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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster H16 - Development of NanoBRET Model as a Screening Tool in Mycobacteria

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Keya Patel

Student
University of Illinois at Urbana-Champaign, United States

Co-Author(s)

CL

Chuan Liang

Post-Doctoral Researcher
Texas A&M School of Medicine, United States

Primary Investigator(s)

JC

Jeffery Cirillo

Regents' Professor
Texas A&M School of Medicine, United States

Introduction: Tuberculosis (TB) is a serious bacterial infection that targets the lungs and is one of the top causes of death globally, causing 10.6 million infections and 1.3 million deaths according to the World Health Organization (2021). Despite the extensive research done on TB, there is still little understood about the genetic makeup and how the bacteria evades the immune system. Recently, a novel putative autoinducer molecule was identified in the genus Mycobacterium, but the mechanisms of gene expression and regulation remain relatively unknown. To elucidate many of these functions, we constructed a promoter-less NanoLuc/NanoBRET reporter assay, which uses light to measure gene expression and a transposase as the mechanism of integration.

Materials and

Methods: In order to validate the construct, pJDC332 was electroporated into a strain of m. smegmatis, mc(2)155. After incubation at 37°C for 4 days, chromosomal DNA was isolated. Using a 1:100 dilution series in a 96 well plate, 10 μM furimazine was added and visualized in IVIS Illumina. qPCR was run to determine expression level of the construct against pJDC332 and wild type m. smegmatis and validated by an agarose gel.

Results, Conclusions, and Discussions: All transformed mycobacterial colonies expressed similar levels of bioluminescence with an increase in radiance at a greater CFU, suggesting genes that are controlled by this molecule are expressed at a higher density. Furthermore, the presence of the reporter construct was observed in all colonies, confirmed by PCR of the inserted kanamycin resistance gene in the construct we built. This confirms the NanoLuc/NanoBRET system is a viable tool that will be used to investigate which genes are regulated by the autoinducer molecule. This enables us to create a transformant library in the future to screen for differential expression.

Acknowledgements (Optional): KP was supported by Texas A&M College of Medicine.