5 The role of quorum-sensing in the virulence of Pseudomonas ...

Leicester). E105-leuX was the first genomic island captured from a clinical isolate
using the yeast-based genomic island capture technique. This section will proceed
by analysing the preliminary data generated for the genomic island. This is followed
by analysis to determine that the insertion at the leuX tRNA locus is a genomic
island (islet). This is followed by analysis of the genomic structure of E105-leuX
and concludes with the possible evolution of the genomic island.
Preliminary experiments were preformed on the genomic island capture clone to
verify the correct recombination event had occurred. Firstly, PCR was performed on
the bacterial plasmid preparation to confirm that the plasmid contained TS1 and
TS2 as well the positive screening region. The plasmid was also subjected to
restriction digestion with I-SceI to get an approximation of the size of the captured
insert (the genomic island plus the conserved flanks). As the captured insert was
only approximately 11kb, it was within the range that could be amplified using
long-range PCR. The capture insert was amplified from the genomic DNA of E.
coli E105 using the leuX TS1 U primer and the leuX TS2 D primer. The size of the
insert from long-range PCR and the I-SceI restriction digest are shown in Figure
3-6. The SGSP data provided by Ali Thani were re-analysed; construction of the
SGSP library, screening and sequencing was performed by Ali Thani. The
following analysis was performed independently.
A 1.4kb SGSP amplicon was generated from the PstI genomic library of E. coli
E105 using the leuX U primer and the T3 universal primer. The amplicon was sent
for sequencing with the KS universal primer. Results of the analysis are depicted in
Figure 3-13.
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