基于AFM力谱与SPR技术的启动子强度高效表征策略的研究

Promoters are important regulatory elements for microorganism metabolism. The promoter characterization method applied right now is based on the reporting gene expression level. However, there are some problems with this method, such as requiring multiple procedures, time consuming, (usually several days), being affected by the microorganism growth stage and other regulatory elements, etc. As a result, the quantification of promoter activity is inefficient, and the rational design of well-controlled and accurate promoter is quite challenging. The most fundamental and key step of promoter regulation reaction is the molecular interaction between promoters and RNA polymerase, the recognition and binding between them determines the activity of the promoters, thus the molecular interaction between these two molecules can provide a new perspective to characterize promoter activity. Hence, in this study, via atomic force microscope (AFM) based force spectrometry and surface plasmon resonance (SPR) analysis, the interaction between promoters of E. Coli and Corynebacterium glutamicum and RNA polymerase will be studied in vitro. The information regarding the biophysics and kinetics of this biochemical reaction including binding force, binding probability, affinity, and binding/dissociation rate constant etc will be collected. The above results will be validated by measuring the transcription and expression level of the reporting gene; and furthermore, the promoter information obtained at different levels with multiple approaches will be correlated with each other via principle component analysis. This study will provide a new approach for promoter sequence rational design and promoter activity screening.

启动子是重要的微生物代谢调控单元，也是合成生物学的重要元件之一。采取报告基因表达间接表征其强度的方法存在操作步骤多、周期长、易受宿主生理状态及非转录水平调控干扰等问题，不利于启动子的理性设计、高通量筛选及其元件化处理。启动子与RNA聚合酶的交互作用是启动子调控反应中最为基础和关键的环节，决定了启动子的强度，可能成为表征其性质的新角度。本项目拟通过体外原子力显微镜（AFM）力谱分析和表面等离子共振技术（SPR），从生物物理学和生化反应动力学角度直观的解析大肠杆菌和谷氨酸棒杆菌不同启动子与RNA聚合酶之间结合/解离过程中的交互作用；进而采集其结合力、结合概率、亲和度、结合/解离速度常数等基本参数；并利用主成分分析等策略关联其体内报告基因的转录及表达量，构建基于体外交互作用分析的启动子强度表征新策略，为不同强度的启动子元件设计、初步筛选提供高效的方法。

启动子是调控转录反应的重要的基因表达顺式调控元件。随着代谢工程及合成生物学的发展，对启动子强度表征方法的准确性和效率提出了更高的要求。由于目前常用的基于报告基因表达量的表征方法操作步骤繁琐，受到其他调控元件的干扰以及细胞生理状态的影响，效率较低，准确性差，因此亟待建立一种新型的、直接的、高效的启动子定量分析方法。项目首先以噬菌体T7转录系统为模式研究对象，建立了可用于AFM力谱分析的基底及探针的修饰方法，提高交互作用分析的准确性。在此基础上，对T7启动子与T7 RNA聚合酶(RNAp)交互作用开展基于原子力显微镜(Atomic Force Microscope, AFM)力谱分析以及表面等离子体共振（Surface Plasmon Resonance，SPR）技术的分子交互作用研究，建立体外分析启动子强度的流程。基于此平台，以大肠杆菌的启动子为研究对象，通过定点改造，得到在启动子核心区域关键位点存在突变的一系列启动子，分析这些启动子与大肠杆菌RNAp之间的交互作用。项目实施中采集大量AFM力-距离曲线，结合统计学分析，分析了启动子一系列突变启动子与RNAp间的交互作用力、解离距离及结合概率、亲和度等参数。通过与体内报告基因表达量的强度表征结果进行对比，验证了启动子体外交互作用分析的有效性。项目开发的这一方法将补充目前启动子表征方法，提供直接的、体外的启动子强度分析方法，为启动子等标准化基因表达元件的表征提供新策略。