TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Descriptiona | Source or reference |
|---|---|---|
| E. coli strains | ||
| DH5α | λ− φ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK−) supE44 thi-1 gyrA relA1 | Bethesda Research Laboratories |
| RV308 | Standard recombinant production strain used for HCDC | ATCC 31608 |
| Plasmids | ||
| pJT19bla | RK2-based expression vector containing the Pm promoter with the gene encoding the regulatory protein XylS and bla as a reporter gene for Pm; Kmr; 8.1 kb | 34 |
| pJB785TT | Vector containing the strong transcriptional terminator rrnBT1T2 | 28 |
| pLITMUS28 | Apr; 2.8 kb | New England Biolabs |
| pHE158 | The rrnBT1T2 transcription terminator from pJB785TT was cloned into the BglII/EcoRI sites of pLITMUS28; the EcoRI site was removed, and a SalI linker was cloned into the BglII site; Apr; 3.2 kb | This study |
| pJB658 | RK2-based expression vector containing the Pm promoter and the gene encoding the regulatory protein XylS; Kmr; 7.1 kb | 3 |
| pIB1 | pJB658 derivative in which a XhoI linker was introduced in the blunted AgeI site; Apr; 6.8 kb | This study |
| pIB2 | The SalI/PstI fragment from pHE158 containing the rrnBT1T2 transcription terminator was cloned into the XhoI/NsiI sites of pIB1; Apr; 6.6 kb | This study |
| pIB3 | The XbaI/MunI fragment from pIB2 containing rrnBT1T2 was cloned into the same sites of pJT19bla; Kmr; 8.1 kb | This study |
| pIB4 | pIB3 derivative with a translational down-mutation in the Pm mRNA leader sequence; Kmr; 8.1 kb | This study |
| pIB5 | An AflIII site was introduced in pIB4 upstream of the Pm promoter by site-specific mutagenesis; Kmr; 8.1 kb | This study |
| pIB6 | The SpeI site downstream of the Pm promoter of pIB5 was changed to a BspLU11I site by site-specific mutagenesis; Kmr; 8.1 kb | This study |
| pIB11 | Identical to pIB6 except that the translational down-mutation in the Pm leader sequence is exchanged with nucleotide of the wild-type sequence; Kmr; 8.1 kb | This study |
| pJT19luc | RK2-based expression vector containing the Pm promoter with the gene encoding the regulatory protein XylS and luc as a reporter gene for Pm; Kmr; 8.1 kb | 34 |
| pOY9 | pIB6 derivative in which the NdeI/BamHI fragment containing bla was substituted with NdeI/BamHI fragment containing luc from pJT19luc; Kmr; 8.8 kb | This study |
| pJB658CelB | Derivative of pJB658 with celB cloned in the NdeI site downstream of Pm; Kmr; 8.7 kb | 3 |
| pLB10 | pIB6 derivative in which the NdeI/BamHI fragment containing bla was substituted with the NdeI/BamHI fragment containing celB from pJB658CelB; Kmr; 9.0 kb | This study |
| pBR322 | ColE1 replicon; Apr; 4.4 kb | New England Biolabs |
| pIB17 | Derivative of pIB6 in which xylS was deleted by BglII/DraIII digestion, filled in and relegated; Kmr; 6.4 kb | This study |
| pGM29pelB | pJB658-based expression vector with a pelB signal sequence preceding the coding sequence of the GM-CSF gene; Apr; 8.8 kb | 31 |
| pAT50 | Identical to pGM29pelB except that an NdeI site was removed from the trfA coding sequence; Apr 8.8 kb | This study |
| pAT52 | Derivative of pAT50 in which the NdeI/XbaI Pm region was exchanged with the corresponding region from pIB11; Apr; 8.8 kb | This study |
| pMV1 | Identical to pAT52, except that a BspLU11I/XbaI fragment (wild-type Pm sequence) was exchanged with Pm mutant sequence ML1-18 (Fig. 3); Apr; 8.8 kb | This study |
| pMV2 | Identical to pAT52, except that a BspLU11I/XbaI fragment (wild-type Pm sequence) was exchanged with Pm mutant sequence ML1-16 Fig. 3); Apr; 8.8 kb | This study |
| pMV3 | Identical to pAT52, except that a BspLU11I/XbaI fragment (wild-type Pm sequence) was exchanged with Pm mutant sequence ML1-14 (Fig. 3); Apr; 8.8 kb | This study |
a
Apr, ampicillin resistance; Kmr, kanamycin resistance.