TABLE 1.
Plasmids and strains used in this study
| Plasmid or strain | Relevant characteristics | Source or reference |
|---|---|---|
| Plasmids | ||
| pCR2.1-TOPO | bla kan; TOPO TA cloning vector | Invitrogen |
| pLOI2302 | AscI linkers inserted into pUC19 at NdeI and SapI sites | 40 |
| pLOI2224 | kan; R6K conditional integration vector | 25 |
| pLOI2065 | bla; FRT-tet-FRT cassette | 40 |
| pGID150 | ery, source of ldhL gene from P. acidilactici | 15 |
| pLOI2392 | kan bla ldhA; ldhA (PCR) from E. coli W3110 cloned into pCR2.1-TOPO | This study |
| pLOI2393 | kan bla ldhL; ldhL (PCR) from pGID150 cloned into pCR2.1-TOPO vector | This study |
| pLOI2394 | bla; pLOI2302 after deletion of multiple cloning site (KpnI-HindIII) | This study |
| pLOI2395 | bla; EcoRI fragment (ldhA) from pLOI2392 cloned into pLOI2394 at EcoRI | This study |
| pLOI2396 | kan bla; ldhL-FRT-tet-FRT KpnI-SmaI fragment (FRT-tet-FRT) from pLOI2065 cloned into KpnI-BamHI (blunted) sites of pLOI2393 | This study |
| pLOI2397 | kan bla; ldhA′-ldhL-FRT-tet-FRT-′ldhA ApaI (blunted)-KpnI fragment (ldhL-FRT-tet-FRT) from pLOI2396 cloned into ldhA at HincII-KpnI sites of pLOI2395 | This study |
| pLOI2398 | kan; ldhA′-ldhL-FRT-tet-FRT-′ldhA AscI fragment (ldhA′-ldhL-FRT-tet-FRT-′ldhA) from pLOI2397 cloned into AscI site of pLOI2224 | This study |
| Strains | ||
| DH5α | ΔlacZM15 recA | Bethesda Research Laboratory |
| TOP10F′ | lacIq (episome) | Invitrogen |
| S17-1 | thi pro recA hsdR RP4-2 tet::Mu aphA::Tn7 λpir | 35 |
| SZ63 | W3110 ΔfocA-pflB::FRT ΔfrdBC ΔadhE::FRT ackA::FRT | 40 |
| SZ75, SZ76, SZ77, and SZ78 | tet W3110 ΔfocA-pflB::FRT ΔfrdBC ΔadhE::FRT ackA::FRT ΔldhA::(ldhL-frt-tet-frt) | This study |
| SZ79, SZ80, SZ81, and SZ82 | W3110 ΔfocA-pflB::FRT ΔfrdBC ΔadhE::FRT ackA::FRT ΔldhA::(ldhL-frt) strains SZ75 to SZ79, respectively, after deletion of tet | This study |
| SZ83, SZ84, SZ85, SZ86, SZ87, SZ88, SZ89, SZ90, SZ91, and SZ92 | Mutants of SZ79 with increased expression of ldhL | This study |