Difference between revisions of "Part:BBa K2571001:Experience"

Latest revision as of 01:42, 22 October 2021

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Proposed assay method of BBa_K2571001

iGEM21_UNSW_Australia has proposed the use of the GSH-Glo™ assay (Promega) to detect and quantify the concentration of glutathione produced by gshF. This will verify whether the bifunctional gamma-glutamate cysteine ligase is catalysing the production of glutathione.

The GSH-Glo™ assay (Promega) is a luminescent-based assay used to detect and quantify glutathione in biological samples. We will use this assay to estimate the concentration of glutathione produced by the glutathione system we designed. This assay would be carried out using an E. coli chassis that expresses gshF and control (fluorescent protein).

The GSH-Glo™ assay is performed in two steps. First, the cells are lysed while in the presence of luciferinNT substrate and glutathione S-transferase. The glutathione in the lysed cells interacts with the luciferinNT to form luciferin. Once the luciferin is formed, the Luciferin Detection reagent is added, and light is produced. The amount of light produced is directly proportional to the amount of GSH involved in the reaction (Promega, 2015). Expected results from this assay would be more luminescence produced in the sample expressing gshF, indicating the elevated level of GSH, compared to the control.

References
PROMEGA 2015. GSH-Glo™ Glutathione Assay, Instructions for Use of Products.

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