (TX-TL) is adjusted by adding macromolecular crowding agents and salts. However, the effects of these cosolutes on the dynamics of individual gene expression processes have not been quantified. Here, we carry out kinetic mRNA and protein level measurements on libraries of genetic constructs using the common cosolutes PEG-8000, Ficoll-400, and magnesium glutamate. By combining these measurements with biophysical modeling, we show that cosolutes have differing effects on transcription initiation, translation initiation, and translation elongation rates with trade-offs between time delays, expression tunability, and maximum expression productivity. We also confirm that biophysical models can predict translation initiation rates in TX–TL using Escherichia coli lysate. We discuss how cosolute composition can be tuned to maximize performance across different cell-free applications, including biosensing, diagnostics, and biomanufacturing.

中文翻译：

调整无细胞成分可控制 TX-TL 表达的时间延迟、动力学和生产力

无细胞表达系统 (TX-TL) 的组成通过添加大分子拥挤剂和盐来调整。然而，这些共溶物对个体基因表达过程动力学的影响尚未量化。在这里，我们使用常见的共溶物 PEG-8000、Ficoll-400 和谷氨酸镁对基因构建体库进行动态 mRNA 和蛋白质水平测量。通过将这些测量与生物物理建模相结合，我们表明共溶物对转录起始、翻译起始和翻译延伸率具有不同的影响，并在时间延迟、表达可调性和最大表达生产力之间进行权衡。我们还证实，生物物理模型可以使用大肠杆菌预测 TX-TL 中的翻译起始率裂解物。我们讨论了如何调整共溶质成分以最大限度地提高不同无细胞应用的性能，包括生物传感、诊断和生物制造。

更新日期：2021-10-15

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