S1 Table. Primers used in the present work. Primers used for ... - PLOS

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Bam. GTGGATCCCCCTCCTTACTATAGAAAATTCG. Primers used for the amplification of promoters of C. thermocellum DSM 1313 cellulosomal genes during ...
S1 Table. Primers used in the present work. Primers used for the construction of pLOXErysigIrsgIBs plasmid # Name Sequence 5'→3' Brief description a CGGTACCCGGGGATCGAAGATCGGCGGACTTGAC P1 Fw.ykoY.fus Amplification upstream region of Bs sigI a TCCTTTCTCGCCTGCCGTGTTTTTTGTAACAAAG P2 Rv.ykoY.fus a GTGACAGGAGCCTCGAGACCTGAATTTATTTAGTTGTGC Amplification downstream region of Bs rsgI P3 Fw.sspD.fus a CGACTCTAGAGGATCTGCAGGAGATCGCCGAACAG P4 Rv.ykrK.fus a GCAGGCGAGAAAGGAGAG P5 Fw.loxerm Amplification lox71-erm-lox66 cassette a CGAGGCTCCTGTCACTGCTTC P6 Rv.loxerm Primers used for the amplification of C. thermocellum DSM 1313 sigI6 and sigI3 genes during the construction of pAX01 derived plasmids # Name Sequence 5'→3' Brief description a GGGGGAAATGGGATCATGGATTGGCATTTTCAAGG P7 Fw.sigI6.fus Amplification of Ct sigI6 gene a CGCGGGAGCTCGATCTCACCGCAAATCCACCTCC P8 Rv.sigI6.fus a GGGGGAAATGGGATCATGCATGGGTTGTTTGTAAATA P9 Fw.sigI3.fus Amplification of Ct sigI3 gene a CGCGGGAGCTCGGATCCTTAGTTATCCATGATACTTTC P10 Rv.sigI3.fus Primers used for the amplification of the promoters of C. thermocellum DSM 1313 sigI factors during the construction of pBS1ClacZ derived plasmids # Name Sequence 5'→3' Brief description GAGAATTCAGCAAGAGCCGATATTAATCG P11 Fw.PsigI1.Eco Amplification promoter of Ct sigI1 TTGGATCCCCAGATCATTCTCCCTTCAG P12 Rv.PsigI1.Bam TAGAATTCCGAATTGAATGGGTTATTTGATAG P13 Fw.PsigI2.Eco Amplification promoter of Ct sigI2 TAGGATCCGGGCTTGAAAGCACAATC P14 Rv.PsigI2.Bam CAGAATTCTAACCGACAAGATTTACATAAC P15 Fw.PsigI3.Eco Amplification promoter of Ct sigI3 GCGGATCCGCCTGTTCACTCCTTCAATAC P16 Rv.PsigI3.Bam GCGAATTCCAAATTTTTAATGCTAAACGTCCAG P17 Fw.PsigI4.Eco Amplification promoter of Ct sigI4

GTGGATCCCAAGTTGGGTTCAAACAAATATAC P18 Rv.PsigI4.Bam CTGAATTCCCATCCGATTCGCTGCAC P19 Fw.PsigI6.Eco Amplification promoter of Ct sigI6 TCGGATCCCACCCCTTATTCTTATATCGATTC P20 Rv.PsigI6.Bam GAGAATTCAAATGGTATCAGCCAAAATTC P21 Fw.PsigI7.Eco Amplification promoter of Ct sigI7 GTGGATCCTCACCCAGTTGCATCAGCAAAG P22 Rv.PsigI7.Bam GTGAATTCTTTCCCGTCACTTTCCGAATC P23 Fw.PsigI8.Eco Amplification promoter of Ct sigI8 GTGGATCCCCCTCCTTACTATAGAAAATTCG P24 Rv.PsigI8.Bam Primers used for the amplification of promoters of C. thermocellum DSM 1313 cellulosomal genes during the construction of pBS1ClacZ derived plasmids # Name Sequence 5'→3' Brief description GTGAATTCGCTCAAGTAAATCAACTCCCAC P25 Fw.Pxyn10Z.Eco Amplification promoter of Ct xyn10Z GAGGATCCGGACCTCATGTAGCAAAC P26 Rv.Pxyn10Z.Bam GTGAATTCTAATAAACGGCAACATAACAC P27 Fw.Pxyn11B.Eco Amplification promoter of Ct xyn11B GCGGATCCCTTTCCCTTCGCCTGTAAATTGAC P28 Rv.Pxyn11B.Bam CAGAATTCATGTGGTCATATTGTCAATGTAC P29 Fw.Pce12A.Eco Amplification promoter of Ct cel2A GCGGATCCTCCCCTCCAACCTTAATATTCCAAAC P30 Rv.Pce12A.Bam GTGAATTCCCGGGTTTCCAACAAAGTGC P31 Fw.PcelE.Eco Amplification promoter of Ct celE GTGGATCCCCCTTCCTATTTGGCTTG P32 Rv.PcelE.Bam GAGAATTCGGATGAAATTTAGTCAATATTACCC P33 Fw.Pcel8A.Eco Amplification promoter of Ct cel8A GAGGATCCCTTCAAACAGTTAATTTTCTCCC P34 Rv.Pcel8A.Bam GTGAATTCGTAATTATTGCATTTTGCCCCC P35 Fw.Pcel9J.Eco Amplification promoter of Ct cel9J GCGGATCCTGCACAATCCATACGC P36 Rv.Pcel9J.Bam GTGAATTCCAATATCCACCTTACTCCGAAC P37 Fw.Pcel9P.Eco Amplification promoter of Ct cel9P GAGGATCCCATGCTTTTACCCCTTTC P38 Rv.Pcel9P.Bam GCGAATTCAACGGGAAACAGGATTTG P39 Fw.Pcel9Q.Eco Amplification promoter of Ct cel9Q GAGGATCCCAAATATTATTATATCATAACTCCATCTC P40 Rv.Pcel9Q.Bam GTGAATTCCCGTGTATTACACTTCGTATG P41 Fw.Pcel9U.Eco Amplification promoter of Ct cel9U GCGGATCCTATTTATTATATTATTTATCTATATATATT P42 Rv.Pcel9U.Bam

P43 P44 P45 P46 P47 P48 P49 P50 P51 P52 P53 P54 P55 P56 P57 P58 P59 P60 P61 P62 P63 P64 P65 P66 P67 P68

Fw.Pcel9V.Eco Rv.Pcel9V.Bam Fw.Pcel48S.Eco Rv.Pcel48S.Bam Fw.Pce8.Eco Rv.Pce8.Bam Fw.PcenC.Eco Rv.PcenC.Bam Fw.PcipA.Eco Rv.PcipA.Bam Fw.PcseP.Eco Rv.PcseP.Bam Fw.Ppelb2.Eco Rv.Ppelb2.Bam Fw.PpilZ.Eco Rv.PpilZ.Bam Fw.Ppl11.Eco Rv.Ppl11.Bam Fw.PrsgI5.Eco Rv.PrsgI5.Bam Fw.PrsgI9.Eco Rv.PrsgI9.Bam Fw.PsdbA.Eco Rv.PsdbA.Bam Fw.Pxgh7A.Eco Rv.Pxgh7A.Bam

CGAAAG GTGAATTCTCCCGAAAATCTACAAATTCC GTGGATCCCGCAAATTAACATAAACTCGC TAGAATTCGCACAAGAACTTCAAATGTTTCC TGGGATCCACGTCAAATGCAGCTGAATCAC GCGAATTCAAATCCCCGCTCAAATGTTG GCGGATCCCATGTTCTCATCTCCATTTT GCGAATTCAGTGTATTTATTAAATGCCTGG GTGGATCCCATAAGTATTAAACTATACCGTACC GCGAATTCATTATTTAAAAAAATGCCCCTC GCGGATCCCTGGGCATCTTCTTTCTTG GAGAATTCGTTCGCTCTACTTTAAGCCAC GTGGATCCTATTGCTTCCGGTTTGATTCG GCGAATTCCTCCCAATGAAATACGACCC CAGGATCCGACACAATCATCCCTCCC TGGAATTCTATTTTTATTTATCAGGTTTCAGG GAGGATCCGCAACTCAATTGCCATAAG CAGAATTCATATGCCAACTGAATAATGGTGT CAGGATCCAATTCACTGATATGTCCCTC GCGAATTCAATAAATAGAAATTTTATGGACC GCGGATCCCCGTTGCACATATTGAATATC GCGAATTCCCTGGTAGCTGAAAATCG GAGGATCCATCCCCCATTCACGAATAAAC GTGAATTCTTAACGTCCAGGTTGCATGGC GTGGATCCCACCCTTGCTTCATATGTTTTTG GTGAATTCTACGGGTACATCAAAGGAAAG GTGGATCCTAATTATGTCCCTCTTCGC

Amplification promoter of Ct cel9V Amplification promoter of Ct cel48 Amplification promoter of Ct ce8 Amplification promoter of Ct cenC Amplification promoter of Ct cipA Amplification promoter of Ct cseP Amplification promoter of Ct pelb2 Amplification promoter of Ct pilZ Amplification promoter of Ct pl11 Amplification promoter of Ct rsgI5 Amplification promoter of Ct rsgI9 Amplification promoter of Ct sdbA Amplification promoter of Ct xgh7

TCGAATTCAGCACGAATGCTTATATTCAAC P69 Fw.Pxyn10D.Eco TCGGATCCCAACCCCTCCCCTAAAAAAG P70 Rv.Pxyn10D.Bam GTGAATTCTATGCTGTTTGCCAATATG P71 Fw.Pxyn10Y.Eco GTGGATCCTAAAGGGTGTTTTAAAGTTTCTTAG P72 Rv.Pxyn10Y.Bam GCGAATTCAAGGCTACAACAGAATACCG P73 Fw.Pclo1313_0563.Eco GTGGATCCGCAAATTTAATACTTCTGTTTGTG P74 Rv.Pclo1313_0563.Bam GTGAATTCTGGCTTTGCATATATCCCTTG P75 Fw.Pclo1313_0987.Eco GTGGATCCTTTCTGTTCAATTTATTTGTACG P76 Rv.Pclo1313_0987.Bam GTGAATTCTGAGTTGGCATGATATATGG P77 Fw.Pclo1313_1436.Eco GCGGATCCTTCCTCCCACATTGTTCCAG P78 Rv.Pclo1313_1436.Bam GTGAATTCTTAGTTTTGTTCGGATTAC P79 Fw.Pclo1313_1494.Eco GAGGATCCCCCTTGATTAAATTTGCCATTTTG P80 Rv.Pclo1313_1494.Bam TGGAATTCGCAGATGTAGCAGTCTATTTC P81 Fw.Pclo1313_2216.Eco GAGGATCCCCAATCCCCTCCATTATCTC P82 Rv.Pclo1313_2216.Bam GTGAATTCTAAGATTGTCAGGTAATTTCTC P83 Fw.Pclo1313_2793.Eco GTGGATCCTTTGCCTCTAAGTACCTG P84 Rv.Pclo1313_2793.Bam GCGAATTCATGCTAAGCCGAATGCAG P85 Fw.Pclo1313_2794.Eco GCGGATCCCCTTTATCGTAATTAATCCCTC P86 Rv.Pclo1313_2794.Bam GTGAATTCCCTGTCAAATTCAAATCAAGTC P87 Fw.Pclo1313_2861.Eco GTGGATCCCTTTACTTGTTTGCCCCCTG P88 Rv.Pclo1313_2861.Bam GTGAATTCGGATTGAAATGGATACAAGTC P89 Fw.Pclo1313_2866.Eco GAGGATCCACAATATCGCCTCTGTTTC P90 Rv.Pclo1313_2866.Bam Primers used for site directed mutagenesis of xyn10Z promoter # Name Sequenceb 5'→3' GCGAATTCTATGTATATGATGCCGGCCGACCGACAC P91 Fw.xynZ.short GCGGATCCCCTTATTTATATATTTATTGTTTCGTGAACG P92 Rv.xynZ.short C

Amplification promoter of Ct xynD Amplification promoter of Ct xyn10Y Amplification promoter of Ct Clo1313_1563 Amplification promoter of Ct Clo1313_0987 Amplification promoter of Ct Clo1313_1436 Amplification promoter of Ct Clo1313_1494 Amplification promoter of Ct Clo1313_2216 Amplification promoter of Ct Clo1313_2793 Amplification promoter of Ct Clo1313_2794 Amplification promoter of Ct Clo1313_2861 Amplification promoter of Ct Clo1313_2866

Brief description Amplification of a short version of xyn10Z promoter

P93

Fw.xynZ.mut1

GCGAATTCTATGTATATGATGCCGGCCGACGGACACAAA AATG

P94

Fw.xynZ.mut2

GCGAATTCTATGTATATGATGCCGGCCGACCCACACAAA AATG

P95

Fw.xynZ.mut3

GCGAATTCTATGTATATGATGCCGGCCGACCGTCACAAA AATG

P96

Fw.xynZ.mut4

GCGAATTCTATGTATATGATGCCGGCCGACCGAGACAAA AATG

P97

Fw.xynZ.mut5

GCGAATTCTATGTATATGATGCCGGCCGACCGACACTAA AATG

P98

Fw.xynZ.mut6

GCGAATTCTATGTATATGATGCCGGCCGACCGACACATA AATGTG

P99

Fw.xynZ.mut7

GCGAATTCTATGTATATGATGCCGGCCGACCGACACAAT AATGTG

P100

Rv.xynZ.mut8

GCGGATCCCCTTATTTATATATTTATTGTTTCCTGAACG CTC

Mutation from C to G in the -35 element (5' CGACacAAA 3' mutation in the underline nucleotide). This primer was used with Rv.xynZ.short. Mutation from G to C in the -35 element. (5' CGACacAAA 3' mutation in the underline nucleotide). This primer was used with Rv.xynZ.short. Mutation from A to T in the -35 element (5' CGACacAAA 3' mutation in the underline nucleotide). This primer was used with Rv.xynZ.short. Mutation from C to G in the -35 element (5' CGACacAAA 3' mutation in the underline nucleotides). This primer was used with Rv.xynZ.short. Mutation from A to T in the -35 element (5' CGACacAAA 3' mutation in the underline nucleotides). This primer was used with Rv.xynZ.short. Mutation from A to T in the -35 element (5' CGACacAAA 3' mutation in the underline nucleotides). This primer was used with Rv.xynZ.short. Mutation from A to T in the -35 element (5' CGACacAAA 3' mutation in the underline nucleotides). This primer was used with Rv.xynZ.short. Mutation from C to G in the -10 element (5' CGAAacaAT 3' mutation in the underline

P101

Rv.xynZ.mut9

GCGGATCCCCTTATTTATATATTTATTGTTTGGTGAACG CTC

P102

Rv.xynZ.mut10

GCGGATCCCCTTATTTATATATTTATTGTTACGTGAACG CTC

P103

Rv.xynZ.mut11

GCGGATCCCCTTATTTATATATTTATTGTATCGTGAACG CTC

P104

Rv.xynZ.mut12

GCGGATCCCCTTATTTATATATTTTATGTTTCGTGAACG CTC

P105

Rv.xynZ.mut13

GCGGATCCCCTTATTTATATATTTGGTGTTTCGTGAACG CTC

Primers used for confirmation of chromosomal integration in B. subtilis # Name Sequence 5'→3' ATATACATTGCCCGTCGGTC P106 GanQ-Cnf GCAATGAAACACGCCAAAG P107 Erm-Cnf P108 P109

GanB-Cnf XylR-Cnf

CAATGGCAGCGGCATATCC GGAGCGGTTTCTATCGTTATTGATTC

nucleotide). This primer was used with Fw.xynA.short. Mutation from G to C in the -10 element (5' CGAAacaAT 3' mutation in the underline nucleotide). This primer was used with Fw.xynA.short. Mutation from A to T in the -10 element (5' CGAAacaAT 3'mutation in the underline nucleotide). This primer was used with Fw.xynA.short. Mutation from A per T in the -10 element (5' CGAAacaAT 3' mutation in the underline nucleotide). This primer was used with Fw.xynA.short. Mutation from AT per TA in the -10 element (5' CGAAacaAT 3' mutation in the underline nucleotide). This primer was used with Fw.xynA.short. Mutation from AT to CC in the -10 element (5' CGAAacaAT 3' mutation in the underline nucleotide). This primer was used with Fw.xynA.short. Brief description Confirmation of chromosomal integration at Bs lacA locus. Primers binds to ganQ upstream of lacA (ganA), and to the erm gene of pAX01. Confirmation of chromosomal integration at Bs lacA locus. Primers binds to ganB

P110 P111

Ycg-Cnf LacZ-Cnf

GGAAGCGTTCACAGTTTCG TCCTGGAGCCCGTCAGTATC

P112 P113

Ldh-Cnf Cat-Cnf

CAATGACCACAAGCTCATCTG CTATTCAGGAATTGTCAGATAGGC

P114 P115

YkoY-Cnf YkrK-Cnf

CGGGCGTAATCTGAAAGACTC ACAACAATTGCCAGCATAAATAAAC

Restriction site sequences are underlined. Bs, Bacillus subtilis; Ct, Clostridium thermocellum. a

These primers were designed for cloning with the In-Fusion HD Cloning Kit.

b

The nucleotides used for site-directed mutagenesis are indicated in red and underlined.

downstream of lacA (ganA), and to the xylR gene of pAX01. Confirmation of chromosomal integration at Bs amyE locus. Primers binds to ycgB upstream of amyE, and to the lacZ gene of pBS1ClacZ. Confirmation of chromosomal integration at Bs amyE locus. Primers binds to ldh downstream of amyE, and to the cat gene of pBS1ClacZ. Confirmation of Bs ΔsigI-rsgI genotype. Primers binds to sigI-rsgI flanking genes ykoY and ykrK.