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.
