TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Properties | Source or reference |
|---|---|---|
| Strains | ||
| E. coli BL21(DE3) | F−ompT hsdSB(rB− mB−) dcm gal λ(DE3) | Merck Biosciences |
| S. aureus RN4220 | 23 | |
| Plasmids | ||
| pIL2 | Wild-type IS256::icaC insertion cloned into Gram-positive/Gram-negative shuttle vector pRB473 | 27 |
| pIL2 IS256Δtnp | pIL2 carrying IS256Δtnp::icaC with an internal 20-bp deletion of the transposase gene | 27 |
| pIL2-NCR LΔ2 | pIL2 vectors carrying IS256 copies with 2- and 4-bp deletions at the left-hand (NCR L) and right-hand (NCR R) IS256 termini, respectively | This study |
| pIL2-NCR LΔ4 | ||
| pIL2-NCR RΔ2 | ||
| pIL2-NCR RΔ4 | ||
| pIL2-NCR LΔ2/NCR RΔ2 | ||
| pIL2-NCR LΔ2/NCR RΔ4 | ||
| pIL2-NCR LΔ4/NCR RΔ2 | ||
| pIL2-NCR LΔ4/NCR RΔ4 | ||
| pCAL-n-FLAG | Expression vector encoding the CBP and FLAG epitope; ori pBR322 blaZ | Stratagene |
| pCAL-n-FLAG-tnp | pCAL-n-FLAG expressing IS256 transposase | This study |
| pCAL-n-FLAG-tnp1-130 | pCAL-n-FLAG expressing the N terminus of the IS256 transposase (aa 1 to 130) | This study |
| pCAL-n-FLAG-tnp100-230 | pCAL-n-FLAG expressing an internal fragment of the IS256 transposase (aa 100 to 230) | This study |
| pCAL-n-FLAG-tnp200-390 | pCAL-n-FLAG expressing the C terminus of the IS256 transposase (aa 200 to 390) | This study |
| pCAL-n-FLAG-tnp L103P | pCAL-n-FLAG vectors expressing IS256 transposase derivatives with various amino acid substitutions | This study |
| pCAL-n-FLAG-tnp Y111A | ||
| pCAL-n-FLAG-tnp G114W | ||
| pCAL-n-FLAG-tnp T117A | ||
| pCAL-n-FLAG-tnp R118A | ||
| pCAL-n-FLAG-tnp L127P | ||
| pACYC184 | Cloning vector, ori p15A cat tet; compatible with ColE1- and pMB-1-related plasmids | Fermentas |
| pACYC184-Δtnp | pACYC184 vector carrying the transposase inactivated IS256 copy from pIL2 IS256Δtnp | This study |