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Development of a whole cell green fluorescent sensor for lysine quantification

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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 μg/ml and 30 μg/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.

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Acknowledgements

This research was supported by Hatch grant H8311 administered by the Texas Agricultural Experiment Station and Texas Advanced Technology Program, grant #: 000517-0220-2001 (Texas Higher Education Board, Austin, TX, USA). The authors would like to express their thanks to Julia Sonka (Center for Food Safety and Microbiology, IFSE, University of Arkansas) for her assistance in the preparation of this manuscript.

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  1. Vesela I. Chalova

    Present address: Department of Food Science, University of Arkansas, 2650 North Young Avenue, Fayetteville, AR, 72704-5690, USA

Authors and Affiliations

  1. Department of Poultry Science, Texas A&M University, College Station, TX, 77843, USA

    Vesela I. Chalova, Cassie L. Woodward & Steven C. Ricke

  2. Department of Poultry Science, University of Arkansas, Fayetteville, AR, 72701, USA

    Vesela I. Chalova & Steven C. Ricke

  3. Department of Food Science, University of Arkansas, 2650 North Young Avenue, Fayetteville, AR, 72704-5690, USA

    Steven C. Ricke

  4. Departamento de Biologia, Facultad Experimental de Ciencias, Universidad del Zulia, Maracaibo, Venezuela

    Irene B. Zabala-Díaz

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  1. Vesela I. Chalova
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  4. Steven C. Ricke
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Correspondence to Steven C. Ricke.

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Chalova, V.I., Zabala-Díaz, I.B., Woodward, C.L. et al. Development of a whole cell green fluorescent sensor for lysine quantification. World J Microbiol Biotechnol 24, 353–359 (2008). https://doi.org/10.1007/s11274-007-9479-3

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  • DOI: https://doi.org/10.1007/s11274-007-9479-3

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