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A cad‐gfp mut3 plasmid construct in Escherichia coli for gene induction‐based quantification of lysine in acid hydrolysates of feedstuffs
Author(s) -
Chalova V.I.,
Woodward C.L.,
Ricke S.C.
Publication year - 2008
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/j.1472-765x.2007.02273.x
Subject(s) - lysine , escherichia coli , hydrolysate , biology , plasmid , soybean meal , recombinant dna , biochemistry , amino acid , gene , raw material , ecology , hydrolysis
Aims: To generate an inducible plasmid‐borne cad‐gfp mut3 transcriptional fusion and develop a method for quantification of total lysine. Methods and Results: The cad‐gfp mut3 transcriptional fusion was constructed by cloning the cad promoter (P cad ) upstream of a promotorless gfp mut3 located on a high‐copy plasmid. The construct was electroporated into Escherichia coli ZK126 and the transformed strain was subsequently used to quantify lysine in feed ingredients. Lysine standard curves based on gene induction of the bacterial cells were used for estimating acid hydrolysate lysine concentrations in four feed ingredients. Except for sorghum, no substantial differences were observed when the data for lysine in soybean (2·49 ± 0·37%), cottonseed (1·82 ± 0·15%), and meat and bone meal (2·31 ± 0·24%) generated by the newly developed construct were compared with previously published data. Conclusions: Using the cad‐gfp mut3 fusion, feed derived lysine induction was measured easily and accurately, and could be a useful tool for the estimation of lysine in acid hydrolysates of feed ingredients. Significance and Impact of the Study: The described approach for lysine quantification in feed ingredients represents a cost‐ and time‐efficient method offering rapid and accurate lysine quantification of multiple samples.