Table 2 Strains, plasmids, and oligonucleotides

Strain

Relevant characteristics

Source or reference

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

Plasmid

Relevant characteristics

Source or reference

pET-20b(+)

E. coli pET-series expression vector (Ap^R; ColE1 ori)

Novagen

pETKnFRT

Derived by inserting the FRT-flanked kan gene of pKD4 into the MCS of pET-20b(+) (Ap^R; ColE1 ori; FRT-Kn^R-FRT)

This study

pFnuDIIM

The M.FnuDII methyltransferase gene of Fusobacterium nucleatum inserted into the tet gene of pACYC184 (p15A ori; Cm^R)

[65]

pFnuDIIMKn

Derived by inserting the FRT-flanked kan gene of pETKnFRT into the cat gene of pFnuDIIM (p15A ori; Kn^R)

This study

pHT3

E. coli-C. pasteurianum shuttle vector containing lacZ from Thermoanaerobacterium thermosulfurogenes EM1 (Ap^R; ColE1 ori; Erm^R; pIM13 ori)

[49]

pIMP1

E. coli-C. pasteurianum shuttle vector (Ap^R; ColE1 ori; Erm^R; pIM13 ori)

[26]

pKD4

Template vector (Ap^R; pR6K ori; FRT-Kn^R-FRT)

[70]

pMTL82151

E. coli-C. pasteurianum shuttle vector (Cm^R; ColE1 ori; pBP1 ori)

[34]

pMTL83151

E. coli-C. pasteurianum shuttle vector (Cm^R; ColE1 ori; pCB102 ori)

[34]

pMTL84151

E. coli-C. pasteurianum shuttle vector (Cm^R; ColE1 ori; pCD6 ori)

[34]

pMTL85141

E. coli-C. pasteurianum shuttle vector (Cm^R; ColE1 ori; pIM13 ori)

[34]

pMTL85141ermB

Derived by insertion of the ermB gene of pIMP1 into pMTL85141

This study

pSC12

E. coli-C. pasteurianum shuttle vector (Cm^R; ColE1 ori; pIM13 ori)

[66]

pSY6

E. coli-C. pasteurianum expression vector carrying the L. lactis ltrB group II intron under control of the C. acetobutylicum ptb promoter, and ltrA ORF (Ap^R; ColE1 ori; Erm^R; pIM13 ori)

[47]

pSY6catP

Derived by replacing the ermB gene of pSY6 with the catP gene from pSC12

This study

Oligonucleotide

Sequence (5’-3’) ^*

KnFRT.BlpI.S

ACACGTGCTCAGC GATTGTGTAGGCTGGAGCTGCTTCG

KnFRT.XhoI.AS

GCCATGCTCGAG ATGAATATCCTCCTTAGTTCCTATTCC

ermB.NdeI.S

ATTACGCATATG TTTGGCTAACACACACGCCATTCC

ermB.PvuI.AS

CTTTTTCGATCG TTTCCGACGCTTATTCGCTTCGCT

catP.BclI.S

GTTTGATCA TGGTCTTTGTACTAACCTGTGG

pSC12.SOE.AS

tacagcatgaccgttaaagtgg

pSC12.SOE.S

ccactttaacggtcatgctgtaAGTGCAAGGTACACTTGCAAAGTAGTGG

catP.ClaI.AS

GGATCGAT CCAACTTAATCGCCTTGCAGCACA

pMTL.seq.S

GGGAGGTCAATCTATGAAATGCG

pMTL.seq.AS

CGGAGCATTTGGCTTTCCTTCCAT