TABLE S4 Bacterial strains, plasmids, and oligonucleotide primers used in this study

Strain, plasmid, and primer / Relevant characteristicsa / Source
C. acetobutylicum
ATCC 824 / Wild type / ATCC
PKW / ptb::intron / This study
BKW / buk::intron / This study
EKW / pta::intron / This study
AKW / ack::intron / This study
CKW / ctfB::intron / This study
BEKW / buk::intron pta::intron / This study
CEKW / pta::intron ctfB::intron / This study
PEKW / ptb::intron pta::intron / This study
BBEKW / buk::intron bukII::intron pta::intron / This study
CBEKW / buk::intron pta::intron ctfB::intron / This study
BEKW(pPptbAAD) / BEKW harboring pPptbAAD plasmid / This study
BEKW(pPthlAAD**) / BEKW harboring pPthlAAD** plasmid / This study
E. coli
Top10 / Host for cloning / Invitrogenb
ER2925(pAN1) / ER2925 harboring pAN1 / This study
Plasmids
pAN1 / Cmr Tcr Φ3T I / (1)
pSOS94-Cm / Apr Cmr, PCR template for amplifying catD gene / (2)
pACYC184 / Cmr PCR template for amplifying cat gene / (3)
pJIR750ai / Plasmid containing Clostridium perfringens alpha toxin gene targeted Ll.ltrB / (4)
pIMP1 / Apr Emr C. acetobutylicum-E. coli shuttle vector / (5)
pJY2 / Apr Cmr C. acetobutylicum-E. coli shuttle vector / This study
pTHL1-Cm / Apr Emr Cmr Pthl C. acetobutylicum expression vector / This study
pIMPH / Apr Emr pIMP1 derivative, no HindIII site / This study
pCACYS1 / Apr Emr Pcpb2::Ll.ltrB / This study
pCACYS2 / Apr Emr Pthl::Ll.ltrB / This study
pCACYS3 / Apr Emr Cmr Ll.ltrB cat::ltrA, knockout vector / This study
pCACYS3-PtbKO / Apr Emr ptb-targeted-Ll.ltrB / This study
pCACYS3-BukKO / Apr Emr buk-targeted-Ll.ltrB / This study
pCACYS3-BukIIKO / Apr Emr bukII-targeted-Ll.ltrB / This study
pCACYS3-PtaKO / Apr Emr pta-targeted-Ll.ltrB / This study
pCACYS3-AckAKO / Apr Emr ack-targeted-Ll.ltrB / This study
pCACYS3-CtfBKO / Apr Emr ctfB-targeted-Ll.ltrB / This study
pJY2-PtaKO / Apr Thr, pta-targeted-Ll.ltrB / This study
pJY2-BukKO / Apr Thr, buk-targeted-Ll.ltrB / This study
pJY2-CtfBKO / Apr Thr, ctfB-targeted-Ll.ltrB / This study
pIMP1exter / Apr Emr Pptb TTctfB C. acetobutylicum expression vector / This study
pPptbAAD / Apr Emr Pptb::adhE1::TTctfB / This study
pPthlAAD** / Apr Emr Cmr Pthl::adhE1D485G / This study
Primers / (Sequence; 5’ to 3’) / (Description)
Universal-EBS / CGAAATTAGAAACTTGCGTTCAGTAAAC / Internal intron sequence
ptb87s-IBS / AAAAAAGCTTATAATTATCCTTACAAGACGAGCCAGTGCGCCCAGATAGGGTG / Containing ptb targeted IBS1 and IBS2 sequences, HindIII
ptb87s-EBS2 / TGAACGCAAGTTTCTAATTTCGGTTTCTTGTCGATAGAGGAAAGTGTCT / Containing ptb targeted EBS2 sequence
ptb87s-EBS1d / CAGATTGTACAAATGTGGTGATAACAGATAAGTCGAGCCAGTTAACTTACCTTTCTTTGT / Containing ptb targeted EBS1 sequence, BsrGI
buk426s-IBS / AAAAAAGCTTATAATTATCCTTATCAAGCATATCAGTGCGCCCAGATAGGGTG / Containing buk targeted IBS1 and IBS2 sequences, HindIII
buk426s-EBS2 / TGAACGCAAGTTTCTAATTTCGGTTCTTGATCGATAGAGGAAAGTGTCT / Containing buk targeted EBS2 sequence
buk426s-EBS1d / CAGATTGTACAAATGTGGTGATAACAGATAAGTCATATCAGGTAACTTACCTTTCTTTGT / Containing buk targeted EBS1 sequence, BsrGI
bukII421s-IBS / AAAAAAGCTTATAATTATCCTTAGAGGACATAGCTGTGCGCCCAGATAGGGTG / Containing bukII targeted IBS1 and IBS2 sequences, HindIII
bukII421s-EBS2 / TGAACGCAAGTTTCTAATTTCGATTTCCTCTCGATAGAGGAAAGTGTCT / Containing bukII targeted EBS2 sequence
bukII421s-EBS1d / CAGATTGTACAAATGTGGTGATAACAGATAAGTCATAGCTAGTAACTTACCTTTCTTTGT / Containing bukII targeted EBS1 sequence, BsrGI
pta81s-IBS / AAAAAAGCTTATAATTATCCTTAGAAGACAAAAGAGTGCGCCCAGATAGGGTG / Containing pta targeted IBS1 and IBS2 sequences, HindIII
pta81s-EBS2 / TGAACGCAAGTTTCTAATTTCGATTTCTTCTCGATAGAGGAAAGTGTCT / Containing pta targeted EBS2 sequence
pta81s-EBS1d / CAGATTGTACAAATGTGGTGATAACAGATAAGTCAAAAGAAATAACTTACCTTTCTTTGT / Containing pta targeted EBS1 sequence, BsrGI
ackA1027s-IBS / AAAAAAGCTTATAATTATCCTTATGTTTCTGCATAGTGCGCCCAGATAGGGTG / Containing ack targeted IBS1 and IBS2 sequences, HindIII
ackA1027s-EBS2 / TGAACGCAAGTTTCTAATTTCGGTTAAACATCGATAGAGGAAAGTGTCT / Containing ack targeted EBS2 sequence
ackA1027s-EBS1d / CAGATTGTACAAATGTGGTGATAACAGATAAGTCTGCATATTTAACTTACCTTTCTTTGT / Containing ack targeted EBS1 sequence, BsrGI
ctfB357s-IBS / AAAAAAGCTTATAATTATCCTTAAATATCGCCAATGTGCGCCCAGATAGGGTG / Containing ctfB targeted IBS1 and IBS2 sequences, HindIII
ctfB357s-EBS2 / TGAACGCAAGTTTCTAATTTCGGTTATATTCCGATAGAGGAAAGTGTCT / Containing ctfB targeted EBS1 sequence
ctfB357s-EBS1d / CAGATTGTACAAATGTGGTGATAACAGATAAGTCGCCAATTGTAACTTACCTTTCTTTGT / Containing ctfB targeted EBS2 sequence, BsrGI
Cdf-cat-F / TATGAGCTCTGTGGATGGAGTTAAGTCAG / SacI
Cdf-cat-R / CGCATCGATTTAACTATTTATCAATTCCTG / ClaI
ptb-confirm-F / CATGAAGGTAAAGAGCAAAG / Knockout confirm for ptb
ptb-confirm-R / GCTCTTGAAGTTAAAACAAC / Knockout confirm for ptb
buk-confirm-F / ATAATCAATCCTGGCTCG / Knockout confirm for buk
buk-confirm-R / ACCTTCTGCAAGTGCAAG / Knockout confirm for buk
bukII-confirm-F / AAATGGAGGACTTTATTATG / Knockout confirm for bukII
bukII-confirm-R / TTTAGCTTCTTCTTGTCCAC / Knockout confirm for bukII
pta-confirm-F / GGAGTGTGCTAAGCAAGAC / Knockout confirm for pta
pta-confirm-R / GCCTGAACAACAGTTATAGC / Knockout confirm for pta
ackA-confirm-F / TGCGGTAGTTCATCAATC / Knockout confirm for ack
ackA-confirm-R / CTTGCCTACTATATCTTTGG / Knockout confirm for ack
ctfB-confirm-F / CCTAGCGAAAGAAATAATAGCC / Knockout confirm for ctfB
ctfB-confirm-R / CTGCAGCAGTTAAAGACCTTATTTC / Knockout confirm for ctfB
Ll.LtrB-F / CCCCCGGGGGAATTCCATTATTAACATGATAAATCC / AvaI
Ll.LtrB-R / CCCCCGGGGGGGCTTGTAGATATGACGACAGG / AvaI
Pthl-F / GCTCTAGAAGAATGAAGTTTCTTATGCACAAG / XbaI
Pthl-R / GGCCCCAAGCTTTCTAACTAACCTCCTAAATTTTGATAC / HindIII
Cat-F / AAAAAAGCTTTTATTCAGGCGTAGCACCAG / HindIII
Cat-R / CAGATTGTACATCCTGGTGTCCCTGTTGATAC / BsrGI
AdhE1-F / ATAGTCGACATGAAAGTCACAACAGTAAAGG / SalI
AdhE1-R / CGCGAATTCTTAAGGTTGTTTTTTAAAACA / EcoRI
Pptb-F / TATCTGCAGTGTGGATGGAGTTAA / PstI
Pptb-R / ATTGTCGACTTTAATCCCTCCTTT / SalI
TTthl-F / CGCGAATTCGGGCCCATATCCAATGAACTTAGACC / EcoRI
TTthl-R / CACCATATGGCCTAGAGCTGAAGTTAT / NdeI
AdhE1-F0 / AAAACTGCAGTTTATGAAAGTCACAACAGTAAAGG / PstI
D485G-R / TAAATTATAGGGGTCACTACCAGTAACTATAAAGGCTC / Bold “ACC” presents the codon mutated
D485G-F / GAGCCTTTATAGTTACTGGTAGTGACCCCTATAATTTA / Bold “GGT” presents the codon mutated
AdhE1-R0 / CCCCCGGGGGTTGAAATATGAAGGTTTAAGGTTG / AvaI

a Ap, ampicillin; Em, erythromycin, Th, thiamphenocol

b Invitrogen, Invitrogen Corporation, Carlsbad, Calif.

SI References

1. Mermelstein LD, Papoutsakis ET. 1993. In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 59:1077-1081.

2. Sillers R, Chow A, Tracy B, Papoutsakis ET. 2008. Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance. Metab. Eng. 10:321-332.

3. Rose RE. 1988. The nucleotide sequence of pACYC184. Nucleic Acids Res 16:355.

4. Chen Y, McClane BA, Fisher DJ, Rood JI, Gupta P. 2005. Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron. Appl. Environ. Microbiol. 71:7542-7547.

5. Lee JY, Jang YS, Lee J, Papoutsakis ET, Lee SY. 2009. Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production. Biotechnol. J. 4:1432-1440.

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