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Table 1 Strains and plasmids employed in this study

From: Expansion of the genetic toolkit for metabolic engineering of Clostridium pasteurianum: chromosomal gene disruption of the endogenous CpaAI restriction enzyme

Strains or plasmids Relevant characteristics Source or reference
Strains   
Escherichia coli DH5α FendA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(r K m K +), λ Lab stock
Escherichia coli ER1821 FendA1 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   
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
  1. ApR: ampicillin resistant; CmR: chloramphenicol resistant; EmR: erythromycin resistant; KmR: kanamycin resistant; TmR: thiamphenicol resistant.