TABLE 1.
Strains and plasmids used in this study
| Plasmid or strain | Relevant genotype or characteristics | Source or reference |
|---|---|---|
| Plasmids | ||
| pDTG514 | pGem3Z with a 2.75-kb EcoRI-SmaI fragment from pDTG350 containing the tod promoter, Ptod; Apr | 14 |
| pUCD615 | Promoterless luxCDABE cassette containing ori pSa and ori pBR322; Apr Kmr | 18 |
| pKK223-3 | Expression vector containing the 5S ribosomal terminator rrnB T1T2 | Pharmacia |
| pBSKS | pBluescript II KS+ with MCS KpnI-SacI; Apr | Stratagene |
| pBSMCS(−) | pBluescript without MCS (BssHII-BssHII fragment removed); Apr | This study |
| pLJS | pBSMCS(−) with added XbaI, NheI, AvrII, and SpeI sites; Apr | This study |
| pLJS-tod | pLJS containing the 1.8-kb SmaI-XhoI tod promoter fragment from pDTG514; Apr | This study |
| pLJS-lux | pLJS containing the 8.35-kb KpnI-PstI luxCDABE cassette from pUCD615; Apr | This study |
| pLJST2 | pLJS containing the 0.77-kb HindIII-HincII fragment from pKK223-3 cloned into HindIII-SmaI site; Apr | This study |
| pUC18Not | Cloning vector containing MCS flanked by NotI sites; Apr | 11 |
| pUC18Not-lux | Contains the 8.35-kb XbaI-PstI fragment from pLJS-lux; Apr | This study |
| pUC18Not-todlux | Contains the 1.8-kb SpeI-XhoI fragment from pLJS-tod; Apr | This study |
| pUT | 5.2-kb cloning vector containing mob RP4, ori R6K and Tn5 tnp lacking NotI sites; Apr | 11 |
| pCR II | 3.9-kb cloning vector for PCR products with 3′ A overhangs; Apr Kmr | Invitrogen |
| pUTK209 | pCR II containing mini-Tn5KmNX with unique NotI and XbaI sites; Apr Kmr | This study |
| pUTK210 | pUT containing mini-Tn5KmNX; Apr Kmr | This study |
| pUTK211 | pUT/mini-Tn5KmT2 containing the 0.8-kb NotI-AvrII rrnB T1T2 fragment; Apr Kmr | This study |
| pUTK214 | pUT/mini-Tn5Kmtod-lux containing the 10.2-kb NotI-XbaI fragment from pUC18Not-todlux; Apr Kmr | This study |
| Strains | ||
| E. coli | ||
| DH5α | F−ϕ80lacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK− mK+) phoA supE44 λ− thi-1 gyrA96 relA1 | Gibco BRL |
| S17-1(λpir) | λpir recA thi pro hsdR M+ RP4:2-Tc:Mu:Km Tn7TprSmr; mobilizing strain for pUT/mini-Tn5 derivatives | 6 |
| INVαF′ | Strain used with TA cloning vector, pCR II; F′ ϕ80lacZαΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK− mK+) phoA supE44 λ− thi-1 gyrA96 relA1 | Invitrogen |
| P. putida | ||
| F1 | Contains a chromosomally encoded tod operon for toluene degradation | 28 |
| TVA8 | F1 containing a mini-Tn5Kmtod-lux insertion in the chromosome; Kmr | This study |