Applied Microbiology and Biotechnology A high efficiency
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 ‘-’ References Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H, Saenger W, Bernhard F, Reinhardt R, Schmidt M, Ullrich C, Stein T, Leenders F, Vater J (1999) The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. 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