Development of high-performance copper biosensor based on synthetic biology


Overview

With the progress of industrialization and urbanization, water pollution has become increasingly severe. Fish ponds, as crucial sites for aquaculture, frequently face issues of wastewater influx and drug overuse. This not only leads to excessive copper levels in the water but also poses serious threats to the health of both the farmed fish and the humans who ultimately consume them[1]. We are particularly concerned about heavy metal pollution in water bodies, especially in fisheries.

Currently, chemical detection methods for heavy metal ions, despite their low detection limits and high specificity, are costly and require specialized equipment, making them often inaccessible in underdeveloped regions[2]. Synthetic biology-based heavy metal biosensors, on the other hand, offer low-cost and easy-to-use alternatives with broad application prospects. However, the current biosensors for heavy metal detection lack sensitivity and accuracy. In this project, we focus on developing a high-performance biosensor for detecting copper.

In previous iGEM competitions, several teams have employed promoters directly or indirectly sensitive to copper, such as promoters of copA, marO, cueO genes[3], the CueR regulated copper inducible promoter (BBa_K190017)[4], and copAP[7]. Additionally, many proteins with copper-specific binding have been utilized, such as the high-affinity copper-binding protein CueR[4, 7], yeast metallothionein CUP1[3], and MymT[6]. We aim to construct a high-performance copper biosensing system by synthesizing a high-throughput library of copper-sensitive promoter mutants and characterizing different promoter/RBS combinations for their regulation of copper-binding protein expression. This system will be characterized by its specificity for copper binding, high sensitivity, and fast response time.


Plasmid Diagram
Transcriptional unit 1
Fig1(a) Schematic map of Transcriptional unit 1 : BBa_J23119 - BBa_B0030 - cueR - BBa_B1006.
Transcriptional unit 2
Fig1(b) Schematic map of Transcriptional unit 2 : BBa_K190017 - BBa_B0034 - mVenusNB -BBa_B0015.
Transcription unit 3
Fig1(c) Schematic map of Transcription unit 3: Promoter variants - RBS variants - cueR - BBa_B1006

References

[1] Izydorczyk, G., Mikula, K., Skrzypczak, D., Moustakas, K., Witek-Krowiak, A. and Chojnacka, K. (2021). Potential environmental pollution from copper metallurgy and methods of management. Environmental Research, 197, p. 111050. doi:10.1016/j.envres.2021.111050.

[2] Rani, S., Kathuria, I., Kumar, A. et al. (2023). Valorised polypropylene waste based reversible sensor for copper ion detection in blood and water. Environmental Research, 228, p. 115928. doi:10.1016/j.envres.2023.115928.

[3] http://2014.igem.org/Team:NJAU_China

[4] http://2017.igem.org/Team:Peshawar

[5] http://2015.igem.org/Team:UMBC-Maryland

[6] http://2009.igem.org/Team:Groningen

[7] https://2019.igem.org/Team:UPNAvarra_Spain