Development of a whole cell green fluorescent sensor for lysine quantification

Abstract

As an essential amino acid, lysine is an important component of animal and human diets and its bioavailability can depend on a variety of factors. Therefore, an accurate pre-determination of bioavailable lysine in foods and feeds is important. In this study a whole cell fluorescent biosensor for the quantification of lysine in protein sources was constructed. A gene encoding for green fluorescent protein (GFPmut3) was introduced into an E. coli lysine auxotroph genome as a part of a mini-Tn5-Km transposon. The location of the transposon was determined and the growth kinetics of the newly constructed biosensor were examined. The transposon disrupted the ybhM gene, which encodes for the synthesis of a protein with an unknown function. No effect of the transposon's location in the genome or the expression of gfp on bacterial growth rates was observed. Based on the fluorescence emitted by GFPmut3, a standard curve after 6-h growth of the strain was generated. A correlation coefficient of 0.95 was observed when the fluorescence method was compared to the conventional optical density (OD) growth-based lysine assay. Using the newly developed lysine fluorescent whole cell sensor we determined the total lysine in casein acid hydrolyzate (7.13 ± 0.34%). When lysine added to 12 lg/ml and 30 lg/ml of casein acid hydrolyzate was quantified, recoveries of 97 ± 1.65% and 103 ± 4.66% respectively were detected. The results suggest that the microbial assay using GFP fluorescence represents a promising alternative method for the potential estimation of lysine in protein sources.

Autofluorescence and green fluorescent protein-derived fluorescence in Listeria innocua

“…Oscar [38] also observed that constitutive expression of gfp altered growth characteristics of transformed Salmonella as the changes affected more growth rates and maximum population densities than lag times. In our study, however, L. innocua M1 transformation and gfp expression did not alter the growth rate which agreed with the reports of [39][40][41] for gfp-labeled Bordetella pertussis, Lactococcus lactis and E. coli K-12 strains respectively.…”

Microbial Biosensors for Environmental Monitoring and Food Analysis

“…In another study, a whole-cell fluorescent biosensor was constructed to quantify lysine in protein sources, which introduced a GFP-encoding gene (GFPmut3) into an E. coli lysine auxotroph genome as a part of a mini-Tn5-Km transposon. (114) Microbial biosensors have also been developed to examine the vitamin content in food products, including vitamin B6, B12, (3) and B1. Recently, a new whole-cell microbial biosensor based on S. cerevisiae was developed to measure vitamin B1 in under 3 minutes and with a detection limit between 0.005 and 0.1 μM.…”

Optical tracking of a stress-responsive gene amplifier applied to cell-based biosensing and the study of synthetic architectures

“…A popular approach is based on tracking promoter activity in response to cell-analyte interactions using endogenously produced reporter molecules (Daunert et al, 2000). Particular analytes of interest include mutagens, heavy metals, phenols, naphthalene, toluene, ammonia, methane, and various biologicals (Chalova et al, 2008;Ron, 2007;Yagi, 2007). Applications that stand to gain from improved whole-cell biosensing technology include air and water quality assessment, bio-warfare defense, drug screening, chemical mutagenicity assessment, and space biology and hazard assessment.…”