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

are competent for DNA uptake. This results in homologous recombination at the
site fixed by the amplicons within the yeast cell as shown in Figure 1-4. The
transformed yeast cells are then plated onto URA medium; this does not
differentiate between cells containing re-circularised pLLX13 and those containing
the complete capture vector as explained in Figure 1-3.
Figure 1-4 Schematic depicting how the four DNA components recombine to generate the
complete capture vector
The capture vector is transferred to E. coli, its structure is verified and a DNA
preparation is created. For genomic island capture to occur, the yeast cell wall is
partially digested with a specialised enzyme known as Zymolyase (Zymo Research
Corporation, USA), to allow exogenous DNA to enter. A yeast cell with a partially
digested cell wall is known as a spheroplast. TAR cloning is stimulated by
transforming the capture vector that has been linearised to stimulate DNA damage
along with the genomic DNA of choice. A successful outcome results in the yeast
‘repairing’ the capture vector via homologous recombination between the vector
and the co-transformed genomic DNA (Figure 1-5). This would result in the capture
vector containing the targeted genomic island.
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