Lysine is one of the more limiting amino acids in protein sources for chickens.
Since lysine is also an essential amino acid for animals, it is an important component of
animal dietary formulation. Therefore, an accurate pre-determination of bioavailable
lysine in feedstuffs is important. An optical density (OD) based microbiological assay
for lysine determination using E. coli lysine auxotroph has been previously developed.
However, because the assay is based on bacterial growth response to extracellular lysine
measured as OD, it can be relatively time consuming (10-12h). Therefore, more rapid
assays are needed if pre-formulation estimates are required. In this dissertation whole
cell fluorescent biosensors for the quantification of bioavailable and total lysine in feed
protein sources were developed. The biosensor for quantification of bioavailable lysine
was based on the growth response of E. coli to an external source of lysine and lysinecontaining
small peptides. Green fluorescent protein (GFP) was inserted in the genome
of E. coli lysine auxotroph as a part of a mini-Tn5- transposon by conjugation. Bacterial
growth response to external lysine and small peptides was monitored and recorded by
measuring the fluorescence emitted by GFP. The second type biosensor developed was
designed for the quantification of total lysine. It was based on the measurement of a promoter activity, which was induced and modulated by extracellular concentration of
lysine. Cad promoter was amplified from E. coli K-12 genome and was cloned into
promoterless gfp plasmid. The construct was electroporated into electrocompetent E. coli
cells. The promoter activity was induced under the conditions of low pH and graded
concentrations of lysine. Lysine-dose response was measured by the fluorescence of
GFP. Both methods were characterized as having a high potential for practical
application.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/4786 |
| Date | 25 April 2007 |
| Creators | Chalova-Zhekova, Vesela I. |
| Contributors | Ricke, Steven C. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 951941 bytes, electronic, application/pdf, born digital |
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