|
E. coli strains
|
| XL1-Blue |
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)] |
Agilent |
| XL10-Gold |
Tetr Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F′ proAB lacIqZΔM15 Tn10 (Tetr) Amy Camr] |
Agilent |
| JM109 |
endA1, recA1, gyrA96, thi, hsdR17 (rk–, mk+), relA1, supE44, Δ(lac-proAB), [F′ traD36, proAB, laqIqZΔM15]. |
Takara Bio |
| BL21 CodonPlus (DE3)-RIL |
E. coli B F− ompT hsdS(rBmB) dcm+ Tetr gal λ(DE3) endA Hte [argU ileY leuW Camr]
|
Agilent |
|
C. glutamicum strains
|
|
Cg ATCC 13032 |
Wild-type parental strain |
(20) |
|
Cg (ΔnucS) |
Cg 13032 (ΔnucS::aphA-3) (replacement of nucS by a non-polar kanamycin resistance cassette (aphA-3))
|
This work |
|
Cg (nucSΔCter) |
Cg 13032 (nucSΔCter::aphA-3) (replacement of nucS by a copy of nucS with its 15 last nucleotides deleted, followed by a non-polar kanamycin resistance cassette)
|
This work |
|
Cg (ΔnucS) + pXMJ19 |
Cg 13032 (ΔnucS::aphA-3) transformed with the empty pXMJ19 vector |
This work |
|
Cg (ΔnucS) + pHM473 |
Cg 13032 (ΔnucS::aphA-3) transformed with the pHM473 plasmid carrying a wild-type copy of the nucS gene |
This work |
|
Plasmids
|
| pMCS5 |
ori pMB1 (colE1), plac, lacZα, Ampr, MCS of pUC18 / Unable to replicate in C. glutamicum (suicide vector) |
MoBiTec |
| pXMJ19 |
ori pMB1 (colE1), ptac, lacIq, Camr, MCS of pUC18 / E. coli-C. glutamicum (shuttle vector) |
(37) |
| pQE80L |
ori colE1, pT5, lacIq, Ampr, 6xHis tag (N-ter) |
Qiagen |
| pG-KJE8 |
ori p15A, paraB, dnaK, dnaJ, grpE, Camr
|
Takara Bio |
| pUC18K |
ori pMB1 (colE1), plac, lacZα, Ampr, Kanr / Promotor less non-polar kanamycin cassette (aphA-3) cloned into pUC18 |
(38) |
| pHM449 |
nucS of Cg ATCC 13032 cloned into pQE80L (BamHI / PstI) |
This work |
| pHM455 |
aphA-3 cassette flanked with the upstream and downstream regions of nucS and cloned into pMCS5 (Ampr,Kanr) |
This work |
| pHM473 |
nucS of Cg ATCC 13032 cloned into pXMJ19 (HindIII / EcoRI) |
This work |
| pHM476 |
nucS with its 15 last nucleotides deleted followed by the aphA-3 cassette and cloned into pMCS5 (Ampr,Kanr) |
This work |
| pHM477 |
A stop codon was introduced into nucS in pHM449 to delete five amino acids from the C-terminus of CglEndoMS |
This work |
| pET-Cglβ-clamp |
dnaN of Cg ATCC 13032 cloned into a modified version of pET28a (NdeI / NotI), ori pMB1 (colE1),pT7, Ampr, 6xHis tag (N-ter) |
This work |
