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Figure 2 | BMC Genomics

Figure 2

From: A new platform for ultra-high density Staphylococcus aureus transposon libraries

Figure 2

Elimination of non-transposase catalyzed transposon integration in strain HG003. (A) The ratio of erythromycin resistant colonies arising from non-transposase catalyzed (hatched) to transposase dependent (solid shading) events was determined in the S. aureus RN4220 (green) or HG003 (blue) recipient strain background by comparing the number of colonies resulting from transduction of the transposon into the full transposase or truncated transposase expressing strains (Additional file 1: Figure S1). The presence of the phage attachment site in the bacterial chromosome (attB), the phage attachment site in the donor lysate (attP-int), and a Φ11 prophage in the recipient is indicated for each combination. (B) Putative mechanisms for integration of the ermR cassette of the transposon into the recipient chromosome include transposase catalyzed (top), integrase mediated site specific recombination (middle), and homologous recombination (bottom) of phage-transposon hybrids resolved from concatemeric transposon donor DNA. (C) The integrase pathway was blocked by replacing the integrase (int) gene and the attL sequence with a FRT element by allelic replacement. To cure the resulting prophage, the attR site was also replaced with a second FRT site and a phage donor lacking int-attP was isolated by introducing the FLP recombinase. In the process, a recipient HG003 strain was generated from which the Φ11 prophage was specifically cured and replaced with a single FRT element, preventing homologous recombination.

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