Booth Id:
PLNT037
Category:
Plant Sciences
Year:
2021
Finalist Names:
Krishnan, Aravind (School: Hillsborough High School)
Abstract:
Harvesting Limulus polyphemus (horseshoe crabs) to produce the Limulus amoebocyte lysate endotoxin assay for medical devices, pharmaceuticals, and drinking water is ravaging coastal ecosystems. This project develops a more sustainable and cost-effective quantitative endotoxin assay. The PAMP-Triggered Immunity response of Arabidopsis thaliana to pathogen-associated molecular patterns (PAMPs) was harnessed for quantitative determination of endotoxin presence based on induction of the FRK1 gene. Transgenic FRK1-LUC A. thaliana were used to express luciferase (LUC) upon activation of FRK1 by exposure to gram-negative bacteria. Luciferase enzyme control was first tested via plate reader, and luminescence produced by varying enzyme quantities was recorded. Next, E. coli ranging from 6*10^5 to 10^3 CFU/mL were infiltrated into the leaf apoplastic space of FRK1-LUC and wild-type plants. Luminescence of infiltrated leaf discs was measured after adding luciferin substrate to reconstitute functional luciferase. The results showed a direct relationship between bacteria concentration and luminescence. The FRK1-LUC luminescence versus endotoxin concentration results yielded a formula of y = 1518e^0.0196x. (R^2 = 0.937). Data suggest this assay achieves a sensitivity down to 18 endotoxin units/mL (p < 0.001, SEM = 1.76%). To determine specificity, the SeeSAR software was used to calculate binding affinities of endotoxin (LPS) and flg22 with several receptors. Results indicate high specificity in LORE-LPS binding, signifying the luminesce results were caused by LPS concentration. This method’s sensitivity and specificity combined with its elimination of environmental impacts and low cost make it a promising new bacterial endotoxin assay for pharmaceutical and drinking water testing.
Awards Won:
Serving Society Through Science: Second Award of $500
Third Award of $1,000