Najamudin, Khairul Ezani
(2007)
Application Of Aequorea Victoria
Green Fluorescent Protein (Gfp) For Expression In
Mycobacterium bovis BCG.
Masters thesis, Perpustakaan Hamzah Sendut.
Abstract
A potential marker, green fluorescent protein (GFP) derived from the jellyfish
Aequorea victoria offer many advantages as a viability reporter, as it requires no
external source of substrate nor cofactors to fluoresce but is dependent on the host
cell being alive. Its use has been reported in M. tuberculosis, but the incorporation of
potentially pathogenic strains for high throughput screening would raise issues of
safety. Therefore, a safe strain such as the vaccine strains M. bovis BCG if made to
express an easily detectable viability * reporter marker would be useful tool as a
screening assay. In this study, a synthetic version of the wild type GFP gene
sequence incorporating mycobacterial codon bias, was used to explore its potential
as a marker of viability in the mycobacterial vaccine strain, M. bovis BCG. The
synthetic gene designated as EzyGFP was successfully constructed by using
assembly polymerase chain reaction (PCR). In the second approach, a modified
version of GFP, GFPuv, was also tested as an alternative candidate. To propagate
these genes in Escherichia coli and then deliver these genes into M. bovis BCG,
mycobacterial shuttle vectors were constructed. To create mycobacterial shuttle
vectors, the mycobacterial origin of replication was excised from a source plasmid,
pNMN012, and ligated into plasmids designated as pEzyGFP, pGFPuvK and
pGFPuvK65 respectively which contain the GFP gene of interest.
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