Publication: Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter
Issued Date
2003-12-01
Resource Type
ISSN
00664804
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2-s2.0-0344443250
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Mahidol University
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SCOPUS
Bibliographic Citation
Antimicrobial Agents and Chemotherapy. Vol.47, No.12 (2003), 3682-3687
Suggested Citation
Chartchai Changsen, Scott G. Franzblau, Prasit Palittapongarnpim Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter. Antimicrobial Agents and Chemotherapy. Vol.47, No.12 (2003), 3682-3687. doi:10.1128/AAC.47.12.3682-3687.2003 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/21000
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Title
Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter
Abstract
The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc2155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.