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

University Of Colorado At Boulder, Boulder CO

Investigators

Abstract

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.