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FRK1-LUC Arabidopsis thaliana: Novel Endotoxin Assay Harnessing PAMP-Triggered Immunity

JSHS · 2020

Aravind Krishnan, Githa Krishnan, Eric Lam · Hillsborough High School Hillsborough, NJ

Overview

The current practice of harvesting Limulus polyphemus (horseshoe crab) to use in the Limulus amoebocyte lysate (LAL) assay for the detection of bacterial endotoxins in medications and medical devices is ravaging coastal ecosystems. Pharmaceutical standards require these products to have an endotoxin concentration below specified thresholds to avoid human septic shock. This project creates an endotoxin assay with similar specificity and sensitivity to existing methods while minimizing cost and environmental impact. The PAMP-Triggered Immunity (PTI) response of Arabidopsis thaliana to pathogen-associated molecular patterns (PAMPs) allows quantitative determination of endotoxin presence based on induction of the FRK1 promoter. Transgenic FRK1-LUC Arabidopsis thaliana were germinated to express luciferase upon activation by exposure to gram-negative bacteria. Luciferase control was first tested via plate reader, luminescence recorded with varying enzyme quantities. This provided expected luminescence levels based on the amount of luciferase produced. Next, E. coli ranging from 6*105 to 103 CFU/mL, as well as flg22 peptide (positive control), were infiltrated into the leaf apoplastic space of FRK1-LUC and wild-type plants. Luminescence of infiltrated leaf discs from plant samples was measured after adding luciferin substrate to treated tissues to reconstitute functional luciferase. This determined correlation of infiltrated bacteria concentration and luminescence, a product of the luciferin-luciferase reaction. Data suggests this assay achieves endotoxin detection specificity down to ~20 endotoxin units (EU)/mL. This provides a promising indication that this method can be further investigated to yield results for lower endotoxin concentrations as well as to demonstrate greater specificity.

Competition history

  • JSHS 2020 Category not listed

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Source: Junior Science and Humanities Symposium

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