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

3.1 Capture experiments involving genome sequenced bacterial
strains
This section will describe the initial capture experiments using reference genomes.
The experiments were designed to develop the methodology associated with
genomic island capture technique and to test the size limits of the captured insert.
For E. coli, the reference genome was K12 MG1655 and for P. aeruginosa PAO1
and PA14.
The yeast cell spheroplasts are transformed with the capture vector plus the genomic
DNA. Homologous recombination between the capture vector and the genomic
DNA results in the genomic island now being present within the vector. The main
principle of the yeast capture method is depicted in Figure 3-1. The term captured
insert refers the DNA sequence running from TS1 to TS2, which consists of the
genomic island with conserved flank genomic DNA.
Figure 3-1 depicts the principles of genomic island capture technique. TS1 and TS2 are the
conserved targeting regions found upstream and downstream of the genomic island. –
Reproduced from the MobilomeFinder website (http://mml.sjtu.edu.cn/MobilomeFINDER/ycv.htm)
The targets for the initial capture experiments were chosen to vary in size based on
the capture vectors available. The targets are listed in Table 3-1. The size of the
captured insert was generated from the GenBank sequence of the reference genomes
using the TS1 U and TS2 D primer sequences. The results also show the number of
positive colonies determined by PCR discovered for each target. The results are
supported by the literature which suggests that the larger the size of captured insert,
the lower the recovery of positive colonies (Leem et al. 2003).
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