Table 1.
Bacterial strains and plasmids.
| Genotype/Description | Source/Reference | |
|---|---|---|
| Bacterial strains | ||
| XL1 Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZ∆M15 Tn10 (TetR)] | Agilent Technologies |
| BL21(DE3) | E. coli B F–ompT gal [E. coli B is naturally dcm and lon] hsdSB with DE3, a λ prophage carrying the T7 RNA polymerase gene and lacIQ | Sigma-Aldrich |
| MG1655 | ilvG rpb-50 rph-1 DlacU169 | E. coli Genetic Stock Center |
| WL400 | CamR StrepRaraD139 Δ (argF-lac)U169, ptsF25 deoC1 relA1 flbB5301 rpsL150 ΔselD204::cat, conjugation recipient | W. Leinfelder, unpublished data |
| Plasmids | ||
| pLEMO | pACYC184-PrhaBAD-lysY (CamR) | 40 |
| pHT | KanR; pET24d derivative T7 expression vector with N-terminal 6xHis-tag and TEV protease cleavage site | 16 |
| pHT TraE | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE | 16 |
| pKM101ΔTraE | SpecR; pKM101 containing a deletion of the traE locus | Jay E. Gordon & Peter J. Christie, unpublished data |
| pHT TraE R37A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change R37A | This work |
| pHT TraE W40A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change W40A | This work |
| pHT TraE Y106A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change Y106A | This work |
| pHT TraE R110A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change R110A | This work |
| pHT TraE Y113A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change Y113A | This work |
| pHT TraE R176A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change R176A | This work |
| pHT TraE F177A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change F177A | This work |
| pHT TraE W195A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change W195A | This work |
| pHT TraE Y201A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change Y201A | This work |
| pHT TraE Y203A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change Y203A | This work |
| pHT TraE R213A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change R213A | This work |
| pHT TraE F220A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change F220A | This work |
| pHT TraE Y225A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change Y225A | This work |
| pHT TraE R226A | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraE with amino change R226A | This work |
| pHT TraD | KanR; T7 promoter vector for the expression of 6xHis-tagged full-length domain of pKM101 TraD | 16 |
| pMSP1D1 | KanR; pET28a derivate containing mutant (1–11) of MSP1; His-tag on N-terminus followed by spacer sequence and TEV protease site |
Addgene #20,061 |
| pMSP1E3D1 | KanR; pET28a derivate containing extended MSP1D1; contains repeats of helices 4, 5 and 6; N-terminal 7-his tag followed by spacer sequence and TEV protease cleavage site |
Addgene #20,066 |
| pMSP2N2 | KanR; pET28a derivate containing tandem MSP; His-tag on N-terminus followed by spacer sequence and TEV protease site |
Addgene #29,520 |
pMSP1D1, pMSP1E3D1 and pMSP2N2 was a gift from Stephen Sligar (Addgene plasmid #20061, #20066 and #29520, respectively).