Fig. 1

Schematic representation of attB-bla system and the conjugative assay used to test att sites. a In the selective tool, the bla gene is fragmented such that the 5′ promoter and signal sequence are associated with an attL site, and the partner attR is associated with the 3′ region. Each component is placed at separate loci, either on the genome or a plasmid, depending on the application. b Conjugation of the attB plasmid into a recipient strain containing the attP and integrase plasmids to form the attR and attL partners with bla gene fragments. c Sequence of the HK022 attB site. We tested attB _HK sites of three different lengths to avoid potential interference with bla function and protein export, 51 bp (violet), 33 bp (teal), and 23 bp (black). To increase the number of potential open reading frames, we introduced a T ➔ A nucleotide change into the attB sequence, indicated in red. The BOB’ core region is demonstrated by black lines. Stars indicate bases in common with attP _HK. Recombination points flank the core O region. d Recombination results of attB _HK sequences. These six sequences were tested using a plasmid conjugation assay in a context independent of the bla gene [29]. This demonstrated that the introduced mutation did not interfere with recombination efficiency and the length of the attB site had a negative correlation with recombination frequency. As we wished to use a shorter sequence to avoid interfering with bla functionality following attB site insertion, we based our subsequent ORF constructions on the 23 bp mut form, despite the fact that it recombines at a lower frequency than the 51 and 33 bp wt sequences