Utilizing Whole-Cell Biosensors to Measure Ionic Mercury in Water Samples

Summary

This article presents protocols for utilizing two whole-cell biosensors (Mer-Blue and Mer-RFP) to detect ionic mercury. By providing detailed procedures for their operation and output analysis, the study aims to facilitate broader adoption and further development of these technologies for monitoring specific pollutants.

Abstract

Whole-cell biosensors (WCBs) are instrumental platforms for discovering and characterizing regulatory elements and advancing bioengineering. They also hold immense promise for environmental and food monitoring. While significant efforts have been invested in enhancing their sensitivity and portability, the standardization of their handling and data analysis remains relatively underdeveloped. This article presents a comprehensive guide to utilizing two recently developed WCBs, Mer-Blue and Mer-RFP, proven capable of detecting ionic mercury at levels below the World Health Organization's drinking water limits. The protocols detailed herein encompass microbial culture preparation, sensor calibration, data acquisition, and analysis. For the fluorometric Mer-RFP biosensor, a novel biosynthesis allocation theorem is employed to identify the time interval for reliable and accurate dose-response measurements. For the colorimetric Mer-Blue biosensor, a low-cost camera setup enables rigorous measurements in settings lacking expensive spectrophotometers and fluorimeters, facilitating decentralized pollution monitoring. The procedures used for testing freshwater samples are described, and the limitations of these biosensors with respect to sample types are discussed. By sharing these handling and analysis techniques, we encourage broader research groups to adopt and improve these biological devices for developing effective environmental monitoring solutions. Ultimately, this research aims to facilitate the widespread adoption of biosensor technologies within the environmental science community, contributing to more effective and efficient monitoring of trace element pollution in various ecosystems.

Introduction

As concerns grow over environmental pollution and the safety of the water and food supply, biosensors have emerged as an attractive, affordable alternative to traditional analytical chemistry methods. Heavy metals are highly toxic pollutants that have become increasingly prevalent due to human activities^1,2,3,4. However, conventional detection methods⁵ are often costly and inaccessible.

Mercury-sensitive WCBs harness the remarkable specificity and sensitivity of the MerR transcription facto....

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Preparing ionic mercury solutions

1. Weigh a small mass of HgBr₂ powder (typically between 0.1 and 0.2 g) using appropriate protective gloves and a mask to prevent inhalation and skin exposure.
2. Calculate the amount of water needed to obtain a 4 mM solution by dissolving the measured mass of solid powder in ddH₂O using the formula:
   

Results

Both biosensors exhibit no detectable baseline expression of their respective reporter proteins under mercury-free conditions. Mer-Blue cultures typically begin to display visible coloration to the naked eye between 6-8 h post-inoculation at HgBr₂ concentrations of 25 nM and above. When properly conducted, titration experiments using either Mer-RFP or Mer-Blue yield responses that fit a Hill function with a Hill coefficient close to 1, indicating non-cooperative binding behavior²³.

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Discussion

The biosensor circuits utilized here are identical, only differing in the reporter protein. Both are entirely encoded on high-copy plasmids, enabling their deployment in Escherichia coli cells of various strains. While functional in E. coli B strain cells, a stronger colored signal from Mer-Blue was observed in K-12 strains, specifically DH5α and chemically competent E. coli cells¹⁵.

We observed that achieving a reliable output requires a subs.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3 mm thick multiplex wood	Many distributors
5 V USB cable	Many distributors
96-well Clear Flat Bottom Polystyrene TC-treated Microplates	Corning	3599
Agar powder	HiMedia	GRM026
Ampicillin	Gold Biotechnology, Inc.	A-301-100
CaCl2	Millipore	208290
Casamino Acids	Calbiochem	2040OP
D-(+)-Glucose	Central Drug House (P) Ltd.	506250
ESP32-CAM-MB 2640 Bluetooth WiFi Camera Module USB to Serial Port Development Board Auto Download (Type C)	Many distributors
Glass culture tubes	Corning	9825
HgBr2, Mercury(II) bromide	Merck	200085
HgCl2, Mercury(II) chloride	Merck	21465
Luria broth (Miller)	Sigma-Aldrich	L3522
M9 minimum salts	Sigma-Aldrich	M6030
MgSO4	HiMedia	GRM684
NeoPixel Stick - 8 x 5050 RGB LED with Integrated Drivers	Adafruit	1426
Plasmid: pUC-Mer-Blue	Our group	-	Full sequence available in https://www.mdpi.com/article/10.3390/bios14050246/s1
Plasmid: pUC-Mer-RFP	Our group	-	Full sequence available in https://www.mdpi.com/article/10.3390/bios14050246/s1
Syringe filters, hydrophilic, pore size 0.22 microns	Millipore	SLGV004SL	For sterilizing environmental samples.