Cloning of Fluorescent Fusion Proteins

Combinations of different fluorescent markers fusioned to trwB, trwC, and trwK genes were created using plasmid pIM09 as a template (I. Matilla, doctoral thesis). pIM09 contains a fusion of trwB, GFP, and kanamicine resistance genes cloned in a pBR322 derivative vector (Supplementary Figure 6). The FRT Kanamycin resistance cassette was obtained from pKD4 plasmid (Datsenko and Wanner, 2000). For TrwC fluorescent constructs, trwC gene was obtained from R388 plasmid by PCR, adding HindIII and XhoI restriction sites at both ends (oligonucleotides 1 and 2 from Supplementary Table 2). For TrwK constructs, oligonucleotides 3 and 4 with the same restriction sites were used (Supplementary Table 2).

In order to create different fluorescent variants, mCherry, mKate2, and mEOS genes were obtained from pROD25 plasmid (Reyes-Lamothe et al., 2008) (oligonucleotides 5 and 6), pBAD33_mKate2 plasmid (Addgene) (Shcherbo et al., 2007) (oligonucleotides 7 and 8), and from a pRSETa_mEos4b vector (Paez-Segala et al., 2015) (Addgene) (oligonucleotides 9 and 10). In all cases, the fluorescent variants were inserted into XhoI and BamH1 restriction sites from pIM09 plasmid. TrwBmCherry was also cloned in a pHis vector to estimate membrane co-location with the fluorescent dye Nonyl-Acridine Orange (NAO), which is a membrane specific stain.

Fluorescent fusion constructs in plasmid pIM09 were then amplified by PCR, by using oligonucleotides containing 50 bases that perfectly matched the flanking R388 sequence where the constructs had to be inserted. Oligonucleotides 11 and 12 were used for trwB constructs, oligonucleotides 13 and 14 for trwC constructs and oligonucleotides 15 and 16 for trwK constructs (Supplementary Table 2). The amplified fragments were purified in an agarose gel and transformed into a E. coli TB10 strain containing R388 plasmid for homologous recombination. Recombination was activated by growing cells at 42°C, as previously reported. Cells were plated on LB agar containing kanamycin (50 μg/ml) and trimethoprim (20 μg/ml) to select the recombinant R388 plasmids. The selected plasmids were subsequently analysed by sequencing.