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Design and Optimization of a Cell-Free Atrazine Biosensor.
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2020-03-03 , DOI: 10.1021/acssynbio.9b00388
Adam D Silverman , Umut Akova , Khalid K Alam , Michael C Jewett 1 , Julius B Lucks 1, 2, 3
Affiliation  

Recent advances in cell-free synthetic biology have spurred the development of in vitro molecular diagnostics that serve as effective alternatives to whole-cell biosensors. However, cell-free sensors for detecting manmade organic water contaminants such as pesticides are sparse, partially because few characterized natural biological sensors can directly detect such pollutants. Here, we present a platform for the cell-free detection of one critical water contaminant, atrazine, by combining a previously characterized cyanuric acid biosensor with a reconstituted atrazine-to-cyanuric acid metabolic pathway composed of several protein-enriched bacterial extracts mixed in a one pot reaction. Our cell-free sensor detects atrazine within an hour of incubation at an activation ratio superior to previously reported whole-cell atrazine sensors. We also show that the response characteristics of the atrazine sensor can be tuned by manipulating the ratios of enriched extracts in the cell-free reaction mixture. Our approach of utilizing multiple metabolic steps, encoded in protein-enriched cell-free extracts, to convert a target of interest into a molecule that can be sensed by a transcription factor is modular. Our work thus serves as an effective proof-of-concept for a scheme of "metabolic biosensing", which should enable rapid, field-deployable detection of complex organic water contaminants.

中文翻译:

无细胞At去津生物传感器的设计和优化。

无细胞合成生物学的最新进展刺激了体外分子诊断技术的发展,该技术可作为全细胞生物传感器的有效替代品。但是,用于检测人为有机水污染物(例如农药)的无细胞传感器稀疏,部分原因是很少有经过表征的天然生物传感器可以直接检测此类污染物。在这里,我们提出了一个平台,该平台通过结合先前表征的氰尿酸生物传感器与重构的阿特拉津-氰尿酸代谢途径(由几种富含蛋白质的细菌提取物混合而成),用于无细胞检测一种关键水污染物阿特拉津。一锅反应。我们的无细胞传感器在孵育一小时内即可检测到at去津,其活化率优于先前报道的全细胞at去津传感器。我们还表明,可以通过控制无细胞反应混合物中浓缩提取物的比例来调节the去津传感器的响应特性。我们利用富含蛋白质的无细胞提取物中编码的多个代谢步骤将目标靶标转化为可以被转录因子感知的分子的方法是模块化的。因此,我们的工作是“代谢生物传感”方案的有效概念证明,该方案应能够快速,现场部署地检测复杂的有机水污染物。将感兴趣的目标转化为可以被转录因子感知的分子是模块化的。因此,我们的工作是“代谢生物传感”方案的有效概念证明,该方案应能够快速,现场部署地检测复杂的有机水污染物。将目标靶标转化为可以被转录因子感知的分子是模块化的。因此,我们的工作是“代谢生物传感”方案的有效概念证明,该方案应能够快速,现场部署地检测复杂的有机水污染物。
更新日期:2020-03-03
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