TABLE 1.
Bacterial strains and plasmids used in this studya
| Strain or plasmid | Lab IDb | GenBank ID no. | Relevant properties | Source or reference(s) |
|---|---|---|---|---|
| Strains | ||||
| E. coli | ||||
| EPMax10B-pir116 Δasd Δtrp::Gmrmob-Kanr | E1354 | Gmr Kanr F− λ−mcrAΔ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara leu)7697 galU galK rpsL nupG Tn-pir116-FRT2 Δasd::wFRT Δtrp::Gmr-FRT5 mob[recA::RP4-2 Tc::Mu-Kanr] | Available lab strain | |
| EPMax10B-lacIqpir | E1869 | F− λ−mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ(ara leu)7697 galU galK rpsL nupG lacIq-FRT8 pir-FRT4 | Available lab strain | |
| B. pseudomallei | ||||
| 1026b | B0004 | Clinical melioidosis isolate | 10 | |
| 1026b/att-Tn7-gat-cfp | B0036 | GSr, 1026b with mini-Tn7-gat-cfp inserted | This study | |
| 1026b/att-Tn7-gat-gfp | B0037 | GSr, 1026b with mini-Tn7-gat-gfp inserted | This study | |
| 1026b/att-Tn7-gat-rfp | B0038 | GSr, 1026b with mini-Tn7-gat-rfp inserted | This study | |
| 1026b/att-Tn7-gat-yfp | B0039 | GSr, 1026b with mini-Tn7-gat-yfp inserted | This study | |
| B. thailandensis | ||||
| E264 | E1298 | Prototroph, environmental isolate | 2 | |
| E264/att-Tn7-kan-cfp | E2552 | Kanr, E264 with mini-Tn7-kan-cfp inserted | This study | |
| E264/att-Tn7-kan-gfp | E2491 | Kanr, E264 with mini-Tn7-kan-gfp inserted | This study | |
| E264/att-Tn7-kan-rfp | E2553 | Kanr, E264 with mini-Tn7-kan-rfp inserted | This study | |
| E264/att-Tn7-kan-yfp | E2554 | Kanr, E264 with mini-Tn7-kan-yfp inserted | This study | |
| Plasmidsc | ||||
| mini-Tn7-gat | E1981 | GSr, mini-Tn7 integration vector based on gat | 21 | |
| mini-Tn7-gat-cfp | E2460 | HM150704 | GSr, mini-Tn7-gat harboring cfp | This study |
| mini-Tn7-gat-gfp | E2462 | HM150705 | GSr, mini-Tn7-gat harboring gfp | This study |
| mini-Tn7-gat-rfp | E2326 | HM150706 | GSr, mini-Tn7-gat harboring rfp | This study |
| mini-Tn7-gat-yfp | E2468 | HM150707 | GSr, mini-Tn7-gat harboring yfp | This study |
| mini-Tn7-kan-cfp | E2480 | HM150708 | Kanr, mini-Tn7-kan harboring cfp | This study |
| mini-Tn7-kan-gfp | E2482 | HM150709 | Kanr, mini-Tn7-kan harboring gfp | This study |
| mini-Tn7-kan-rfp | E2486 | HM150710 | Kanr, mini-Tn7-kan harboring rfp | This study |
| mini-Tn7-kan-yfp | E2488 | HM150711 | Kanr, mini-Tn7-kan harboring yfp | This study |
| pPS747 | E0042 | Apr, plasmid harboring eGFP | 9, 15 | |
| pRK2013 | E1358 | Kanr, helper plasmid encoding conjugative proteins | 11 | |
| pTNS3-asdEc | E2237 | Suicidal helper plasmid containing E. coli asd and transposase for the Tn7 site-specific transposition system | 17 | |
| pUC57-PS12-cfp | E1739 | HM150701 | Apr, plasmid harboring B. pseudomallei codon-optimized cfp driven by PS12 | This study |
| pUC57-PS12-rfp | E1735 | HM150702 | Apr, plasmid harboring B. pseudomallei codon-optimized rfp driven by PS12 | This study |
| pUC57-PS12-yfp | E1738 | HM150703 | Apr, plasmid harboring B. pseudomallei codon-optimized yfp driven by PS12 | This study |
Abbreviations: Apr, ampicillin resistant; asdEc, E. coli asd gene; gat, gene encoding glyphosate acetyltransferase; Gmr, gentamicin resistant; GSr, glyphosate resistant; kan, gene encoding kanamycin resistance; Kanr, kanamycin resistant; PS12, rpsL promoter from B. pseudomallei; w, wild type.
Please use the laboratory identification number (Lab ID) when requesting strains and plasmids.
The mini-Tn7 plasmid backbone is based on pUC18R6KT-mini-Tn7T (7). Fluorescent protein genes (cfp, rfp, yfp) were synthesized by GeneScript and cloned into the EcoRV site of pUC57. These genes were removed by digestion with KpnI and PstI and then subcloned into mini-Tn7-gat cut with the same enzymes. Two-step PCR was performed to incorporate the PS12 promoter upstream of the gfp gene, which was digested with KpnI and PstI and cloned into mini-Tn7-gat that had been digested with the same enzymes. The mini-Tn7-kan constructs were created by replacing the gat cassette with the kan cassette.