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-genR sequence 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.