Extended Data Table 5.
Plasmids
| Plasmid | Description | Restriction sites used | Primers | Source, reference |
|---|---|---|---|---|
| pRARE | E. coli Rosetta (DE3) plasmid, encodes rare tRNAs, CamR | Novagen | ||
| pET-52b(+) | T7 RNA polymerase based expression vector, AmpR | Novagen | ||
| pWUR627 | T. thermophilus HB8 ago with N-term. strep(II)-tag in pET-52b(+) expression vector for TtAgo | KpnI | AgoFW | This study |
| NotI | AgoRV | |||
| pWUR641 | pWUR627, ago active site residue substituted (D546A) | - | BG3456 BG3457 | This study |
| pWUR642 | pWUR641, ago active site residue substituted (D478A) | - | BG3454 | This study |
| Expression vector for TtAgoDM(D478A,D546A) | BG3455 | |||
| pCDF-1b | T7 RNA polymerase based expression vector, SmR | Novagen | ||
| pWUR702 | strep(II)-ago insert from pWUR627 inserted in pCDF-1b | AvrII | BG4207 | This study |
| Expression vector for TtAgo | NcoI | BG4208 | ||
| pWUR703 | strep(II)-agodm(D478A,D546A) insert from pWUR642 inserted in pCDF-1b | AvrII | BG4207 | This study |
| Expression vector for TtAgoDM(D478A,D546A) | NcoI | BG4208 | ||
| pUC18 | AmpR | Thermo scientific | ||
| pUC19 | AmpR | Thermo scientific | ||
| pWUR673 | 2.4kb downstream sequence of ago inserted in pUC18 | XbaI | BG3528 | This study |
| EcoRI | BG3529 | |||
| pWUR674 | 1kb upstream sequence of ago inserted in pWUR673 | HindIII | BG3524 | This study |
| SalI | BG3525 | |||
| pWUR675 | ago with N-terminal strep(II)-tag inserted in pWUR674 | SalI | BG3526 | This study |
| XbaI | BG3527 | |||
| pWUR676 | KanR marker with pSLPa promoter inserted in pWUR675 | XbaI | BG3563 | This study |
| XbaI | BG3564 | |||
| pK18 | Recombination vector | 27 | ||
| pWUR701 | Insert from pWUR674 transferred to pK18 | HindIII | This study | |
| EcoRI | ||||
| pMHPnqosGFP | E. coli / T. thermophilus shuttle vector, HygR, sGFP under control of Pnqo promoter | 30 | ||
| pMKPnqosGFP | E. coli / T. thermophilus shuttle vector, KanR, sGFP under control of Pnqo promoter | 30 | ||
| pMK184 | E. coli / T. thermophilus shuttle vector, KanR | 31 | ||
| pFU98 | pSC101 ori, rbs-luxCDABE, CamR | 29 | ||
| pWUR677 | pFU98, CamR marker replaced by HygR marker | SacI | BG3870 | This study |
| NheI | BG3871 | |||
| pWUR704 | pWUR677, rbs-luxCDABE replaced by annealed BG4262-BG4263 | NotI | BG4262 | This study |
| SalI | BG4263 | |||
| pWUR705 | pWUR677, rbs-luxCDABE replaced by annealed BG4264-BG4265 | NotI | BG4264 | This study |
| SalI | BG4265 | |||
| PWUR708 | pWUR677, rbs-luxCDABE replaced by annealed BG3467-3468 insert | XbaI | BG3467 | This study |
| HindIII | BG3468 | |||