Strains or plasmids | Relevant characteristics | Source or reference |
---|---|---|
Strains | Â | Â |
Escherichia coli DH5α | F−endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(r K −m K +), λ− | Lab stock |
Escherichia coli ER1821 | F−endA1 glnV44 thi-1 relA1? e14−(mcrA−) rfbD1? spoT1? Δ(mcrC-mrr)114::IS10 | Lab stock; New England Biolabs |
Clostridium pasteurianum ATCC 6013 | Wild-type | American Type Culture Collection |
Clostridium pasteurianum ΔcpaAIR | Disruption mutant generated by inserting the Ll.ltrB intron into position 176a of the cpaAIR gene encoding the CpaAI RENase | This study |
Plasmids | Â | Â |
pFnuDIIMKn | M.FnuDII methyltransferase plasmid for methylation of E. coli-C. pasteurianum shuttle vector s (KmR ; p15A ori) | [15] |
pIMP1 | E. coli-Clostridium shuttle vector (ApR; ColE1 ori; EmR; repL ori) | [44] |
pMTL85141 | E. coli-Clostridium shuttle vector (CmR/TmR; ColE1 ori; repL ori) | [45] |
pMTL85141ermB | E. coli-Clostridium shuttle vector (CmR/TmR; EmR; ColE1 ori; repL ori) | [15] |
pSY6catP | E. coli-Clostridium shuttle vector expressing the Ll.ltrB intron and ltrA IEP from the Clostridium acetobutylicum ptb promoter (ApR ; CmR/TmR; ColE1 ori; repL ori) | [15] |
pMTL007C-E2 | ClosTron vector expressing the Ll.ltrB intron with RAM and ltrA IEP from the Clostridium sporogenes fdx promoter (CmR/TmR; ColE1 ori; repH ori; EmR RAM) | [18] |
pMTL007C-E6 | repL derivative of pMTL007C-E2 | This study |
pltrB | ltrA-deletion derivative of pSY6catP | This study |
pltrA | Ll.ltrB-deletion derivative of pSY6catP | This study |
pDelPptb | Derived by deleting the −35 and −10 signals of the C. acetobutylicum ptb promoter from plasmid pSY6catP | This study |
pDel2dcm | Derived by mutating the two E. coli Dcm restriction recognition sites downstream of the ltrA coding sequence within plasmid pSY6catP | This study |
pMB | Derived by replacing a 1,661 bp MfeI + BstAPI restriction fragment of pSY6catP with a 48 bp stuffer fragment | This study |
pNM | Derived by replacing a 688 bp NheI + MfeI restriction fragment of pSY6catP with a 48 bp stuffer fragment | This study |
pNS | Derived by replacing a 1,434 bp NheI + SacII restriction fragment of pSY6catP with a 48 bp stuffer fragment | This study |
pFrag1 | Derived by replacing a 1,332 bp BglII + EcoO109I restriction fragment of pSY6catP with a 589 bp ltrA region | This study |
pFrag2 | Derived by replacing a 1,332 bp BglII + EcoO109I restriction fragment of pSY6catP with a 363 bp ltrA region | This study |
pFrag3 | Derived by replacing a 1,332 bp BglII + EcoO109I restriction fragment of pSY6catP with a 574 bp ltrA region | This study |
pSY334 | Derived by subcloning a 334 bp SacII + AatII fragment of the ltrA coding sequence into plasmid pMTL85141 | This study |
pMut98 | pSY6catP derivative possessing 98 silent mutations in the ltrA coding sequence | This study |
pMTLCP-E2 | Derived by subcloning a 1,427 bp MscI + AclI restriction fragment of pMut98 into plasmid pMTL007C-E2 | This study |
pMTLCP-E6 | Derived by subcloning a 1,427 bp MscI + AclI restriction fragment of pMut98 into plasmid pMTL007C-E6 | This study |
pDelCpaAII | Deletion of the unique CpaAII recognition site within pSY6catP by introducing three silent point mutations | This study |
pCpaAII | Introduction of a unique CpaAII recognition site within pMTL85141 by introducing two point mutations | This study |
pSYCP-cpaAIR | Derived by replacing the ptb promoter of pMut98 with a thl promoter and targeting the Ll.ltrB intron to position 176a of the cpaAIR gene | This study |
pMTLCP-E2-cpaAIR | Targeting construct of plasmid pMTLCP-E2 for disruption of the cpaAIR gene at position 176a | This study |
pMTLCP-E6-cpaAIR | Targeting construct of plasmid pMTLCP-E6 for disruption of the cpaAIR gene at position 176a | This study |