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

揭示顺式反义转录的设计原理以构建稳健的基因开关

• 批准号: 1714564
• 负责人: Anushree Chatterjee
• 金额: $ 50万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1714564&HistoricalAwards=false
• 关键词: Uncovering; Design; Principles; cis; Antisense

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.

翻译后摘要：生物系统具有巨大的潜力，调节自己，以应对他们的环境。这是通过复杂的基因调控电路在多年的进化中实现的。 一种研究相对较少但占主导地位的基因调控形式涉及在所有生命王国中发现的顺式反义转录。这包括在人类基因组中发现的数千个顺式-反义基因对，其中许多与危及生命的疾病有关。当DNA相对链上的两个基因具有部分重叠的DNA时，发生顺式反义转录。这种构型可以产生基因产物，这些基因产物可以通过称为顺式反义RNA调节（cis-AR）的机制潜在地沉默彼此的表达。此外，蛋白质在该区域的DNA上的移动和运输也可以通过称为转录干扰（TI）的机制影响基因表达。该项目通过工程化顺式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组合，以探索用于高阶生物计算的新型遗传装置的设计。该项目中构建的遗传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的事件触发蛋白质表达，以及针对超级细菌的新型抗生素的创造。该项目还将培养一项计划，使K-12和本科生获得研究机会，为研究生提供指导，培训和经验，并提高两个群体对应用工程教育解决生物技术问题的认识。本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，并揭示顺式反义转录过程中基因调控机制的可调性和生物鲁棒性的设计原则。该项目将探索TI和顺式AR的不同机制，以识别和表征新的感觉和调节元件，从而使用生物部件创建新的合成遗传电路和装置。首先，通过RNA聚合酶碰撞、坐鸭干扰和DNA路障等TI机制自下而上构建遗传电路，创造新的生物部件。其次，将通过工程化的翻译抑制、转录衰减和转录物共降解的顺式AR机制来构建新的合成遗传模块。第三，基于TI和顺式AR的合成遗传模块将被结合以开发能够实现更高阶计算的复杂和通用的逻辑门器件。最后，通过结合基于TI、顺式-AR的正反馈和负反馈回路以及来自由TI和顺式-AR调节的蛋白质的反馈，将开发出具有更少生物学部分的稳健的合成记忆开关。这个项目将提供答案的问题是否顺式反义转录为基础的基因调控可以引起一个高度可调的系统输出，从一个简单的一阶响应，生物学复杂的高阶响应，如开关。拟议研究的结果也将导致顺式反义转录调控的生物开关的鲁棒性和可调性的基本理解，这将是生物学家以及研究复杂生物基因网络的工程师感兴趣的。教育目标将包括制定导师计划以培训高中生，为大一和大二学生举办研讨会以使他们了解STEM职业，利用现有技术工具开发研究屏幕，以及进行评估以促进多样性并增加女性在科学领域的入学率。

项目成果

Engineering Transcriptional Interference through RNA Polymerase Processivity Control

通过 RNA 聚合酶持续过程控制工程转录干扰

• DOI: 10.1021/acssynbio.0c00534
• 发表时间: 2021
• 期刊: ACS Synthetic Biology
• 影响因子: 4.7
• 作者: O’Connor, Nolan J.;Bordoy, Antoni E.;Chatterjee, Anushree
• 通讯作者: Chatterjee, Anushree

Design of a De Novo Aggregating Antimicrobial Peptide and a Bacterial Conjugation-Based Delivery System

从头聚集抗菌肽和基于细菌缀合的递送系统的设计

• DOI: 10.1021/acs.biochem.8b00888
• 发表时间: 2019
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Collins, Logan T.;Otoupal, Peter B.;Campos, Jocelyn K.;Courtney, Colleen M.;Chatterjee, Anushree
• 通讯作者: Chatterjee, Anushree

Construction of Two-Input Logic Gates Using Transcriptional Interference

• DOI: 10.1021/acssynbio.9b00321
• 发表时间: 2019-10-01
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Bordoy, Antoni E.;O'Connor, Nolan J.;Chatterjee, Anushree
• 通讯作者: Chatterjee, Anushree