TABLE 4.
Strains and plasmids used in this work
| Strain or plasmid | Features | Source |
|---|---|---|
| Burkholderia cenocepacia K56-2 | Clinical isolate from cystic fibrosis patient; ET12 lineage | (36) |
| K56-2 fliF::pAH26 | Derived from K56-2; pAH26 integrated into fliF; Tpr | (12) |
| K56-2 phbC::pAH27 | Derived from K56-2; pAH27 integrated into phbC; Tpr | (12) |
| K56-2 paaA::CAST-Tp (3390) | Derived from K56-2; cargo containing Tpr integrated into paaA; made with crRNA 3390 | This study |
| K56-2 paaA::CAST-Tp (3422) | Derived from K56-2; cargo containing Tpr integrated into paaA; made with crRNA 3422 | This study |
| K56-2 paaA::CAST-Tp (3423) | Derived from K56-2; cargo containing Tpr integrated into paaA; made with crRNA 3423 | This study |
| K56-2 fliF::CAST-Tp (3463) | Derived from K56-2; cargo containing Tpr integrated into fliF; made with crRNA 3463 | This study |
| K56-2 fliF::CAST-Tp (3466) | Derived from K56-2; cargo containing Tpr integrated into fliF; made with crRNA 3466 | This study |
| K56-2 phbC::CAST-Tp (3469) | Derived from K56-2; cargo containing Tpr integrated into phbC; made with crRNA 3466 | This study |
| K56-2 phbC::CAST-Tp (3472) | Derived from K56-2; cargo containing Tpr integrated into phbC; made with crRNA 3472 | This study |
| K56-2::CAST-fugfp-Tp (3475) | Derived from K56-2; cargo containing fugfp and Tpr integrated into intergenic region RS16895-90; made with crRNA 3475 | This study |
| K56-2::CAST-fugfp-Tp (3478) | Derived from K56-2; cargo containing fugfp and Tpr integrated into intergenic region RS16895-90; made with crRNA 3478 | This study |
| K56-2::CAST-fugfp | Derived from K56-2::CAST-fugfp-Tp (3475); Tpr flanking by the FRT region excised via Flp recombination using pFLP3 | This study |
| K56-2::CAST-fugfp fliF::CAST-Tp | Derived from K56-2::CAST-fugfp (3475); cargo containing Tpr integrated into fliF; made with crRNA 3463 | This study |
|
Burkholderia multivorans ATCC 17616 |
Soil sample | ATCC |
| ATCC 17616 paaA::CAST-Tp (3390) | Derived from ATCC17616; cargo containing Tpr integrated into paaA; made with crRNA 3390 | This study |
| ATCC 17616 paaA::CAST-Tp (3422) | Derived from ATCC17616; cargo containing Tpr integrated into paaA; made with crRNA 3422 | This study |
| ATCC 17616 paaA::CAST-Tp (3424) | Derived from ATCC17616; cargo containing Tpr integrated into paaA; made with crRNA 3424 | This study |
| ATCC 17616 fliF::CAST-Tp (3463) | Derived from ATCC17616; cargo containing Tpr integrated into fliF; made with crRNA 3463 | This study |
| E. coli DH5α |
F- Φ80lacZΔM15 Δ(lacZYAargF) U169 recA1 endA1 hsdR17 (rH−, mK+) phoA supE44 λ− thi-1 gyrA96 relA1 |
Cardona lab collection |
| E. coli Stbl2 | F- mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1lon gyrA96 thi supE44 relA1 λ− Δ(lac-proAB) | Invitrogen |
| E. coli PIR1 | F- ∆lac169 rpoS(Am) robA1 creC510 hsdR514 endA recA1 uidA(∆MluI)::pir-116 | Cardona lab collection |
| E. coli SY327 | F- araD Δ(lac-proAB) argE(Am) recA56 Rifr nalA λpir | (37) |
| pHelper_ShCAST_crRNA | AmpR, oricolE1, Sh-cas12K-tnsBC-tniQ, PJ23119-crRNA | (17) |
| pDonor_ShCAST_kanR | KmR, oriR6K, Tn-neo | (17) |
| pSCrhaB2plus | ripBBR1
rhaR, rhaS, PrhaB TpR mob+, rhaI1-rhaI1 permutation of rhaS binding sites upstream of PrhaBAD and bacteriophage T7 gene 10 stem-loop inserted upstream of native rhaBAD 5′ UTR |
(10) |
| pKD3 | Cmr oriR6K rgnB | (38) |
| pRK2013 | ricolE1 RK2 derivative Kanr mob+ tra+ | (39) |
| pZLrhaB2plus | pSCrhaB2plus with modifications of resistance cassette to CmR | This study |
| pDonorTp | pDonor_ShCAST_kanR with modification of KmR to TpR | This study |
| pDonorTp-fugfp | pDonorTp with the addition of fugfp gene from pUS252 outside of the FRT sites | This study |
| pUS252 | ripUC, KmR, fuGFP | Addgene plasmid #127674 |
| pFLP3 | Ampr, Tetr, Flp | (30) |
| pRhaCAST | pZLrhaB2plus with Sh-cas12K-tnsBC-tniQ cloned into, which is driven by the rhamnose-inducible promoter | This study |