SUPPLEMENTARY MATERIAL
Applied Microbiology and Biotechnology
A high efficiency recombineering system with PCR-based ssDNA in Bacillus
subtilis mediated by the native phage recombinase GP35
Zhaopeng Sun1, 2, Aihua Deng1, Ting Hu1, Jie Wu1, 2, Qinyun Sun1, 2, Hua Bai1, 2, Guoqiang Zhang1,
Tingyi Wen1, *
1
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100101, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
* To whom correspondence should be addressed. Tel: +86 10 64806119; Fax: +86 10 64806157. Email:
[email protected]
Fig. S1 Effects of the field strength on the transformation efficiency of B. subtilis W168.
All of the experiments were carried out in triplicate.
Fig. S2 Electrophoresis of the ssDNA constructs of deoD generated by a two-step asymmetric PCR.
The ssDNA was single-stranded DNA generated using the WY031 primer; the dsDNA was a
double-stranded PCR product generated using the WY031 and WY034 primers.
Fig. S3 The relationship between the recombineering frequency of the recombinases and the amino
acid sequences and recombinase expression levels. (a) Using the GP35 amino acid sequence as a
standard, the amino acid sequences of nine other recombinases were aligned with GP35. (b) The mean
GFP value represents the recombinase expression level.
Fig. S4 Recombineering mediated by GP35 in B. subtilis ATCC 6633 with poor natural competence. (a)
Natural competence of B. subtilis W168, ATCC 6633 and its derivative strain. The plasmids pWYE799
and pWYE837 are the integrated plasmids for knocking out the upp gene of W168 and ATCC 6633,
respectively; pWB980 is a replicative plasmid; ※ represents no transformants by transforming the
plasmid pWB980. (b) Recombineering mediated by GP35. All of the experiments were carried out in
triplicate.
Table S1 Strains and plasmids used in this study.
Strains and plasmids
Characteristics
Reference or source
General cloning host strain
(Zhang et al. 2012)
Tryptophan auxotrophic
(Zacchi et al. 2010)
W168
Prototroph
(Zeigler et al. 2008)
BS045
W168△mutS::P43-gp34.1-35-36-CmR
this study
BS046
W168△mutS::Pspac-gp34.1-35-36-CmR
this study
BS047
W168△mutS::PxylA-gp34.1-35-36-CmR
this study
BS056
W168△lacA::PxylA-beta-MLSR
this study
BS057
W168△lacA::PxylA-s65-MLSR
this study
BS058
W168△lacA::PxylA-recT-MLSR
this study
BS059
W168△lacA::PxylA-plu2935-MLSR
this study
BS060
W168△lacA::PxylA-gp35-MLSR
this study
BS061
W168△lacA::PxylA-orf 48-MLSR
this study
BS062
W168△lacA::PxylA-orf 245-MLSR
this study
BS063
W168△lacA::PxylA-gp61-MLSR
this study
BS064
W168△lacA::PxylA-gp20-MLSR
this study
BS067
BS060△deoD::kanR
this study
BS068
W168△lacA::PxylA-orf C-MLSR
this study
BS069
BS060△upp::kanR
this study
BS070
BS060△amyE::CmR
this study
BS141
W168△lacA::PxylA-gp35-gfpmut3a-MLSR
this study
E. coli strain
EC135
Saccharomyces strain
S. cerevisiae DAY414
B. subtilis strains
BS142
W168△lacA::PxylA-beta-gfpmut3a-MLSR
this study
BS143
W168△lacA::PxylA-s65-gfpmut3a-MLSR
this study
BS144
W168△lacA::PxylA-recT-gfpmut3a-MLSR
this study
BS145
W168△lacA::PxylA-plu2935-gfpmut3a-MLSR
this study
BS146
W168△lacA::PxylA-orf48-gfpmut3a-MLSR
this study
BS147
W168△lacA::PxylA-orf245-gfpmut3a-MLSR
this study
BS148
W168△lacA::PxylA-orfC-gfpmut3a-MLSR
this study
BS149
W168△lacA::PxylA-gp61-gfpmut3a-MLSR
this study
BS150
W168△lacA::PxylA-gp20-gfpmut3a-MLSR
this study
ATCC6633
Wild-type, produces mycosubtilin
(Duitman EH et al.
1999)
WYB263
ATCC6633/pHCMC04-gp35
this study
pAX01
B. subtilis integrative vector, xylose-inducible promoter
(Hartl et al. 2001)
pAD43-25
pAD123 derivative, gfpmut3a controlled by upp promoter
BGSC a
pUB110
kanamycin resistance
BGSC
pHCMC04
E.
Plasmids
coli-Bacillus
shuttle
plasmid,
chloramphenicol
BGSC
resistance
pWB980
kanamycin resistance
BGSC
pWYE597
pWYE724 carrying P43 promoter and gp35
this study
pWYE598
pWYE724 carrying PxylA promoter and gp35
this study
pWYE599
pWYE724 carrying Pspac promoter and gp35
this study
pWYE724
pBAD43 derivative, E. coli-S. cerevisiae shuttle plasmid, (Zhang et al. 2012)
CEN6 ARS4 ori and TRP1 marker in S. cerevisiae
pWYE753
Integrative vector for deoD of W168, conferring kanR
(Li et al. 2011)
pWYE782
gp35 cloned into pAX01
this study
pWYE783
beta cloned into pAX01
this study
pWYE784
s65 cloned into pAX01
this study
pWYE785
recT cloned into pAX01
this study
pWYE786
plu2935 cloned into pAX01
this study
pWYE787
orf 48 cloned into pAX01
this study
pWYE788
orf 245 cloned into pAX01
this study
pWYE789
orf C cloned into pAX01
this study
pWYE790
gp61 cloned into pAX01
this study
pWYE791
gp20 cloned into pAX01
this study
pWYE799
Integrative vector for upp of W168, conferring kanR
this study
pWYE800
orf 48-gfpmut3a cloned into pAX01
this study
pWYE801
orf 245-gfpmut3a cloned into pAX01
this study
pWYE802
gp20-gfpmut3a cloned into pAX01
this study
pWYE803
recT-gfpmut3a cloned into pAX01
this study
pWYE804
plu2935-gfpmut3a cloned into pAX01
this study
pWYE805
orf C-gfpmut3a cloned into pAX01
this study
pWYE806
gp35-gfpmut3a cloned into pAX01
this study
pWYE807
beta-gfpmut3a cloned into pAX01
this study
pWYE809
s65-gfpmut3a cloned into pAX01
this study
pWYE810
gp61-gfpmut3a cloned into pAX01
this study
pWYE836
gp35 cloned into pHCMC04
this study
pWYE837
Integrative vector for upp of ATCC6633, conferring kanR
this study
a
Bacillus Genetic Stock Center;
Table S2 Primers used in this study
Primer
Sequences (5’-3’, restriction sites are underlined)
WB325
ATGCCATAGCATTTTTATCC
WB326
GATTTAATCTGTATCAGG
WB662
ACTCAATAATGCTGAGCTCGAATTCAAGAGACTTGGGGGT
Description
ACTGTCTCCC
WB663
GGGAGACAGTACCCCCAAGTCTCTTGAATTCGAGCTCAGC
ATTATTGAGT
WB664
CCCTCGCCATTTTAACCCCTCCTAAGTGTACATTCCTCTCTT
ACCTATAATG
WB665
CATTATAGGTAAGAGAGGAATGTACACTTAGGAGGGGTTA
AAATGGCGAGGG
WB666
WB667
WB668
GAATTAATTAATCATCGCGACTGCATTAGAAGGGAAGGTCT
GATTCATTTATG
Primers
for
CATAAATGAATCAGACCTTCCCTTCTAATGCAGTCGCGATG
the
vivo
ATTAATTAATTC
assembly
GACTTTTGCCACATTCATCACCCCGTTCTTCAACTAAAGCA
pWYE598,
CCCATTAGTTC
WB669
GAACTAATGGGTGCTTTAGTTGAAGAACGGGGTGATGAAT
GTGGCAAAAGTC
WB670
GAATTAATTAATCATCGCGACTGCAGAAGAGACTTGGGGG
TACTGTCTCCC
WB671
GGGAGACAGTACCCCCAAGTCTCTTCTGCAGTCGCGATGA
TTAATTAATTC
WB672
GCAAAATTTTTCAGGAATTTTAGAAGAGACTTGGGGGTAC
TGTCTCCC
WB673
GGGAGACAGTACCCCCAAGTCTCTTCTAAAATTCCTGAAA
AATTTTGC
in
of
pWYE597,
pWYE599
WB674
GAATTAATTAATCATCGCGACTGCATCACTGCCCGCTTTCC
AGTCGGGAAACCTG
WB675
CAGGTTTCCCGACTGGAAAGCGGGCAGTGATGCAGTCGC
GATGATTAATTAATTC
WB789
CGGGATCCCGATGGCAACTAAAAAACAAGAGG
Primers for the construction of
WB790
CCCCCGGGGG CTATTCATTTGTTTCCCCTC
pWYE836
WB968
CGGGATCCATGGCAACTAAAAAACAAGA
gp35 forward
WB969
CGGGATCCCTATTCATTTGTTTCCCCTC
gp35 reverse
WB970
CGGGATCCATGAGTACTGCACTCGCAAC
beta forward
WB971
CGGGATCCTCATGCTGCCACCTTCTGCT
beta reverse
WB972
CGGGATCCATGGAAAAACCAAAGCTAAT
s065 forward
WB973
CGGGATCCCTAAGAAGCTAAAGGCTGTG
s065 reverse
WB974
CGGGATCCATGACTAAGC AACCACCAAT
recT forward
WB975
CGGGATCCTTATTCCTCTGAATTATCGA
recT reverse
WB976
CGGGATCCATGAGCACAGCAGTACAAAAAG
plu2935 forward
WB977
CGGGATCCTTATGATGCCTTTTTCCTTA
plu2935 reverse
WB978
CGGGATCCATGTCAACTA ACGACGAATT
orf 48 forward
WB979
CGGGATCCTTAGATCATTGACCCTTGAAC
orf 48 reverse
WB980
CGGGATCCATGGCAAATG AATTAGGAAT
orf 245 forward
WB981
CGGGATCCTTAGAATCCCTCCAAAGGCTC
orf 245 reverse
WB982
CGGGATCCATGGCAACAC AAAAAGTTGA
orf C forward
WB983
CGGGATCCTTAAGCCTTATCCTGATTAG
orf C reverse
WB984
TCCCCGCGGATGGCTGAAA ATGCTGTCAC
gp61 forward
WB985
TCCCCGCGGTCATGCGTTGGGCCCGTCGA
gp61 reverse
WB986
CGGGATCCATGACTGAAAATAATAAATT AC
gp20 forward
WB987
CGGGATCCTTAAAATGGCTCTTCTTCGC
gp20 reverse
WB1150
GGCATTATGT TTGAATTTCC G
WB1150S
G-G-C-A-TTATGT TTGAATTTCC G
WB1151
GTGCTTTAGT TGAAGTCTTG ACACTCCTTA
amyE upstream forward
amyE upstream reverse
WB1152
TAAGGAGTGT CAAGACTTCA ACTAAAGCAC
cat forward
WB1153
CGTCTAGCCT TGCCCTGCAG TCGCGATGAT
cat reverse
WB1154
ATCATCGCGA CTGCAGGGCA AGGCTAGACG
amyE downstream forward
WB1155
GGCGCAAATG CAGACAATAT C
WB1155S
G-G-C-G-CAAATG CAGACAATAT C
WB1177
AACAGTTCCC AGAATAAGGC
WB1177S
A-A-C-A-GTTCCC AGAATAAGGC
WB1178
CGCATACCAT TTTGAAAAAT GAAATCCCCA
upp upstream reverse
WB1179
TGGGGATTTC ATTTTTCAAA ATGGTATGCG
kan forward
WB1180
GAGCTGAAAC ACAGTATGAG AATAGTGAAT
kan reverse
WB1181
ATTCACTATT CTCATACTGT GTTTCAGCTC
upp downstream forward
WB1182
TCCTCGTACA ATCGACTTTA AG
WB1182S
T-C-C-T-CGTACA ATCGACTTTA AG
GGTGGCGGTGGCTCTGGAGGTGGTGGGTCCGGTGGCGGT
WB1276
amyE downstream reverse
upp upstream forward
upp downstream reverse
gfpmut3a forward
GGCTCT ATGAGTAAAG GAGA
WB1277
CGGGATCCTTATTTGTAT AGTTCATCCA
WB1281
AGAGCCACCG CCACCTTCATTTGTTTCCCC
WB1282
GGGGAAACAA ATGAA GGTGG CGGTGGCTCT
WB1283
AGAGCCACCG CCACCTGCTGCCACCTTCTG
WB1284
CAGAAGGTGG CAGCA GGTGG CGGTGGCTCT
WB1287
AGAGCCACCG CCACC AGAAGCTAAAGGCTG
WB1288
CAGCCTTTAG CTTCTGGTGG CGGTGGCTCT
WB1289
AGAGCCACCG CCACC TTCCTCTGAATTATC
WB1290
GATAATTCAG AGGAAGGTGG CGGTGGCTCT
WB1291
AGAGCCACCG CCACC TGATGCCTTTTTCCT
WB1292
AGGAAAAAGG CATCAGGTGG CGGTGGCTCT
WB1293
AGAGCCACCG CCACC GATCATTGACCCTTG
WB1294
CAAGGGTCAA TGATCGGTGG CGGTGGCTCT
WB1295
AGAGCCACCG CCACC GAATCCCTCCAAAGG
gfpmut3a reverse
gp35-linker-gfpmut3a primers
beta-linker-gfpmut3a primers
s65-linker-gfpmut3a primers
recT-linker-gfpmut3a primers
plu2935-linker-gfpmut3a primers
orf48-linker-gfpmut3a primers
orf245-linker-gfpmut3a primers
WB1296
CCTTTGGAGG GATTCGGTGG CGGTGGCTCT
WB1297
AGAGCCACCG CCACC AGCCTTATCCTGATT
WB1298
AATCAGGATA AGGCTGGTGG CGGTGGCTCT
WB1299
AGAGCCACCG CCACC TGCGTTGGGCCCGTC
WB1300
GACGGGCCCA ACGCAGGTGG CGGTGGCTCT
WB1301
TCCCCGCGGTTATTTGTAT AGTTCATCCA
WB1302
AGAGCCACCG CCACC AAATGGCTCTTCTTC
WB1303
GAAGAAGAGC CATTTGGTGG CGGTGGCTCT
WB1449
TGAGACGCTCTGGACACG
WB1450
GTCTGTTCCGAATGGTGC
WB1451
GCGTGAGTGATGAAGGTTT
WB1452
GCCGTGGCTTTCTGGTTA
WB1712
TCCCTTTCTCAACGGCTCT
WB1713
CGCATACCATTTTGAAAAAGAAAACCCGAC
WB1714
GTCGGGTTTTCTTTTTCAAAATGGTATGCG
WB1715
GAGCTGAAACACAGTATGAGAATAGTGAATG
WB1716
CATTCACTATTCTCATACTGTGTTTCAGCTC
WB1717
CAGGCGGAACTGACAACC
WY031
ACTGGGATCCAGCGATCATCAAGACG
WY034
GTCATACGAACGGTACCCTGAGACA
WY031S
A-C-T-G-GGATCCAGCGATCATCAAGACG
WY034S
G-T-C-A-TACGAACGGTACCCTGAGACA
orfC-linker-gfpmut3a primers
gp61-linker-gfpmut3a primers
gp20-linker-gfpmut3a primers
RT-qPCR of gp35
RT-qPCR of reference gene (16S
rRNA)
Primers for generating PCR
products or ssDNA of upp::kanR
cassette(ATCC6633)
Primers for generating PCR
products or ssDNA of
deoD::kanR cassette(W168)
Phosphorothioate modification was indicated by ‘-’
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