Engineering Of Orthogonal Synthetic Biology Tools Based On Directed Evolution Approaches

Along with the development of biotechnology, the main research of biology is shifting its emphasis on design and construction of complex biological systems rather than observational experiments. This shifting enables us to program cells and generate much needed architectures and functions. During this time, synthetic biology and systems biology have done many impressive works. As a newly emerged discipline, synthetic biology mainly focuses on how to rationally design cellular reactions based on engineering principles and approaches, and to acquire information as much as possible of how to construct biological systems, or to be specific, mini-pathways. Those design principles are also applicable in biotechnology, biosensing, medical research. At the early stage of synthetic biology, researches on constructing genetic circuits and programming them into cells to perform certain functions were successful. However, the design principles are slightly simple, which are not robust when deal with more complicated gene networks. It is quite necessary to further optimize basic biological regulatory parts to design more complicated gene systems which require more predictable components. Therefore, this work focuses on two basic levels to generate orthogonal synthetic biology tools:1. Rewiring protein degradation system to generate orthogonal pairs Many synthetic biology researches have adopt the protein degradation system ClpXP-SsrA. It is known that degradation systems without interaction between each other help design precise gene networks. Previously, two SsrA variants were indentified to be disconnected with wildtype ClpXP. This work built a ClpX mutant library, and tried to identify needed ClpX variants by dual selection and screening. However, these methods are prone to generate false positive results, which make it hard to perform high-throughput selection. More rational design and laborious work are required to identify needed individuals from a large group of mutants.2. Engineering orthogonal transcriptional factorsThe fundamental investigation of synthetic biology comes from the design and engineering transcriptional factors and cognate genes. Well-defined transcriptional factor-gene pairs will largely promote the work of building high-order and programmable systems. Previous studies have shown that the transcriptional factor LuxR of bacterial quorum sensing system, which is responsible for the virulence, self-organization, social communication of bacteria, especially many kinds of pathogenic bacteria, could recognize many downstream promoters based on the fact that many of those promoters contain one kind of specific DNA sequence called lux box. Therefore, engineering new type of LuxR which operates parallel with the wildtype form will be helpful to construct gene regulatory networks and gain better understanding of how bacteria build the quorum sensing hierarchies. This work evolves the C terminal domain of Lux based on directed evolution, rendering new form of LuxR to recognize lux box variants that are not recognized by wildtype LuxR. Eventually, orthogonal transcriptional factor-gene pairs are generated. These orthogonal pairs will be applicable in constructing synthetic biology networks and understanding the hierarchy of quorum sensing system.This work have engineered some basic biological parts and made them operating parallel with the original ones. Also, this work explores the principles and methods of directed evolution, an important approach in synthetic biology.