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

Figure 1-5 depicts the principles of genomic island capture method – Reproduced from the
MobilomeFinder website (http://mml.sjtu.edu.cn/MobilomeFINDER/ycv.htm)
To increase the likelihood of recovery of a capture vector with a genomic island
inserted, URA media is supplemented with cycloheximide. Any yeast cells that
contain the capture vector only, will be rendered susceptible to cycloheximide,
which is toxic to the cells, due to the dominant allele present on the capture vector.
Successful recombination between the capture vector and the genomic DNA results
in the loss of the cyh gene from the capture vector; therefore if the capture vector
undergoes re-circularisation instead of homologous recombination, the presence of
the cycloheximide within the media should eliminate them.
The resulting colonies are screened to ensure the correct recombination event has
occurred by PCR. The capture vector: genomic island is then transferred to bacteria
for further analysis. This capture vector plus genomic island can now be used for
functional studies as well as sequencing projects. This molecular biology tool is
very powerful as the targeting regions will be conserved among the majority of the
members of the same species. This allows for comparative studies of these regions.
1.3.3 Aims
The aim of the first project was to create a generic genomic island capture technique
to aid discovery and characterisation of novel genomic islands. To achieve this aim,
the project will further develop the genomic island capture technique as described
by Wolfgang et al (2003) by testing the parameters and capturing from bacterial
species other than P. aeruginosa. The method described in this thesis streamlines
the design and creation of capture vectors as well as providing an alternative
protocol for effective screening of transformants. The project encompasses E. coli
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