2.1 Construction of a dual fluorescent reporter gene system
For vector and primer information see Supplementary table 1 and 2, respectively. The insertion of the Plppmcherry-lacIq-gensequence into the chromosomal attTn7 site of the E. coli donor was achieved by Tn7 transposition, as described previously (McKenzie & Craig, 2006), utilizing the vector pGRG36::Plppmcherry-lacIq-genR(Klümper et al., 2015). The construction of the E. coli recipient strain was performed in a similar fashion, with the vector pGRG36::PlppmCherry-kanR . PCR amplification and FACS were used to verify chromosomal integration and loss of vectors. The tagging of R27 with PA1/O4/O3gfpmut3-kanR was performed using the λ-red system as described previously (Anjum et al., 2018; Datsenko & Wanner, 2000). The insert sequence was amplified by PCR from the vector pENT::PA1/O4/O3gfpmut3-kanR(Klümper et al., 2015) with 40 nucleotides 5’ overhangs homologous to the tetR-D region of the Tn 10 tetracycline resistance region of R27. Allelic exchange of this region generated a tetracycline sensitive phenotype. FACS was used to verify expression of the inserted fluorescence reporter gene in the resulting plasmid R27::gfp (referred to as R27). The construct was verified by Sanger and shotgun sequencing (Supplementary figure 1 and 2). For shotgun sequencing, R27 was extracted by Plasmid Mini AX kit (A&A Biotechnology) and sequencing libraries built using the Nextera XT DNA Library Preparation Kit (Illumina Inc.), and sequenced on the Illumina MiSeq Desktop Sequencer (Illumina Inc.) following the manufacturer’s protocol. R27 and pB10::gfp (referred to as pB10) were conjugated into the constructed E. coli donor, respectively.