Supplementary material
Construction of p3CHPT and pYPA2 plasmids
Plasmid of p3CHgld was constructed based on p3CHPT (unpublished) as a
cloning vector developed by Mikkelsen from pUC19 (Invitrogen). Supplementary
table 1 listed the primers used for construction of plasmid p3CHPT which
contains the following cloned DNA fragments: 1) a DNA fragment upstream of
the ldh gene of strain BGl, amplified using primers ldhup1F and ldhup2R; 2) a
gene encoding a highly thermostable kanamycin resistance, amplified from
plasmid pUC18HTK (Hoseki et al. 1999) using primers of htkF and htkR; 3) a
glucose repressed xylose promoter (xyl) from Thermoanaerobacter ethanolicus
(Erbeznik et al. 1998), amplified using primers of xylF and xylR; 4) a DNA
fragment downstream of the ldh gene of strain BGl, amplified using primers of
ldhdown3F and ldhdown4R.
Plasmid of pYPAgld was constructed based on pYPA2 (unpublished) as a
cloning vector developed by Slawomir from pYES2 (invitrogen). Supplementary
table 1 listed the primers used for construction of plasmid pYPA2 which contains
the following cloned DNA fragments: 1) truncated glyceraldehyde 3-phosphate
dehydrogenase gene of BG1, amplified using primers of gapdhF and gapdhR; 2) a
gene encoding a highly thermostable kanamycin resistance, amplified from
plasmid pUC18HTK using primers of htkF and htkR; 3) a complete
phosphoglycerate kinase open reading frame of BG1, amplified using primers of
pgkF1 and pgkR1.
Molecular characterization of T. mathranii BG1G1 and BG1G2
Gene disruption in strain BG1G1 was verified by PCR using genomic DNA
of independent clones as template compared with BG1 wild type and BG1L1
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strains. Supplementary table 1 listed the primers used for analysis of the mutant
strain. The primer pairs, ldhOUTF - ldhOUTR, located outside of the ldh region in
BG1 were used in a PCR reaction and resulted in three fragments with the
expected sizes of approximately 2.7 kb (wild type), 3.5 kb (BG1L1) and 4.6 kb
(BG1G1), respectively (supplementary Fig.1A). All the amplified PCR fragments
were further analyzed by digestion with restriction enzymes of PstI, as shown in
supplementary Fig.1B. The digested pattern of strain BG1G1 with both enzymes
resulted in extra bands with the expected sizes as compared with the control
strains. All the PCR products were sequenced and confirmed the replacement of
the gldA ORF with the htk marker cassette via a double crossover event in strain
BG1G1.
Gene disruption in strain BG1G2 was analyzed by PCR using genomic
DNA of independent clones as template. Supplementary table 1 listed the primers
used for analysis of the mutant strain. The primer of gapexF located outside of
gap region in BG1 and primer of gldAR2 located inside of the inserted gldA gene
were used in a PCR reaction and resulted in a fragment with the expected size of
1.32 kb (supplementary Fig.1C, lane 1-5). The primer of htkmidF located inside
of the inserted htk gene and primer of pgkexR located outside of pgk region in
BG1 were used in a PCR reaction and resulted in a fragment with the expected
size of 1.75 kb (supplementary Fig.1C, lane 6-10). All the PCR products were
also sequenced and confirmed the replacement of the gldA ORF with the htk
marker cassette via a double crossover event in strain BG1G2.
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Supplementary table 1. Primers used for construction of cloning vectors or for analysis of the
mutants.
Primer
ldhup1F
ldhup2R
Sequence
ldhdown3F
ldhdown4R
xylF
xylR
htkF
htkR
gapdhF1
gapdhR1
pgkF1
pgkR1
5’ ATATAAAAAGTCACAGTGTGAA 3’
ldhOUTF
ldhOUTR
gapexF
gldAR2
htkmidF
pgkexR
5’ GAGCTGCTTTAAGTGTCTCAGG 3’
5’ TTCCATATCTGTAAGTCCCGCTAAAG 3’
5’ ATTAATACAATAGTTTTGACAAATCC 3’
5’ CACCTATTTTGCACTTTTTTTC 3’
5’ GCTTTATTTTTTTTATTTTTTTACTCAAAAAAGGAGG 3’
5’ AAATGCCCATATTCCGACACTGTTT 3’
5’ AAGGAGGTAGTATATATGCGGCGTCGACTAGAATTC 3’
5’ GTGGATGTGTCAAAACGCATACCATTTTGA 3’
5’ GGGGTGTTAAAGTTGCTATCAATGG 3’
5’ CCTAGCAAAATATATTGCTGATAGGCTC 3’
5’ AAGGAGGTTTTTGTGATGAAAAAAACC 3’
5’ GCCGGGGATTGATGTTTTAAATGACAAGTAA 3’
5’ CAGAAGCCGTTACAGTTGGTCC 3’
5’ GCAGAAGTAGCAATGTACAGGTGC 3’
5’ CAGATCTTTGGTGGAGAGTGTTCAG 3’
5’ CGATGCGATCTGTGCCCTTATCG 3’
5’ CTTTGCTCACTTCTGTCAAGTCCAC 3’
Source, reference, or purpose
Primers for amplifying ldh
upstream of strain BG1
Primers for amplifying ldh
downstream of strain BG1
Primers for amplifying xyl
promoter (Erbeznik et al.1998)
Primers for amplifying htk gene
for KamR (Hoseki et al. 1999)
Primers for amplifying gapdh
gene fragment of strain BG1
Primers for amplifying pgk gene
of strain BG1
Primers for mutant analysis on
strain BG1G1
Primers for mutant analysis on
strain BG1G2
Primers for mutant analysis on
strain BG1G2
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Figure legend
Supplementary Fig. 1 Verification of strain BG1G1 and BG1G2 chromosome integration: A. PCR
analysis on genomic DNA from BG1 wild type, BG1L1, and independent clones of BG1G1 strains
using primers pairs of ldhOUTF - ldh3OUTR; B. Restriction analysis of the fragments shown in A
using restriction enzymes PstI; C. PCR analysis on genomic DNA of independent clones of
BG1G2 strain using primer pairs of gapexF - gldAR2 (lane 1-5) and primer pairs of htkmidF pgkexR (lane 6-10).
A
B
C
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