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Introduction

Protocol

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Materials

References

Biochemistry

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Published: October 1st, 2016

DOI:

10.3791/54144

1Synthetic Biology & Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology, 2College of Interdisciplinary & Creative Studies, Konyang University, 3Biosystems and Bioengineering Program, University of Science and Technology
* These authors contributed equally

This protocol describes the rapid and highly sensitive quantification of Förster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Recent improvements in Förster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 °C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

Förster resonance energy transfer (FRET) has been widely used as a biometric sensor to detect small molecules such as sugars, calcium ions, and amino acids1-4. FRET biosensors contain fluorescent proteins, cyan fluorescent proteins (CFPs), and yellow fluorescent proteins (YFPs), which are fused to both ends of periplasmic-binding proteins (PBPs). Sugars bind to PBPs located in the middle of the FRET sensor, causing structural changes to the sensor that subsequently alter the distance and transition dipole orientation of the two fluorescent proteins at either end of the PBPs. This change enables quantitative analysis of sugar content by measuring the ra....

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1. Preparation of Biosensor

  1. Construct the plasmid pET21a(+)-CFP-MBP-YFP-His6 by following the previously-established protocol2.
  2. Inoculate 5 ml of Luria broth (LB) with a single colony of an Escherichia coli DE3 strain and incubate at 37 °C for 16 hr with shaking.
  3. Transfer 1 ml of the O/N culture into a 500-ml flask containing 100 ml LB and incubate at 37 °C in a shaking incubator until the optical density at 600 nm (OD600) reaches 0.5 (about 3 hr).<.......

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To perform quantitative analysis of sugar content using the FRET analyzer, it is necessary to build a fitted curve estimating the target sugar concentration from the observed FRET ratio. Let r define the ratio of the emission intensity of CFP at 480 nm and the emission intensity of YFP generated at 530 nm (Eq. 1).

Equation 1

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This protocol allows rapid and efficient quantification of the sugar content in beverage samples, using a custom-made FRET analyzer7 at an optimal temperature for FRET sensors. The analyzer was designed with a recently-developed, inexpensive 405-nm band ultraviolet-LED as the light source and two photodetectors with a silicon photodiode. This device is more cost-effective than other comparable fluorometers. The device showed high detection sensitivity, specifically when measuring the ratio of two emission wave.......

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This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944) and the KRIBB Research Initiative Program.

....

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Name Company Catalog Number Comments
LB BD #244620
isopropyl β-D-thiogalactoside (IPTG) Sigma I6758
Ampicillin Sigma A9518
Tri-HCl Bioneer C-9006-1
PMSF Sigma 78830
EDTA Bioneer C-9007
DTT Sigma D0632
NaCl Junsei 19015-0350
phosphate-buffered saline (PBS) Gibco 70011-044 0.8% NaCl, 0.02% KCl, 0.0144% Na2HPO4, 0.024% KH2OP4, pH 7.4
SOC 2% tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MGCl2, 20 mM Glucose
Resource Q Amersham Biosciences 17-1177-01 6 × 30 mm anion-exchange chromatography column 
HisTrap HP1 Amersham Biosciences 29-0510-21
Quartz cuvette Sigma Z802875
AKÄKTAFPLC Amersham Biosciences 18-1900-26 a fast protein liquid chromatography (FPLC)
Cary Eclipse VarianInc a fluorescence spectrophotometer
VICTOR   PerkinElmer 2030-0050 a multilabel plate reader
E. coli JM109 (DE3) Promega Electrocompetent cells
A (Beverage) Korea Yakult Co. (Korea) Birak Fermented drinks
B (Beverage) Lotte Foods (Korea) Epro Soft drink
C (Beverage) Lotte Foods (Korea) Getoray Sports drink

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