TABLE 1.
Strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Relevant features | Reference or source |
|---|---|---|
| E. coli | ||
| DH5α | supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | Laboratory collection |
| S17-1 | hsdR thi pro recA; RP4 integrated into the chromosome (kan::Tn7 ter::Mu) | 41 |
| BL21(DE3) | F−ompT [lon] hsdSB (rB− mB−; B strain); λDE3 lysogen with T7 RNA polymerase | Novagen |
| P. aeruginosa | ||
| PAO1 | Prototroph | 14 |
| PAO-CB | cysB::Gm | This study |
| CHA-CB | cysB::Gm | 6 |
| AX18 | cysI::miniTn5Tc | 17 |
| AC309 | cysN::miniTn5Tc | 17 |
| SLF2 | msuD::xylE, xylE antiparallel to msuD | This study |
| SLF3 | msuD::xylE, xylE parallel to msuD | This study |
| SLF4 | cysI::miniTn5Tc msuD::xylE | This study |
| SLF5 | cysN::miniTn5Tc msuD::xylE | This study |
| Plasmids | ||
| pBluescript II KS | Cloning vector, Ampr | Stratagene |
| pET24b | Expression vector, Kmr | Novagen |
| pIA3 | pUC18Mob cysB::Gm | 6 |
| pEX100T | ColE1 AmprsacB oriT | 39 |
| pUCGM1 | ColE1 Ampr Ωaac1 | 38 |
| pX1918GT | ColE1 AmprxylE Gmr | 39 |
| pME3087 | ColE1 TcroriT | 29 |
| pME4006 | pLAFR3 cosmid carrying the msu locus | This study |
| pME4088 | pME3087 carrying an 8-kb BamHI/HindIII fragment with msuD::xylE | This study |
| pME4095 | pBluescript KS carrying a 7-kb EcoRV fragment with msuEDC | This study |
| pME4503 | pET24b with msuEDC genes | This study |
| pME4529 | pET24b with the msuC gene | This study |
| pME4531 | pET24b with the msuE gene | This study |
| pME4532 | pET24b with msuED genes | This study |
| Oligonucleotides | ||
| 960377 | 5′-CCGAGACGGTCGGCGAAA-3′ | This study |
| 960873 | 5′-CAGGAAACATGGCTTGGGAAAC-3′ | This study |
| M13 | 5′-GTAAAACGACGGCCAGT-3′ | This study |
| SlfA-N | 5′-CCACAGGGAAATCATATGACCAGCC-3′a | This study |
| SlfC-N | 5′-GAAACCCCATATGAATGCGAAG-3′ | This study |
| orfM500 | 5′-CAASRGCCGYATCCGCC-3′ | This study |
| orfM970rev | 5′-TCTTCYTRRTGCGGATAGCC-3′ | This study |
| PA13F1 | 5′-CAAAGTCGTCGCCGTCTCC-3′ | This study |
| PA13R1 | 5′-AAGTCCACCGCCTCGCC-3′ | This study |
a
Nucleotides in boldface were changed from the template sequence to introduce restriction sites.