All data (main text &amp; supplementary) presented in '<i>In vitro </i> transcription-based biosensing of glycolate for prototyping of a complex enzyme cascade'

<i>This dataset contains all data published in the bioRxiv pre-print: </i><i>In vitro</i> transcription-based biosensing of glycolate for prototyping of a complex enzyme cascade (https://doi.org/10.1101/2024.04.26.591264). See the abstract as description below.<br><i>In vitro</i> metabolic systems allow the reconstitution of natural and new-to-nature pathways outside of their cellular context and are of increasing interest in bottom-up synthetic biology, cell-free manufacturing and metabolic engineering. Yet, the prototyping of such <i>in vitro</i> networks is very often restricted by time- and cost-intensive analytical methods. To overcome these limitations, we sought to develop an <i>in vitro</i> transcription (IVT)-based biosensing workflow that offers fast results at low-cost, minimal volumes and high-throughput. As a proof-of-concept, we present an IVT biosensor for the so-called CETCH cycle, a complex <i>in vitro</i> metabolic system that converts CO₂ into glycolate. To quantify glycolate production, we constructed a sensor module that is based on the glycolate repressor GlcR from <i>Paracoccus denitrificans</i>, and established an IVT biosensing off-line workflow that allows to measure glycolate from CETCH samples from the µM to mM range. We characterized the influence of different cofactors on IVT output and further optimized our IVT biosensor against varying sample conditions. We show that availability of free Mg²⁺ is a critical factor in IVT biosensing and that IVT output is heavily influenced by ATP, NADPH and other phosphorylated metabolites frequently used in <i>in vitro</i> systems. Our final biosensor is highly robust and shows an excellent correlation between IVT output and classical LC-MS quantification, but notably at ~10-fold lowered cost and ~10 times faster turnover time. Our results demonstrate the potential of IVT-based biosensor systems to break current limitations in biological design-build-test cycles for the prototyping of individual enzymes, complex reaction cascades and <i>in vitro</i> metabolic networks.本数据集包含发表于bioRxiv预印本《基于体外转录的乙醇酸生物传感用于复杂酶级联原型开发》（https://doi.org/10.1101/2024.04.26.591264）的全部数据，下文以该预印本摘要作为数据集说明。 体外（in vitro）代谢系统可在细胞外环境中重构天然及人工合成代谢通路，在自下而上合成生物学、无细胞制造及代谢工程领域的研究关注度与日俱增。然而，此类体外代谢网络的原型开发常受限于耗时且成本高昂的分析检测方法。为突破这些瓶颈，我们开发了一种基于体外转录（in vitro transcription, IVT）的生物传感工作流程，可实现快速检测结果、低成本、低反应体积及高通量分析。 作为概念验证，我们针对名为CETCH循环的复杂体外代谢系统构建了一款体外转录生物传感器，该系统可将二氧化碳（CO₂）转化为乙醇酸。为量化乙醇酸产量，我们搭建了基于脱氮副球菌（Paracoccus denitrificans）乙醇酸阻遏蛋白GlcR的传感模块，并建立了体外转录离线生物传感工作流程，可实现从微摩尔（µM）至毫摩尔（mM）浓度范围内的CETCH样品乙醇酸定量检测。 我们表征了不同辅因子对体外转录输出信号的影响，并针对不同样品条件进一步优化了该体外转录生物传感器。研究发现，游离镁离子（Mg²+）的可用性是体外转录生物传感的关键影响因素，且体外转录输出信号显著受ATP、NADPH及其他常用于体外代谢系统的磷酸化代谢物的调控。 最终构建的生物传感器具备优异的鲁棒性，其体外转录输出信号与经典LC-MS定量结果呈现极佳的线性相关性，且检测成本仅为传统方法的约1/10，周转时间缩短约90%。本研究结果证实，基于体外转录的生物传感系统有望突破当前生物设计-构建-测试循环中针对单个酶、复杂反应级联及体外代谢网络进行原型开发的现有局限。