Uncovering Design Principles of cis-Antisense Transcription to Build Robust Genetic Switches

摘要：生物系统具有根据环境进行自我调节的巨大潜力。这是通过多年进化过程中复杂的基因调控电路实现的。一种研究相对较少但占主导地位的基因调控形式涉及顺式反义转录，这种转录存在于所有生命王国中。这包括在人类基因组中发现的数千对顺式反义基因，其中许多与威胁生命的疾病有关。当DNA相反链上的两个基因部分重叠时，就会发生顺式反义转录。这种结构可以产生基因产物，这些基因产物可能通过一种称为顺式反义RNA调节(cis-AR)的机制来沉默彼此的表达。此外，该区域DNA上蛋白质的移动和运输也可以通过一种称为转录干扰(TI)的机制来影响基因表达。本项目通过设计顺-AR和TI的机制来揭示基因调控的设计原理，并以有利的方式研究它们的结合，以探索用于高阶生物计算的新型遗传装置的设计。该项目中建立的基因设备可以解决生物技术和生物医学中的重要应用，包括基因治疗、细胞治疗中的细胞命运控制、生物燃料生产过程中事件触发的蛋白质表达，以及创造针对超级细菌的新型抗生素。该项目还将培养一个项目，让K-12和本科生接触研究机会，为研究生提供指导、培训和经验，并提高这两个群体应用他们的工程教育来解决生物技术问题的意识。该项目的长期目标是对顺式反义转录的功能作用有一个基本的了解，并揭示由于顺式反义转录过程中发生的基因调控机制而产生的可调性和生物稳健性的设计原则。该项目将探索TI和cis-AR的不同机制，以识别和表征新的感觉和调节元件，以利用生物部分创建新的合成遗传电路和设备。首先，通过设计RNA聚合酶碰撞、坐鸭干扰和DNA路障的TI机制，自下而上地建立遗传电路，以创造新的生物部分。其次，将通过设计翻译抑制、转录衰减和转录共降解的顺式AR机制来构建新的合成遗传模块。第三，将基于TI和cis-AR的合成遗传模块相结合，开发出能够实现高阶计算的复杂而通用的逻辑门器件。最后，通过结合基于TI、cis-AR的正负反馈环，以及受TI和cis-AR调控的蛋白质的反馈，将以更少的生物部件开发出健壮的合成双稳态记忆开关。该项目将回答基于顺式反义转录的基因调控是否可以产生高度可调的系统输出，范围从简单的一阶反应到生物上复杂的高阶反应，如双稳开关。这项研究的结果还将有助于从根本上理解顺式反义转录调控的生物开关的稳健性和可调性，这将引起生物学家和研究复杂生物基因网络的工程师的兴趣。教育目标将包括开发培训高中生的导师计划，为一年级和二年级学生举办讲习班，让他们了解STEM职业生涯，利用现有技术工具开发研究截屏，以及进行评估，以促进多样性和增加女性在科学领域的入学率。

Abstract: Biological systems have the immense potential for regulating themselves in response to their environment. This is achieved by the intricate gene regulatory circuitry wired over years of evolution. A relatively less studied but predominant form of gene regulation involves cis-antisense transcription that is found across all kingdoms of life. This includes thousands of cis-antisense gene pairs found in the human genome, many implicated in life-threatening diseases. cis-antisense transcription occurs when two genes on opposite strands of DNA have partially overlapping DNA. Such a configuration can give rise to gene products that can potentially silence expression of each other via a mechanism called cis antisense RNA regulation (cis-AR). Further the movement and traffic of proteins on the DNA in this region can also influence gene expression via a mechanism called transcriptional interference (TI). This project uncovers the design principles of gene regulation by engineering the mechanisms of cis-AR and TI, and examines their combination in advantageous ways to explore design of novel genetic devices for higher-order biological computing. The genetic devices built in this project can address important applications in biotechnology and biomedicine including gene therapy, cell fate control in cell therapy, and event-triggered protein expression during biofuel production, and creation of novel antibiotics against superbugs. This project will also cultivate a program to expose K-12 and undergraduate students to research opportunities, provide graduate students with mentorship, training and experience, and both groups to an improved awareness of applying their engineering education to solve biotechnological problems. The long-term goal of this project is to develop a fundamental understanding of functional role of cis-antisense transcription, and uncover the design principles of tunability and biological robustness as a result of gene regulatory mechanisms occurring during cis-antisense transcription. The project will explore different mechanisms of TI and cis-AR to identify and characterize novel sensory and regulatory elements to create novel synthetic genetic circuits and devices using biological parts. Firstly, genetic circuits will be built bottom up by engineering TI mechanisms of RNA polymerase collision, Sitting Duck interference and DNA roadblock to create novel biological parts. Secondly, novel synthetic genetic modules will be built by engineering cis-AR mechanisms of translational inhibition, transcriptional attenuation, and transcript co-degradation. Thirdly, TI and cis-AR based synthetic genetic modules will be combined to develop sophisticated and versatile logic gate devices that can achieve higher order computation. Finally, by combining positive and negative feedback loops based on TI, cis-AR, and feedback from proteins regulated by TI and cis-AR, robust synthetic bistable memory switches will be developed with fewer biological parts. This project will provide answers to the question whether cis-antisense transcription based gene regulation can give rise to a highly tunable system output, ranging from a simple-first order response to biologically complex higher-order response such as bistable switches. The outcomes of the proposed research will also lead to fundamental understanding of robustness and tunability of biological switches regulated by cis-antisense transcription, which will be of interest to biologists as well as engineers investigating complex biological gene networks. The educational goals will include development of mentorship program to train high school students, workshops for freshman and sophomore students to expose them STEM careers, development of research screencasts utilizing available technological tools, as well as performing assessments to promote diversity and increase enrollment of women in science.