Application of an Escherichia coli green fluorescent protein - based lysine biosensor under nonsterile conditions and autofluorescence background

Abstract

Aims: To examine the utility of an Escherichia coli green fluorescent protein (GFP) containing biosensor for quantification of bioavailable lysine in selected feed samples under nonsterile conditions and to estimate the background fluorescence of analyzed feed samples and evaluate the risk of confounding GFP emission from the lysine assay organism. Methods and Results: Escherichia coli lysine auxotroph GFP based biosensor was used to determine the percentage of bioavailable lysine in two samples of soyabean‐, cottonseed‐, and meat and bone meal under nonsterile conditions. The fluorescence emitted by GFP was successfully measured using a spectrofluorimeter to monitor bacterial growth response to protein‐derived lysine and lysine containing small peptides. The autofluorescence of analyzed feed samples at different concentrations could also be estimated. Conclusions: When feed protein concentrations are decreased, autofluorescence interference can be avoided. Significance: The E. coli lysine auxotroph GFP‐based biosensor can successfully be used for the determination of bioavailable lysine in these selected animal feed proteins under nonsterile conditions. Impact of the Study: E. coli GFP biosensor for lysine has potential for routine application in animal feeds.

Translation-dependent bioassay for amino acid quantification using auxotrophic microbes as biocatalysts of protein synthesis

“…This bioassay allows for rapid determination of amino acid concentrations within 60 min, a remarkably shorter time than necessary for any other conventional bioassays (Bertels et al 2012;Cardinal and Hedrick 1948;Chalova et al 2006;Erickson et al 2000;Kim et al 2010;Li and Ricke 2003;Palanza et al 2016;Steele et al 1949;Tamura et al 1952). The present results also demonstrate the high versatility of this bioassay in terms of both Fig.…”

“…Some recent studies have substituted auxotrophic Escherichia coli mutants for lactic acid bacteria (Li and Ricke 2003), since the former exhibits a lower doubling time. In addition, marker proteins-such as green fluorescent protein (GFP) and β-galactosidase-can be introduced to increase the detection sensitivity for cell growth (Bertels et al 2012;Chalova et al 2006;Chalova et al 2007;Erickson et al 2000;Kim et al 2010). Although these modifications have succeeded in shortening the incubation time when compared to conventional methods, they still require no less than a 4-6-h incubation period due to the above restriction intrinsic to growth-dependent bioassays.…”

Tight Modulation of Escherichia coli Bacterial Biofilm Formation through Controlled Expression of Adhesion Factors

“…S1 in the supplemental material). In vivo approaches using the controlled biofilm capacities of innocuous bacteria could thus be envisaged (i) to challenge deleterious biofilms found in both industrial and medical settings through bacterial interference or competitive adhesion to the surface (77) or through the production of toxic or matrix-dissolving compounds such as cellulase and dispersins, (ii) to optimize the persistence of probiotic strains (15,19,24,27,28,31,33,37,64), and (iii) to improve-via stronger heterologous bacterial interactions-the establishment, in nonsterile soil microcosms or in mixed bioreactors, of strains with desirable or genetically engineered features and their development as biosensors or agents in bioremediation processes (8,22,26,53,65,72).…”

Development of a whole cell green fluorescent sensor for lysine quantification

“…In addition, the use of GFP for the purpose of a lysine assay on animal feeds is of special interest because fluorescence is considered relatively rare in microorganisms (Errampalli et al 1999;Vialette et al 2004). This would allow lysine quantification to be performed on non-sterile animal feeds (Chalova et al 2006). Currently, feeds must be sterilized prior to the assay, which can decrease bioavailable lysine (Hankes et al 1948, Anderson-Hafermann et al 1993, Johnson et al 1998, or must be supplemented with antibiotics to suppress the growth of the indigenous microflora that could contribute to background OD .…”