Transcription factor sensor system for parallel quantification of metabolites on-chip.
Transcription factor sensor system for parallel quantification of metabolites on-chip.
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DOI：
10.1021/ac503269m
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发表时间：
2014-12
影响因子：
7.4
通讯作者：
Simon Ketterer;Désirée Hövermann;R. J. Guebeli;Frauke Bartels-Burgahn;D. Riewe;T. Altmann;Matias D. Zurbriggen;Björn H. Junker;W. Weber;Matthias Meier
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文献类型：
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作者：
Simon Ketterer;Désirée Hövermann;R. J. Guebeli;Frauke Bartels-Burgahn;D. Riewe;T. Altmann;Matias D. Zurbriggen;Björn H. Junker;W. Weber;Matthias Meier
在更高的吞吐量下，对细胞代谢物的鉴定和定量的需求稳步增长，这带来了对新的分析技术的需求。在这里，我们开发了一种合成生物传感器系统，用于定量测定生物细胞样本中的代谢物。为此，细菌转录因子被开发出来，它们与调节DNA元件结合或解离，以响应细胞代谢物浓度范围内的生理变化。具有代表性的细菌丙酮酸脱氢酶(PDHR)、海藻糖(TreR)和L精氨酸(ArgR)阻遏蛋白被功能化以检测溶液中丙酮酸、海藻糖-6-磷酸(T6P)和精氨酸的浓度。对于每个转录因子，确定了代谢物与DNA的相互结合行为、它们的作用范围和正交性。通过在微流控大规模集成(MLSI)芯片平台上集成代谢传感器系统，实现了高通量、并行处理和自动化。为了验证集成代谢传感器系统的功能，我们测量了拟南芥莲座体细胞内丙酮酸和植物信号分子T6P的浓度日变化。转录因子传感器系统具有通用性，可在微流控芯片上扩展。
Steadily growing demands for identification and quantification of cellular metabolites in higher throughput have brought a need for new analytical technologies. Here, we developed a synthetic biological sensor system for quantifying metabolites from biological cell samples. For this, bacterial transcription factors were exploited, which bind to or dissociate from regulatory DNA elements in response to physiological changes in the cellular metabolite concentration range. Representatively, the bacterial pyruvate dehydrogenase (PdhR), trehalose (TreR), and l-arginine (ArgR) repressor proteins were functionalized to detect pyruvate, trehalose-6-phosphate (T6P), and arginine concentration in solution. For each transcription factor the mutual binding behavior between metabolite and DNA, their working range, and othogonality were determined. High-throughput, parallel processing, and automation were achieved through integration of the metabolic sensor system on a microfluidic large-scale integration (mLSI) chip platform. To demonstrate the functionality of the integrated metabolic sensor system, we measured diurnal concentration changes of pyruvate and the plant signaling molecule T6P within cell etxracts of Arabidopsis thaliana rosettes. The transcription factor sensor system is of generic nature and extendable on the microfluidic chip.