Design and construct modular transcriptional repressors to facilitate the development of living diagnostics

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PROJECT SUMMARY/ABSTRACT Developing multi-input, multi-output genetic circuits has to date been a significant challenge due to the fact that natural genetic systems are only capable to connect one single chemical input to one specific promoter to control gene expression, which poses a major barrier to creating engineered organisms with complex signal response behavior for biomedical applications. The long-term goals of this research team are to establish robust strategies for constructing biological parts of genetic circuits, and to use these parts to implement new cellular functions for practical applications. As important steps toward these goals, Aim #1 is to identify functional modules among TetR family homologs for small molecule sensing and DNA recognition. The central hypothesis is that TetR family repressors are composed of discrete and functional modules for detecting ligand molecules and for interacting with promoters to control gene expression, in which swapping these modules leads to hybrid repressors with new combinations of allosteric and DNA-binding properties. The strategy to achieve Aim #1 is to use bioinformatics approaches to analyze sequence and structure information of TetR homologs, predicting functional protein modules, and to experimentally validate the predictions by assessing the performance of these hybrid repressors during in vivo transcription regulation. For Aim #2, the research team proposes to create a genetic circuit platform that facilitates the use of various organisms for monitoring multiple physiological parameters. The working hypothesis is that modular repressors are able to serve as biosensors in many types of organisms to realize a genetic circuit design for simultaneous detection of multiple physiological changes. This aim is independent from Aim #1 because we can achieve the goal by using modular repressors developed previously from another protein family. The strategy to achieve Aim #2 is to engineer promoters in a range of organisms, in which the resulting promoters can be controlled by the desirable repressors. These engineered inducible expression systems can then be used to implement the circuit design for multiple signal detection. The contribution of this project is expected to be the establishment of a design principle for creating modular parts from TetR homologs and also, the creation of a genetic circuit platform for monitoring multiple physiological parameters by using various organisms. This contribution will be significant because it is expected to release many new possibilities in circuit topologies for engineering organisms for biomedical uses, including the biomonitoring platform developed in this project, which has a great potential to be used for diagnosis of biomedical conditions. Therefore, the proposed work is expected to move the field vertically at both the basic and applied levels.