Supporting Information

Átírás

1 Supporting Information Paired design of dcas9 as a systematic platform for the detection of featured nucleic acid sequences in pathogenic strains Yihao Zhang 1,2,8, Long Qian 4,8, Weijia Wei 1,3,7,8, Yu Wang 1, Beining Wang 1, Pingping Lin 1, Wenchao Liu 1, Luze Xu 1, Xiang Li 1, Dongming Liu 1, Sida Cheng 1, Jiaofeng Li 1, Yixuan Ye 1, Hang Li 1, Xiaohan Zhang 1, Yiming Dong 2, Xuejin Zhao 3, Cuihua Liu 5, Haoqian M. Zhang 2, Qi Ouyang 2, 6 *, and Chunbo Lou 3,7* 1 Peking University Team for the International Genetically Engineered Machine Competition (igem), Beijing, , China; 2 Center for Quantitative Biology and Peking-Tsinghua Joint Center for Life Sciences, Peking University, Beijing, , China; 3 CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, , China; 4 Department of Biology and Center for Genomics and Systems Biology, New York University, 12 Waverly Place, New York, New York, 10003, USA. 5 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, , China 6 The State Key Laboratory for Artificial Microstructures and Mesoscopic Physics, School of Physics, Peking University, Beijing, , China 7 University of Chinese Academy of Science, Beijing, , China 8 These authors contributed equally to this work. *Correspondence should be addressed to C.L. (louchunbo@im.ac.com) and Q.O. (qi@pku.edu.cn). S1

2 Supplementary Figure S1. Reconstitution of luciferase activity via rapamycin-induced FRB/FKBP dimerization. Error bars show standard error for three independent experiments. S2

3 Supplementary Figure S2. Protein purification and characterization. (a) SDS-PAGE of purified proteins. Cell lysate showed the efficiency of 200 μm IPTG induction. (b) Characterization of PC reporter system using different protein concentrations. Error bars show standard error from three independent experiments. a b S3

4 Supplementary Figure S3. Array-based detection of B. subtilis genome using PC reporter system. (a) Markers selected from the genome. (b) Output of PC reporter using B. subtilis genomic DNA and E. coli genomic DNA. a b S4

5 Supplementary Figure S4. Distribution of scored segments from Mycobacterium tuberculosis genome. S5

6 Supplementary Figure S5. Amino acid sequences of all proteins. FRB ATCCTCTGGCATGAGATGTGGCATGAAGGCCTGGAAGAGGCATCTCGTTTGTACTTTGGGGAAAG GAACGTGAAAGGCATGTTTGAGGTGCTGGAGCCCTTGCATGCTATGATGGAACGGGGCCCCCAG ACTCTGAAGGAAACATCCTTTAATCAGGCCTATGGTCGAGATTTAATGGAGGCCCAAGAGTGGTG CAGGAAGTACATGAAATCAGGGAATGTCAAGGACCTCaccCAAGCCTGGGACCTCTATTATCATGT GTTCCGACGAATCTCA FKBP GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQM SVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE NFluc MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKR YGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNV QKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVAL PHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDY KIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILIT PEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDG CFluc SGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAG VAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIR EILIKAKKGGKIAV dcas9 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKK LVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAIL SARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD S6

7 QSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFI ERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIE ERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQ KGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVD AIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGG LSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFK TEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSD KLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY KEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD S7

8 Supplementary Figure S6. DNA sequence of sgrna expression cassette [Primer site, T7 promoter, BsaI restriction site, LacZ (reversed), LacZ promoter (reserved), crrna, T7 terminator]. CCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGG CAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCCGATCGTGACA CAAGGTTTAATACGACTCACTATAGGCGAGACCCCATTCGCCATTCAGGCTGCGCAACTGTTGGG AAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAG GCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAATCACGACGTTGTAAAACGACGGCCAGTGAGC GCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCCTCCAGCT TTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGT GAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGG GGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCGGTCTCAGTTTTAGAGCTAGAAAT AGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTAG CATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCGGCCGCTGCAGTCCGGCAAA AAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCA GGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC TCAAAGGCGGTAATA S8

9 Supplementary Figure S7. DNA target sequence for Figure 1 and Figure 2 [sgrna binding site 1, sgrna binding site 2, Spacer (27 bp)]. ATCGATCCATCTTCAGGGAAAAGCTGGGTGTCGCCTGCAGAGAGATATGCAGTTGTTCCTGATGA AACGGGCCTAACGGATGGTT S9

10 Supplementary Table S1. Parameters for BLAST. Parameter Description evalue=10 ExpecPtation value (E) threshold for saving hits, Default word_size=11 Word size for wordfinder algorithm (length of best perfect match) reward=1 Reward for a nucleotide match penalty=-3 Penalty for a nucleotide mismatch gapopen=5 Cost to open a gap gapextend=2 Cost to extend a gap outfmt=5 BLAST output in format.xml S10

11 Supplementary Table S2. Markers used in array-based detection of M. tuberculosis H37Rv genome. Region 1 Region 2 Region 3 Region 4 Marker 1 cccaaagctttgtcgacgcccag cctggtcacggacgttgctccgg Marker 2 ccgttgcataggtcaatgcaagt caactaactaaccaaccggttgg Marker 3 cctggccgtgtcggtcctctcac atgaagaatatgcggagtctagg Marker 4 cctcttggtacggacatgcgcgc ggatgcgaaaactacgtcctcgg Marker 5 ccgccaaaagcagaatggcagga gacccgccggaaccggtttccgg Marker 6 cctgtttggccgtcctggctggc atcggcaaaggaagcgtcgctgg Marker 7 cctactgtggacggcagccgtgg tatgccgggcattgcctcaccgg Marker 8 ccgtacccggggaccagcccgac tttcgtctgcgtcggactcttgg Marker 1 ccttgtctcatgttgccagcacg gactgccggggtcaactcggagg Marker 2 ccggtacaaagggctgcgatgcc aagccggtctcagttcggatcgg Marker 3 ccgaggatttccgaatggggaaa ctacccgcatctgaatatatagg Marker 4 cccggctgagaggcagtcagaaa ctgggatggtctgccgtagacgg Marker 5 cctaaatactcctcgatgaccga gggaatggtgaaaagtaccccgg Marker 6 ccctactaggttactgacgtcag tgtaaagcgtggcagtgagacgg Marker 7 ccgtacgtcgaaagggaaacagc aagcgtgtgctaagtgggaaagg Marker 8 ccacccttgaaagagtgcgtaat tgtgcgccgataatgtagcgggg Marker 1 ccccagctgagtagtcggaggcc gttcgcgcggacccatgtcgagg Marker 2 ccaccacttaatcagcaccaggc tagtcaacctccaagtcagccgg Marker 3 ccacttgactagccactgccaac accccggacccggactggatagg Marker 4 ccaggcccattgtctctcatctg tcgtcgtgcgctacggacaccgg Marker 5 ccagcaagcgatcctgagcgtcg ccctcggcgagccaggcactggg Marker 6 ccgacgctagttgccaggtccca gcatcgggatcctttcggctcgg Marker 7 ccaccgcttgtgggatcaatcgt gaatgccacgctcacttcgtcgg Marker 8 cctctgtgtgcaagtcgatgacg ggatgtgcagtttcccggtcagg Marker 1 ccaccgtggcgccttcgtcgcgc gaccgacgcggagtgcaccgcgg Marker 2 ccgagtttggaaatgtgcaccag cgatacaaaggcaccgaaatcgg Marker 3 ccttgggtccgatgacctccccg ggtggtcacccgcggggcgtagg Marker 4 cctgcgaagggatcccgtcgagc ggtgacgaagtccttggtcccgg Marker 5 ccgaccggtctcgccggtggaga atgtaccgcttcgatgtctccgg Marker 6 ccagctttttggtcagggcacgc ttcgcgaccctcgtaggtgtcgg Marker 7 ccggatccaggctgagaatcgtc cgcagcccgtcgacaaacgacgg Marker 8 ccggccgcatacatgcgctcctc agaagtcgaagtgttctttgggg S11

12 Supplementary Table S3. Markers used in array-based detection of B. subtilis genome. Region 1 Region 2 Region 3 Region 4 Region 5 Marker 1 ccttgaggaattgcagcatattg aaacgaaacagaggacgccgcgg Marker 2 cctgttgtgcttcctggcgagga gacgggaaagagcataatcaggg Marker 3 ccggacagggaaagcggatgaag cattgagctgaagggagacccgg Marker 4 cccgtttcaaacttcaattgagc gacagcacagtgtgtataagggg Marker 5 ccttgtacatgacggtgcccgtc aattgacgaactgatcgcagagg Marker 6 cctacttataacttcgcggtagc aaaatgacacacgtgctgcgcgg Marker 7 ccgggttttagaagtgcgccctt ttgaaatcagtattcaaaggcgg Marker 8 ccgccgttacaggctgaagttgg tcaaacagagtggaaccgcgcgg Marker 1 cctggcgttaagatagaagatga accaagctgtgatttagttgcgg Marker 2 ccggtttagttaagaaactattt aacatgcaaatcgccctccatgg Marker 3 ccttctgttgtggatcattccaa acggaagggttaagaggtgtcgg Marker 4 ccacctataaaatgcggatcgtc tgcagtttggtctccttacgagg Marker 5 ccaaaagggtcgatatcagattt acttggccaagtgcagcatgggg Marker 6 ccgctacatgcttgtagttcatt agatttgctgtaccatgaagcgg Marker 7 cctttgctaggaggaagctggcc ttatatccattgtattcaggcgg Marker 1 ccgatcttcgcagttgcgaactg tagaagcccatatggcgacgtgg Marker 2 ccatccgcatgagacctttagcg caaattgagcgtacgtatagcgg Marker 3 ccaagtaagttggccgcaggcac tttccgctgtattcgaaccgcgg Marker 4 ccccatatacctagcttgatcac tttgatagagaaagaacacaagg Marker 5 ccggtcgggtcatctgcggcgtt ctgatgccgaatttgacatacgg Marker 6 ccgaccaaggcccgtaagctgac cggtttaatctgaacgataacgg Marker 7 cctgtatgcccgctggtttcggg aatgaatgcttgctttgatgtgg Marker 8 ccttttctctgactgcttcaggc ttaccggtatcgataggcaaagg Marker 1 ccaatagagatggtaatgactag tcactaaactgaatatactgagg Marker 2 ccgtcatacgtatacttaccaga agaacgttacagtatctttctgg Marker 3 ccaaatgatatgaggggcttgtc atctggaaggatcactgaagcgg Marker 4 ccggctgcacgggatcttggttt cttgcaggagggcttcttggcgg Marker 5 ccgatcatcggaccacgaacggt aaaagccgttgaaatcagtctgg Marker 6 cctcccattgatgaggggatgat ggcatccaccatctttgtcccgg Marker 7 ccataaccatgggattcatacca tgctgagactgttatcatatagg Marker 8 ccaatacccttaccaaaacctcc gtgatggtcaaggcaacaatagg Marker 1 cctccttaacgttaattctcatc caaaacgaagtagttcaaacagg Marker 2 ccatctcagtatctgaagaagga ggatatttatttcatggctcagg Marker 3 ccccttgcttactgcatcgtcga gtcaccaatgcggaacgggcagg Marker 4 ccatctagtgtaacagaacatcc gcgccgatatccaaaaatgccgg Marker 5 cccgccgaacaagacttcattca gtttctttcgcgcggccaacagg Marker 6 ccgagtaacgcaaacatattgac ggtcaaacggattgacgccaagg S12

13 Region 6 Region 7 Region 8 Region 9 Marker 7 ccggctaaatagttcaagccgtc tcaattgtcagttcatgtttagg Marker 8 ccgcatattggtatgctgggtta caaaatctttgctcgcatccagg Marker 1 cccgtataatatgcgtcgcgctg cgtcaatctgactcgaaatcagg Marker 2 cctttctgagcacctgattaacc tacctcttgtaaaatcctcgtgg Marker 3 cctcaacctttgcgtccgggtcc atgttttgatattagtggcttgg Marker 4 ccagaaagctagttttatttgaa tccttcaaaatggaccggaacgg Marker 5 ccttttctattgagaaaagagaa tcagctattcttgtaaaatgagg Marker 6 ccaagcaatgaagccgtcagcgg cgccgccgaggccggaaccccgg Marker 7 ccacgagatatctaactgccata gctgatcatgaatacaacggcgg Marker 8 cctttaatatggatgctgatgtc gtggttatctgtatgtaagaagg Marker 1 ccgaaagcgtaagaagtccttgg tgtatagcaagacttttggccgg Marker 2 ccgctggacgcacaatcattgta ggcatgggtttccgccttggcgg Marker 3 ccgaacagttataacctgccaag atgagtttcatcataatgtgcgg Marker 4 ccctgtattgtaaggaatatagc aagcggctgaatagccgcgtagg Marker 5 ccggttccaaagtagcggatttc tgataggaaaatccgtagcccgg Marker 6 ccaagcttagggccgattgagac gcatcaatggtgagctgaagagg Marker 7 ccaagacgttcaggtaactgaaa gaaacgcccgcgcgtcatttggg Marker 1 cctcatccagtgggtaaaacgga agaaccgggcatctcctatacgg Marker 2 ccgaatacctgtgcatcaccgag cgcgttccatgtcacttgagcgg Marker 3 ccttcttggaacttcacacctga gcttatgctcaagttgcgctagg Marker 4 ccaaggctcacaaatgcagacaa ccaatgcagcagggacagcaagg Marker 5 ccgccgagcggggtgatcgctcc acttagaatatataagcttccgg Marker 6 cctgaagatacttatcaggtatt cctgaagatacttatcaggtatt Marker 7 cctcaccctttttgattcttctt aaaggccagaccgcccattgcgg Marker 8 ccaccacaagccggctgggcttt gcctttcaaacactgaatgacgg Marker 1 cctattgtaaaggagatatacaa tcattttttatccgaatataagg Marker 2 cctcgctcacgacggtaagagtt tttttgcttcctcaaggcgaagg Marker 3 ccagcttcttaatcgtcagccgc atgcaattgaggcatagctatgg Marker 4 ccgtgtgtatgtccgtttttcgc cattagcaaaccccttcctttgg Marker 5 cccgtcagcagttggtgaggctt cctccttttataggcgtctgcgg Marker 6 ccgcttatcccaagtccaaattt ctcttcaataggaccgattctgg Marker 7 cctgaaacgttccgactgaaaac ttgttcataattcgaatataagg Marker 8 ccccaagccgtcatgtcaataaa aatgcttgagtagattgctcagg S13