785158.fig.002
Figure 2: Schematic of the RecA-mediated recombination method used to develop HVS mORF51. Two plasmids are transformed into competent E. coli cells that already harbour the HVS-GFP-BAC genome. These plasmids are both temperature sensitive and have antibiotic resistance markers for ease of selection. The first plasmid, pDF25-Tet, contains a RecA expression cassette to facilitate recombination, as well as a tetracycline resistance gene. The second, pKOV Kan, contains the mutated region of the HVS genome flanked on either side by regions of homology of at least 500 bp. These homology regions target the recombination event to a specific point in the viral DNA. pKOV Kan also contains a SacB gene, allowing negative selection on sucrose-containing medium, and a kanamycin resistance gene. When both plasmids are transformed into the E. coli, RecA expressed from pDF25-Tet induces a recombination event between one of the homology regions in pKOV Kan and the corresponding region in the HVS genome. Clones containing the pKOV Kan plasmid integrated into the HVS genome are then selected and made competent. These cointegrant clones are then retransformed with pDF25-Tet in order to produce a second recombination event. Depending on whether this recombination is in the same or adjacent homology region to the initial recombination, a revertant clone or a recombinant clone will be formed. Selection is used to identify recombinants, which can then be further analysed and confirmed by restriction digest and sequencing.