Najamudin, Khairal Ezani
(2003)
Construction of the aequorea Victoria green fluorescent protein (gfp) for expression in mycobacterium sp.
Masters thesis, Universiti Sains Malaysia.
Abstract
The development of sensitive methods for observing individual bacterial
cells in a population in experiment and natural environments is very crucial for
rapid development of anti-mycobacterial drugs. This is because more pathogenic
Mycobacteria are slow growing organisms, and therefore the screening
compounds for anti-mycobacterial activity is slow and inefficient. Previously,
studies have been done using fluorescent bacteria that expressing 13-
galactosidase (21 ), and luciferase (3) as a high screening format for antimicrobial
activity. However, one drawback of these systems is that substrates
such as luciferin have to be added at the required time points to induce
fluorescence.
Recently Green Fluorescent Protein (GFP) has become a valuable and
favourite tool as a marker of growth, which could be used for screening of antimicrobial
activity. This is because the marker can be visualised without
interruption or termination of an experiment as is required with the detection of
other commonly used markers such as B-galactosidase and luciferase.
In this study a synthetic gene of GFP was constructed with mycobacteria
codon bias using assembly PCR. A strong mycobacterial promoter from 65 kD
mycobacterial heat shock protein was added to derive the expression. The
constructed gene was cloned into vector and transformed into Escherichia coli.
The ultimate aim is to use the recombinant plasmid carrying the synthetic gene
with mycobacterial codon bias to create a recombinant fluorescing BCG, whichcan be used as a tool for screening compound libraries for anti-mycobacterial
activity.
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