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Whole-Cell MEP Pathway-Selective Antibacterial Screening Assay Service

 
Catalog No.:  T-2000

Description:  **Please Contact Echelon Biosciences for a custom quote or to discuss process of two part screening**

The Whole-Cell Methylerythritol Phosphate (MEP) Pathway-Selective Antibacterial Screening Assay, described in [1], utilizes a genetically engineered strain of Salmonella typhimurium (CT31-7d) that can utilize either the MEP or the mevalonate (MVA)pathways in order to produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the two essential precursors for all isoprenoids. In the absence of arabinose and MVA, the MEP pathway is utilized for growth. When supplemented with arabinose and MVA, the engineered MVA pathway is activated. Compounds inhibiting MEP pathway growth with no effect on MVA pathway growth are selective for the MEP pathway. Validated using known MEP pathway inhibitors and other antibacterials, the screening platform is more sensitive to MEP pathway inhibitors than wild-type S. typhimurium. Antibacterials targeting other pathways demonstrate similar minimum inhibitory concentrations (MICs) for both MEP and MVA pathway growth.
The Whole-Cell MEP Pathway-Selective Antibacterial Screening Assay is performed in a 96 well plate and consists of two stages:

1. Primary Antibacterial Screen: This screen uses our engineered cell line under growth conditions with only the MEP pathway active to identify initial antibacterial “hits”. Compounds are added in a 96-well plate and screened in duplicate. This screen tests a single concentration (usually 100 µg/mL) or a user determined amount against each pathway. We are able to screen up to 80 compounds per plate in duplicate.
2. Secondary MEP Pathway-Selectivity Screen: Antibacterial “hits” are subjected to dose response analysis evaluating MEP growth in one plate and MVA in a second to determine MICs for both growth conditions. Using CT31-7d and different growth conditions allow us to determine if the antibacterial hit displays specificity toward the MEP pathway. Dose-response experiments are performed on 4 compounds per plate in duplicate for the secondary screen. Fosmidomycin, a known MEP pathway inhibitor, is included in both screens as a positive control. The concentration used (33 μM) is insufficient to kill wild-type bacteria, but is effective against CT31-7d due to heightened MEP pathway sensitivity.


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T-2000-1ea $ 1,400.00

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assay and reagents for drug discovery in lipid signaling pathways