Additional file 1 Constriction of the vectors set
Table S1 Construction of pCot and pCge vectors
Plasmid
pTL01
Amplified
DNA
sequence
Primer
name
Tryptophan P2trpC-F
biosynthesis
pathway
gene trpC
with prrnO
P2trpC-R
promoter
pCotC series
pMSW1
Left
flanking
region of
lacA gene
pMSW2
Right
flanking
region of
lacA gene
pMSW3
Tryptophan
biosynthesis
pathway
gene trpC
The
pCotC-C
promoter
KF933399
and the
cotC gene
pCotC-CL The
promoter
KF933400
and the
cotC gene
N-lacA-up
N-lacA-dn
C-lacA-up
C-lacA-dn
trpC-up
trpC-dn
pcotC-up
cotC-dn
pcotC-up
cotClinkdn
Primer sequence 5’-3’
Template
Destination
EcoRI
CCGGAATTCTTTTCAAAAAAGTATTGACCTAGTTAACTAAAAATGTTACTAT
TAAGTAGTCGCCAAGGAGGTGAGACCCATGCTTGAAAAAATCATCAAACA
AAAGAAAGAAGAAGTGAAAACACTGGTTC
Eco105I
CCCTACGTATTACTCCCTAAACAAAGCATGGATTGCTTTACGCTGAGAAGT
TTGTCTCATCAATGATTCACCGATAAGTACAGCTCGCGCCCCATGTTCATTG
ACAAATGTTAAATGTTCTAAAGAACCGATTC
B. subtilis
3610
chromosomal
DNA
pDG1663
[1]
CGGCCCGATATTTTAGCTG
HindIII
CACTCCCGGAAAGCATGC
SphI
TCAGAGCTCCAGCGAGGACACCGTTTC
SalI
ATAGAATTCCTGATCCTGCCTCGCTTG
EcoRI
ACGCTCTAGAAAAAAGTATTGACCTAGTTAAC
XbaI
ATACTGCATGCATTACTCCCTAAACAAAGC
SphI
TACTGAGCTCAGGATAAATCGTTTGGG
SacI
TCAGGGTACCGTAGTGTTTTTTATGCTTTTT
KpnI
TACTGAGCTCAGGATAAATCGTTTGGG
SacI
TCAGGGTACCAGAACCGCCGCCACCGTAGTGTTTTTTATGCTTTTT
KpnI
linker
B. subtilis 168
chromosomal
DNA
pUC19
B. subtilis 168
chromosomal
DNA
pMSW1
pTL01
pMSW2
B. subtilis 168
chromosomal
DNA
pMSW3
B. subtilis 168
chromosomal
DNA
pMSW3
pMSW4
pCotC-N
KF933401
pCotC-NL
KF933402
with linker
at Cterminus
The
promoter
and RBS
sequence of
the cotC
The cotC
gene
The cotC
gene with
linker at Nterminus
pCotG series
pAIW1
Lysine
biosynthesis
pathway
gene lysA
The
pCotG-C
promoter
KF933403
and the
cotG gene
pCotG-CL The
promoter
KF933404
and the
cotG gene
with linker
at Cterminus
pAIW2
The
promoter
and RBS
sequence of
the cotG
pcotC-up
pcotC-dn
cotC-up
cotCtermdn
cotClinkup
cotCtermdn
lysA-trofup
lysA-trofdn
cotG-up
cotG-dn
cotGL-up
cotGL-dn
promoGup
promoGdn
TACTGAGCTCAGGATAAATCGTTTGGG
SacI
TCAGGGTACCGTAGTGTTTTTTATGCTTTTT
KpnI
TACTGGATCCGGTTATTACAAAAAATACAAAG
BamHI
TCAGTCTAGACACAAACAAAAAAGACCC
XbaI
TACTGGATCCGGTGGCGGCGGTTCTGGTTATTACAAAAAATACAAAG
BamHI
linker
TCAGTCTAGACACAAACAAAAAAGACCC
XbaI
TTAAGCATGCGATTTCTTCGATTCTATCTGG
SphI
TTAATCTAGATGTGGCAGGTTCTTGTC
XbaI
CCCGGATCCCGAGAAAAAATCC
BamHI
CTTGGATCCTTTGTATTTCTTTTTGACTAC
BamHI
CCAGGTACCCGAGAAAAAATCC
KpnI
CCAGAGCTCGTTTGTTGAACTAATGGG
SacI
ATAGAATTCCAATTTGAAATCC
EcoRI
ATAGGATCCCCGAGAAAAATC
BamHI
B. subtilis 168
chromosomal
DNA
pMSW3
B. subtilis 168
chromosomal
DNA
pMSW4
B. subtilis 168
chromosomal
DNA
pMSW4
B. subtilis 168
chromosomal
DNA
pPyr-kan
[2]
pKH16
pAIW1
pKH36
pAIW1
B. subtilis 168
chromosomal
DNA
pAIW1
pCotG-N
KF933405
The cotG
gene
pCotG-NL
KF933406
The cotG
gene with
linker at Nterminus
pCotZ series
pAGW1
Tryptophan
biosynthesis
pathway
gene trpC
pAGW2
The
promoter
and the cotZ
gene
pAGW3
The
promoter
and the cotZ
gene with
linker at Cterminus
An insert
pCotZ-C
harboring
KF933407
MCS
flanked by
sticky ends
pCotZ-CL An insert
harboring
KF933408
MCS
flanked by
sticky ends
pCgeA series
pAGW4
The
promoter
cotG2-up
TTAGAATTCTCGAGCTCGGCCACTATTCCCA
EcoRI
cotG2-dn
TTGGTACCCTATTTGTATTTCTTTTTG
KpnI
cotG2-lnk- AATGAATTCTCGAGCTCGGTGGCGGCGGTTCTCACTATTCCCATTC
up
EcoRI
cotG2-dn
TTGGTACCCTATTTGTATTTCTTTTTG
KpnI
trpC-trofF
trpC-trofR
cotZ-F
cotZ-R
cotZlinker-F
cotZlinker-R
MCS-01F
MCS-01R
MCS-01F
MCS-01R
cgeA-F
GGCGCAATTGTTTCAAAAGTCAATTTGATCAACGG
MunI
ACATGCATGCAAAGTACGTATTACTCCCTAAACAAAGC
SphI
CGTAGCGAATTCAGTTATCACTCTTGTCCTC
EcoRI
GCTTAGGATCCATGATGATGTGTACGATTG
BamHI
EcoRI
CCGGAATTCGCAACCCTTATTTCTACAGCAACAAATACACTCGTAGCCATC
CTAGTTATCACTCTTGTCCTCTAGGACC
BamHI
linker
CGCGGATCCTCCTCCACCTTTCGCTGCTGCTTCTCCTCCACCATGATGATGTGT
ACGATTGATTAATCGAGGATTTAAGC
CGGTACCCACGTCAAATCTAGAGTTAACGGTTACCTACGTAAG
(BamHI – SacI)
GATCCTTACGTAGGTAACCGTTAACTCTAGATTTGACGTGGGTACCGAGCT
(BamHI – SacI)
CGGTACCCACGTCAAATCTAGAGTTAACGGTTACCTACGTAAG
(BamHI – SacI)
GATCCTTACGTAGGTAACCGTTAACTCTAGATTTGACGTGGGTACCGAGCT
(BamHI – SacI)
CAGCTTAGAATTCTTGAGAGTGAAACATGAG
EcoRI
B. subtilis 168
chromosomal
DNA
pAIW2
B. subtilis 168
chromosomal
DNA
pAIW2
pTL01
pDL
[3]
B. subtilis 168
chromosomal
DNA
pAGW1
B. subtilis 168
chromosomal
DNA
pAGW1
Self-annealed
primers
pAGW2
Self-annealed
primers
pAGW3
B. subtilis 168
chromosomal
pAGW1
and the
cgeA-R
CGGGGATCCTGAAAAGAACGTAAC
cgeA gene
BamHI
pAGW5
The
cgeAEcoRI
promoter
linker-F
CCGGAATTCAAGCAGAGCCTCTGTCATCATTTAAAAAGCACCCCAGCTTAC
and the
AACACTTGAGAGTGAAACATGAGATCTCG
cgeA gene
cgeABamHI
linker
with linker
linker-R
CGCGGATCCTCCTCCACCTTTCGCTGCTGCTTCTCCTCCACCTGAAAAGAACG
at CTAAACGCTTTCTACTTTGTCTACATC
terminus
An insert
MCS-02F AGCTTAGGGCCCATCTAGAAGGTACCGACTCGAGTGTACAGGTAACCAAG
pCgeA-C
harboring
(BamHI – HindIII)
KF933393
MCS
MCS-02R GATCCTTGGTTACCTGTACACTCGAGTCGGTACCTTCTAGATGGGCCCTA
flanked by
(BamHI – HindIII)
sticky ends
MCS-02F AGCTTAGGGCCCATCTAGAAGGTACCGACTCGAGTGTACAGGTAACCAAG
pCgeA-CL An insert
harboring
(BamHI – HindIII)
KF933394
MCS
MCS-02R GATCCTTGGTTACCTGTACACTCGAGTCGGTACCTTCTAGATGGGCCCTA
flanked by
(BamHI – HindIII)
sticky ends
In bold shown names of plasmids composing vector system. GenBank accession numbers are indicated underneath the names.
DNA
B. subtilis 168
chromosomal
DNA
pAGW1
Self-annealed
primers
pAGW4
Self-annealed
primers
pAGW5
Construction of pCotB series
A DNA fragment containing thrC gene along with the promoter and cotB gene flanked by linker-encoding sequences and multicloning sites at both termini was
commercially synthesized (Life Technologies, USA) and delivered in a plasmid 12AA35IP_cotB_pMS. E. coli DH5 was used for transformation and
propagation. The plasmid was digested with EcoRV restriction enzyme, cleaned up and subsequently digested with SphI and BsiWI restriction enzymes.
Obtained products were separated in agarose gel and appropriate fragment was isolated from the gel. The fragment was cloned into SphI/BsiWI-digested pDL
vector resulting in pIW plasmid.
pCotB-NL
pIW plasmid was digested with EcoRI and XhoI, the sticky ends of the obtained product were filled in with Klenow fragment and re-ligated.
pCotB-N
pCotB-NL plasmid was digested with KpnI to remove linker-encoding sequence and re-ligated.
pCotB-CL
pIW plasmid was digested with KpnI, the sticky ends of the obtained product were filled in with Klenow fragment and re-ligated.
pCotB-C
pCotB-CL plasmid was digested with EcoRI to remove linker-encoding sequence and re-ligated.
References
1. Guérout-Fleury AM, Frandsen N, Stragier P: Plasmids for ectopic integration in Bacillus subtilis. Gene 1996, 180:57-61.
2. Middleton R, Hofmeister A: New shuttle vectors for ectopic insertion of genes into Bacillus subtilis. Plasmid 2004, 51:238-245.
3. Yuan G, Wong SL: Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted
repeat sequence (CIRCE). J Bacteriol 1995, 177:5427-5433.