基于原核生物别构转录因子的全新小分子检测方法研究

小分子生物检测方法在环境监测、食品安全和高产菌株筛选等领域具有广泛需求，申请人在体外利用原核生物别构转录因子（aTF）作为识别元件，开发了全新的小分子检测方法。然而该方法尚存在两个亟待解决的问题：输出为负信号和灵敏度有待提高。为了解决上述问题，本项目拟首先利用锌指蛋白与aTF竞争结合DNA位点的方式，建立负信号转换为正信号的新策略。然后再利用Klenow聚合酶与aTF竞争结合DNA位点的方式，将aTF感应小分子的别构效应转化为Klenow聚合酶和切刻内切酶介导的多轮链替换扩增反应，实现信号的放大。最后将转换或放大的信号通过偶联商业化的Alpha系统，实现检测方法的光信号输出。利用以上策略，本项目选取分别感应4-羟基苯甲酸和土霉素的aTF HosA和OtrR，示范开发一系列不同灵敏度的便捷、高效检测方法。本项目的实施将为利用原核生物丰富的aTF资源，开发其它小分子检测方法提供全新的技术支撑。

The development of novel biosensors for highly sensitive, selective, and rapid detection is of paramount importance for environmental monitoring, food safety and strain screening. We have developed a platform for the construction of novel biosensors using allosteric transcription factors (aTF) as recognition elements. However, there are still two shortcomings: the negative output signal and the relative low sensitivity. This project therefore will solve the above problems to meet different detection demands. To transform the negative signal to the positive, zinc finger protein will be employed to compete with aTF on the designed DNA binding sequence, thus inversing the negtive signal. To further amplify the positive signal, klenow polymerase will be used as competitor to aTF, and then a multicycle strand displacement amplification will be performed by klenow polymerase and nicking endonuclease. By this way, the concentration of target chemical is transformed to the amplified DNA signal. Finally, the transformed positive signal or the amplified DNA signal will be outputed by the commercial Alpha (amplified luminescent proximity homogeneous assays) system. As proof-of-concept, the aTF recognition elements HosA and OtrR, responding to 4-hydroxybenzoic acid and oxytetracycline, respectively, will be used to construct biosensors with various sensitivities. Since numerous chemicals are sensed by known aTFs in bacteria, our project will provide practical technical support for the development of other aTF-based biosensors.