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

estriction 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 14kb, it was within the range that could be amplified using
long-range PCR. The captured insert was amplified from the genomic DNA of E.
coli E106 using the serW TS1 U primer and the serW TS2 D primer. The size of the
insert from long-range PCR and the I-SceI restriction digest are shown in Figure
3-6.
Figure 3-6 shows a 0.6% agarose gel with the restriction profile of E105-leuX and E106-serW
with I-SceI and the long-range PCR product using the primer TS1 U and TS2 D
Lane 1 λHindIII 500ng; Lane 2 E105-leuX long-range PCR product; Lane 3 E105-leuX I-SceI
Lane 4 E106-serW I-SceI; Lane 5 E106-serW long-range PCR product
The SGSP data provided by Ali Thani was re-analysed; construction of the SGSP
library, screening and sequencing was performed by Ali Thani (Thani A, PhD
thesis, University of Leicester). The following analysis was performed
independently. The SGSP data provides a preview of the DNA sequence present at
the genomic island flanks.
23kb
9.4kb
6.8kb
4.4kb
2.3kb
2.0kb
500b
p
1
A 1.9kb SGSP amplicon was generated from the EcoRV genomic library of E. coli
E106 using the serW U primer and the T3 universal primer. The amplicon was sent
for sequencing with the serW U primer and 405bp of DNA sequence was produced.
The most significant nucleotide alignment (blastn) was to the E. coli strains APEC
O1 and UTI89 which produced identical score to one another (S: 719, E: 0, I: 99%).
All other significant alignments were to the first 341bp of the amplicon sequence,
only the E. coli strains APEC O1, B7A and UTI89 covered the first 401/405
nucleotides. The amplicon contained one gene, infA (58-277 nucleotides) and is part
of the conserved U flank. The same SGSP amplicon was also sent for sequencing
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