TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Description | Source or reference |
|---|---|---|
| E. coli | ||
| DH5α | lacZΔM15 recA | Bethesda Research Laboratory |
| TOP10F′ | hsdR mcrA lacZΔM15 endA recA; F′ tet lacI | Invitrogen |
| HB101 | recA lacY | ATCC 37159 |
| S17-1 | thi pro recA hsdR RP4-2-tet::Mu aphA::Tn7 λpir | 12 |
| Z. mobilis CP4 | Prototrophic | 18 |
| K. oxytoca | ||
| M5A1 | Prototrophic | 40 |
| P2 | pfl::pdc adhB cat | 40 |
| SZ6 | pfl::pdc adhB cat; integrated celZ tet | 42 |
| SZ12 | pfl::pdc adhB cat; integrated celZ celY kan | This study |
| SZ21 | pfl::pdc adhB cat; integrated celZ celY | This study |
| SZ22 | pfl::pdc adhB cat; integrated celY celZ::aac | This study |
| Plasmids | ||
| pUC18 | bla cloning vector | New England Biolabs |
| pUC19 | bla cloning vector | New England Biolabs |
| pCR2.1-TOPO | TA cloning vector, bla kan | Invitrogen |
| pMH18 | bla celY from E. chrysanthemi 3937 | 13 |
| pHPΩ45aac | bla aac source of apramycin gene | 4 |
| pBR322 | bla tet cloning vector | New England Biolabs |
| pRK2013 | kan, mobilizing plasmid | American Type Culture Collection |
| pCPP2006 | spm, ∼40-kbp fragment containing out genes from E. chrysanthemi EC16 | 14 |
| pFT-A | bla flp low-copy vector containing recombinase and temperature-conditional pSC101 replicon | 23 |
| pLOI2224 | kan integration vector containing conditional R6K replicon and two FRT sites | 23 |
| pLOI2302 | pUC19 containing AscI linkers inserted into blunt NdeI and SapI sites | 42 |
| pLOI2307 | bla celZ gene and a surrogate promoter from Z. mobilis DNA | 44 |
| pLOI2311 | PCR fragment containing celY gene cloned into pCR2.1-TOPO, expressed from lac promoter | 43 |
| pLOI2316 | pUC18 containing the celY gene on a Klenow-treated EcoRI fragment from pLOI2311 inserted into a HincII site; celY expressed from the lac promoter | This study |
| pLOI2317 | EcoRI-HindIII fragment from pLOI2316 inserted into the corresponding sites of pLOI2302 | This study |
| pLOI2318 | Sau3A1 fragment of Z. mobilis DNA exhibiting promoter activity inserted into the BamHI site of pLOI2317 | This study |
| pLOI2319 | Sau3A1 fragment of Z. mobilis DNA exhibiting promoter activity inserted into the BamHI site of pLOI2317 | This study |
| pLOI2320 | Sau3A1 fragment of Z. mobilis DNA exhibiting promoter activity inserted into the BamHI site of pLOI2317 | This study |
| pLOI2323 | Sau3AI fragment of Z. mobilis DNA exhibiting promoter activity inserted into the BamHI site of pLOI2317 | This study |
| pLOI2342 | Sau3A1 fragment of Z. mobilis DNA exhibiting promoter activity inserted into the BamHI site of pLOI2317 | This study |
| pLOI2348 | Random EcoRI fragment of K. oxytoca M5A1 DNA cloned into EcoRI site of pLOI2323 | This study |
| pLOI2349 | EcoRI linker inserted into the Klenow-treated SphI site of pLOI2307 | This study |
| pLOI2350 | EcoRI fragment (celZ and surrogate promoter) from pLOI2349 inserted into the EcoRI site of pLOI2224 | This study |
| pLOI2352 | AscI fragment (K. oxytoca fragment, Z. mobilis promoter fragment and celY) from pLOI2348 inserted into the AscI site of pLOI2350 | This study |
| pLOI2353 | EcoRI-AvaI fragment (tet gene) from pBR322 inserted into the ClaI site of pFT-A | This study |
| pLOI2354 | pUC19 derivative in which the multiple cloning sites from HindIII to SmaI were deleted by digestion, Klenow treatment, and self-ligation | This study |
| pLOI2355 | EcoRI fragment (celZ gene) from pLOI2349 inserted into the EcoRI site of pLOI2354 | This study |
| pLOI2356 | SmaI fragment containing the apramycin resistance gene (aac gene) from pHPΩ45aac inserted into the T4 DNA polymerase-treated PstI site of pLOI2355, disrupting the celZ gene | This study |
| pLOI2357 | EcoRI fragment (aac and disrupted celZ) from pLOI2356 inserted into the EcoRI site of pLOI2224 | This study |
| pLOI2358 | Subclone of pLOI2323 in which the internal PstI fragment was deleted, used for sequencing | This study |
| pLOI2359 | Subclone of pLOI2323 in which the ClaI-HindIII fragment was deleted, used for sequencing | This study |