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Designing, Constructing, and Testing of a Dual Fluorescent Genetic Circuit for Mycobacteria Promoter Characterization
Guo, Min
Guo, Min
Abstract
Mycobacterium is a large genus of bacteria that are ubiquitous and diverse. While most members of the genus are non-pathogenic, a few species such as Mycobacterium leprae and Mycobacterium tuberculosis are responsible for fatal diseases such as leprosy and tuberculosis. Other species such as Mycobacterium neoaurum, Mycobacterium smegmatis are used for the industrial synthesis of chemicals such as sterols, and Mycobacterium smegmatis is a good platform to study tuberculosis. Mycobacteria also have clinical applications such as vaccine production and drug screening. Synthetic biology can further the study and application of mycobacteria by designing sophisticated genetic circuits. To design functional circuits, quantitative modeling is critical for researchers to select the proper genetic parts. However, mycobacteria lack a comprehensive promoter library that quantifies promoter characteristics such as promoter strength, induction, and repression. This study aims to construct a promoter library for mycobacteria using a dual fluorescence circuit pSUM7. We have designed and constructed pSUM7 that expresses mCherry while having an insertion site for a test promoter that drives the expression of sfGFP. Based on pSUM7, we have constructed genetic circuits to test 18 different promoters from Mycobacterium smegmatis. We then successfully transformed the test circuits into M. smegmatis, and the fluorescence was measured. Ultimately, problems and limitations of the test circuits such as the use of the sfGFP gene and low plasmid vector copy number are identified and solutions are proposed. The ultimate goal is to develop a standard quantitative method to characterize M. smegmatis promoters and construct a comprehensive promoter library.
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2023-05-01
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2033-05-11
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Biology