Organophosphorus compound detection on a cell chip with yeast coexpressing hydrolase and eGFP.
Organophosphorus compounds (OPs) such as pesticides, fungicides, and herbicides are highly toxic but are nevertheless extensively used worldwide. To detect OPs, we constructed a yeast strain that co-displays organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (EGFP) on the cell surface using a Flo1p anchor system. OP degradation releases protons and causes a change in pH. This pH change results in structural deformation of EGFP, which triggers quenching of its fluorescence, thereby making this cell useful for visual detection of OPs. Fluorescence microscopy confirmed the high-intensity fluorescence displayed by EGFP on the cell surface. The yeast strain possessed sufficient OPH hydrolytic activities for degrading OPs, as measured by incubation with 1 mM paraoxon for 24 h at 30 degrees C. In addition, with 20 mM paraoxon at 30 degrees C, fluorescence quenching of EGFP on the single yeast cell was observed within 40 s in a microchamber chip. These observations suggest that engineered yeast cells are suitable for simultaneous degradation and visual detection of OPs.
Fukuda
Takeshi
T
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, Fukui, Japan.
Tsuchiya
Keiji
K
Makishima
Hirokazu
H
Tsuchiyama
Kouta
K
Mulchandani
Ashok
A
Kuroda
Kouichi
K
Ueda
Mitsuyoshi
M
Suye
Shin-ichiro
S
D015374:Biosensing Techniques / Q000379:methods*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D009943:Organophosphorus Compounds / Q000032:analysis* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D009943:Organophosphorus Compounds; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006867:Hydrolases