Table 3.
Bacterial strains, plasmids and primers used in this study
| Strains/Plasmids | Genotype | Reference |
|---|---|---|
| Escherichia coli | ||
| TOP10F' | F' {lacIq, Tn10(TetR)} mcrA Δ(mrr-hsdRMS-mcrBC) f80dlacZDM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG | Invitrogen |
| Pseudomonas aeruginosa | ||
| PAO1 | Wild-type | [47] |
| PKM400 | PAOampP::aacCI; GmR | This study |
| PKM500 | PAOampG::aacCI; GmR | This study |
| PKM300 | PAOampR::aacCI; GmR | [10] |
| PKM301 | PAOattB::PampC-lacZ; TcR | [10] |
| PKM303 | PAOampR::aacCI attB::PampC-lacZ; TcRGmR | [10] |
| PKM104 | PAOattB::PampP-lacZ; TcR | KKF0290, This study |
| PKM105 | PAOattB::PampG-lacZ; TcR | This study |
| PKM312 | PAOampR::aacCI attB::PampG-lacZ; TcRGmR | This study |
| PKM313 | PAOampR::aacCI attB::PampP-lacZ; TcRGmR | This study |
| PKM404 | PAOampP::aacCI attB::PampP-lacZ; TcRGmR | This study |
| PKM405 | PAOampP::aacCI attB::PampG-lacZ; TcRGmR | This study |
| PKM504 | PAOampG::aacCI attB::PampP-lacZ; TcRGmR | This study |
| PKM505 | PAOampG::aacCI attB::PampG-lacZ; TcRGmR | This study |
| PKM506 | PAOampG::aaCI attB::PampC-lacZ; TcRGmR | This study |
| PKM507 | PAOampP::aaCI attB::PampC-lacZ; TcRGmR | This study |
| Plasmids | ||
| pCRII-TOPO | ApR, KmR; ColE1ori lacZα | Invitrogen |
| pUCGm | ApR, GmR; pUC19 derivative containing gentamycin cassette | [38] |
| pEX100T | ApR; sacB oriT | [39] |
| pMF54 | ColE1-SF broad-host replicon | [48] |
| pME6030 | TcR; oriVpVS1 oriVp15A oriT | [41] |
| pRK2013 | KmR; ColE1ori-Tra (RK2)+ | [40] |
| pTrcphoA | ApR; low-copy trc promoter expression vector carrying the lacIq and phoA | [43] |
| pTrclacZ | ApR, low-copy trc promoter expression vector carrying the lacIq and lacZ | [43] |
| pSJ10 | TcR; CTX-lacZ fused with ampC promoter, PampC | [10] |
| pKKF003 | ApR, KmR; pCRII-TOPO with a 2904-bp fragment containing PAO1 coordinates 4921591-4924494 (PA4392-PA4393/ampF-ampG) | This study |
| pKKF004 | ApR, KmR; pCRII-TOPO with a 2779-bp fragment containing PAO1 coordinates 4721496-4724275 (PA4217-PA4218/ampO-ampP) | This study |
| pKKF069 | TcR; pME6030 with a 2904-bp EcoRI flanked fragment containing ampF-ampG | This study |
| pKKF073 | TcR; pME6030 with a 2779-bp EcoRI flanked fragment containing ampO-ampP | This study |
| pKKF087 | TcR; CTX-lacZ fused with ampP promoter, PampP | This study |
| pKKF091 | TcR; CTX-lacZ fused with ampG promoter, PampG | This study |
| pKKF145 | ApR, GmR; pCRII-TOPO derivative with ampP::aacCI | This study |
| pKKF149 | ApR, GmR; pCRII-TOPO derivative with ampG::aacCI | This study |
| pKKF157 | ApR, GmR; pEX100T derivative with ampG::aacCI | This study |
| pKKF161 | ApR, GmR; pEX100T derivative with ampP::aacCI | This study |
| pAA0115 | ApR, pTrcphoA with 1.9 kb PstI DNA from pMF54 containing stabilization fragment | This study |
| pAA0112 | ApR, KmR; pCRII-TOPO containing a 1797 ampG PCR product amplified using primers KKF09 and KKF10 | This study |
| pAA0121 | ApR, pAA0115 containing a 1,813 EcoRI fragment containing ampG from pAA0112 | This study |
| pAA1261 | ApR; pAA0121 digested with BamHI and SalI, treated with Klenow and re-ligated to remove an XbaI site. Used as basis for Erase-a-base system | This study |
| pKKF259 | ApR; pTrcphoA derivative with a 1797-bp fragment containing PA4393 | This study |
| pKKF458 | ApR; pTrcphoA derivative with a 1245-bp fragment containing PA4218 | This study |
| pKKF459 | ApR; pTrclacZ derivative with a 1797-bp fragment containing PA4393 | This study |
| pKKF465 | ApR; pTrclacZ derivative with a 1245-bp fragment containing PA4218 | This study |
| Primers | ||
| KKF01ampGFor | 5'-TCCAGCTTGACGTCGAGATT-3' | This study |
| KKF04ampGRev | 5'-AGAACATTCTCCTGGCCATGG-3' | This study |
| KKF05ampPFor | 5'-AACGGCCACGCTAGCAACAC-3' | This study |
| KKF08ampPRev | 5'-GTGGCGCCTGGAGTCTTG-3' | This study |
| KKF09ampG2For | 5'-GGGAATTCCATATGACTCAGCAATCCTGG-3' | This study |
| KKF10ampG2Rev | 5'-GCTCTAGATGCTCGGCGTTCTGGTGT-3' | This study |
| KKF13ampP2For | 5'-TCTAGATCAGGCCTCTTCCGCCCG-3' | This study |
| KKF14ampP2Rev | 5'-ATGCTTGAGCTGTACCGCCA-3' | This study |
| PA4218_9junctionRTF | 5'-ACCTCACCCTGATCCTCTG-3' | This study |
| PA4218_9junctionRTR | 5'-CAGGTAGAGCAACGCCAG-3' | This study |
| PA4392_3junctionRTF | 5'-CAACGACAGGGTGGACATAC-3' | This study |
| PA4392_3junctionRTR | 5'-GAGACTTGTAGGCGACCAG-3' | This study |
| (NS)5RandomPrimer | 5'-NSNSNSNSNS-3' | [49] |