Table 1.
Primers and plasmids used for assays in the present study
| Ampr, ampicillin resistant; Carr, chloramphenicol resistant. | ||
|---|---|---|
| Bacterial strains, primers and plasmids | Relevant characteristics | Source or reference |
| Strains | ||
| E . coli | ||
| DH5α | l2 f80d/lacZDM15 D(lacZYA-argF)U169 recA1 endA1 hsdR17 (rK2 mK1) supE44 thi-1 gyrA relA1 | Gibco |
| Bl21(DE3)-RIL | B F-ompT hsdS(rB–mB–) dcm + Tetr E. coli gal λ (DE3) endA Hte [argU ileY leuW Camr] | Stratagene |
| Walker cells-RIL (C43) | F-ompT gal hsdSB (rB-mB-) dcm lon _DE3 endA Hte [argU ileY leuW Camr] | [38] |
| S. acidocaldarius | ||
| DSM639 | Hyperthermo-acidophilic crenarchaeon which grows optimally at 80°C and pH 2–3 | DSMZ |
| Primers | ||
| FlaI713 | 5′-GGGGGCATATGAGCTTCGTGGAAGAC-3′; forward primer for FlaI containing an NdeI restriction site (underlined) | The present study |
| FlaI714 | 5′-GGGGGCTCGAGTCAAATATTACTGACATATTTTATC-3′; reverse primer for FlaI containing a XhoI restriction site (underlined) | The present study |
| FlaI728 | 5′- CCCCCGAATTCGATGAGCTTCGTGGAAGAC-3′; forward primer for FlaI containing an EcoRI restriction site (underlined) | The present study |
| FlaI729 | 5′-CCCCCAAGCTTTCAAATATTACTGACATATTTTATC-3′; reverse primer for FlaI containing a HindIII restriction site (underlined) | The present study |
| FlaI742 | 5′-ATCTGGAGCGACAACTAC-3′; overlapping internal forward primer for the K268A mutation in FlaI | The present study |
| FlaI743 | 5′-AATGTAGTTGTCGCTCCAG-3′; overlapping internal reverse primer for the K268A mutation in FlaI | The present study |
| FlaI782 | 5′-TATTGAGGCGACTCCAG-3′; overlapping internal forward primer for the D290A mutation in FlaI | The present study |
| FlaI783 | 5′-TCTGGAGTCGCCTCAATA-3′; overlapping internal reverse primer for the D290A mutation in FlaI | The present study |
| FlaI784 | 5′-ACTTGTGGGAGCAATTAGAG-3′; overlapping internal forward primer for the E336A mutation in FlaI | The present study |
| FlaI785 | 5′-CTTTGTCTCTAATTGCTCCCACAAG-3′; overlapping internal reverse primer for the E336A mutation in FlaI | The present study |
| Plasmids | ||
| pETDuet-1 | Ampr, Carr, expression plasmid containing replicon ColE1 (pBR322) and two MCS (MCS1 and MCS2) | Novagen |
| pSVA251 | Ampr, Carr, pETDuet-1 carrying untagged FlaI in MCS2 using restriction sites NdeI-XhoI | The present study |
| pSVA263 | Ampr, Carr, pETDuet-1 carrying N-terminal His-tagged FlaI in MCS1 using restriction sites EcoRI-HindIII | The present study |
| pSVA265 | Ampr, Carr, pETDuet-1 carrying N-terminal His-tagged FlaIK268A in MCS1 using restriction sites EcoRI-HindIII | The present study |
| pSVA307 | Ampr, Carr, pETDuet-1 carrying N-terminal His-tagged FlaID290A in MCS1 using restriction sites EcoRI-HindIII | The present study |
| pSVA308 | Ampr, Carr, pETDuet-1 carrying N-terminal His-tagged FlaIE336A in MCS1 using restriction sites EcoRI-HindIII | The present study |
| pSVA314 | Ampr, Carr, pETDuet-1 carrying N-terminal His-tagged FlaIK268A/E336A in MCS1 using restriction sites EcoRI-HindIII | The present study |