基因选殖的策略

基因選殖的策略原核生物-細菌萃取chromosomeDNAPartialdigestionDNA(選一種限制酶)Vector(去磷酸根)(降低self-ligation)Ligation,trnasformation藍白篩選活性篩選真核生物?M-Marker:DNA/HindIII1-StreptomycesPMS502chromosome2-1/BamHI(mgDNA/0.18unit)3-1/BamHI(mgDNA/0.06unit)4-1/BamHI(mgDNA/0.02unit)5-1/BamHI(mgDNA/0.007unit)23.19.46.64.42.32.0M12345ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCLigationTransformationPickingwhitecolonies選殖基因的定序Clone確認定序primer的選擇Vector上的forwardreverse序列Ex:pUC18第二次定序自行設計primer序列的組合(正股反股)DeterminingDNAsequences1975FrederickSanger–dideoxynucleosidetriphosphates(ddNTPs)定序完成後的工作上網比對序列blast確認所clone的基因預測ORF(openreadingframe)DNA-STARVECTORNTI9.0ORF的確認Ribosomebindingsite(RBS-6~-12GArich)蛋白的分子量與基因長度相關性TATAbox(-35-10)設計primerssubclone,expressionExpressionvector的選擇(pET,pQE,pGEX4T…)基因產物的純化設計PrimersPCR基因產物前的分析工作解讀限制酶圖譜Primer序列加上限制酶切位選擇哪種限制酶切位Multi-cloningsite有的切位轉錄或轉譯起始位置的考量(與載體有關)primer設計時要注意序列inframe實做練習ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGAGCCGGGGAGCACATTGGCGGCACCGTGAGAGCGCTCTCAAAGGTCCCGCGTCCCCC660GCTCCCCGCTCCCGAGAGGCCACCCATGAGTGATTCCTCCGGCACCCCCAGACCCCGTTC720MSDSSGTPRPRSCCACTCCCGCCCCCGCTCCCGCTCCGTGCGCCGCGCCCTCATGGCGGCCGTCGCGACGTT780HSRPRSRSVRRALMAAVATFCGGCCTCGCCGCGGCCGTCGCCACCGCGGCCACCGGCCCCGCGGACGCCTCCGCCCCGGC840GLAAAVATAATGPADASAPAGCCGCCCTCCGGCTGGTCGCAGGTGTTCCTCGACGACTTCGACGGCGCGGCCGGAAGCAG900PPSGWSQVFLDDFDGAAGSSCGTCAACACCGCCAACTGGCAGTTCGACACCGGCACTTCGTACCCCGGCGGGGCAGGCAA960VNTANWQFDTGTSYPGGAGNCTGGGGCACTGGCGAGGTCGAGTCGATGACGTCGAGCACCAGCAATGTCTCCCTCGACGG1020WGTGEVESMTSSTSNVSLDGCAACGGCGATCTGCTCATCACCCCGCGCCGCGACGCGTCCGGCAACTGGACCTCCGGCCG1080NGDLLITPRRDASGNWTSGRGATCGAGACCACCCGCACCGACTTCCAGCCCCCGGCCGGCGGCAAGCTGCGCGTCGAGGC1140IETTRTDFQPPAGGKLRVEAGCGCCTGCAGATGCCGAACGTGACCGGGGACGCGGCCGCCGGGTACTGGCCCGCGTTCTG1200RLQMPNVTGDAAAGYWPAFWGATGCTGGGCGCGCCCTTCCGCGGCAACTACCAGAACTGGCCCGGCGTCGGCGAGCTGGA1260MLGAPFRGNYQNWPGVGELDCATCATGGAGAACGTCCAGGGCCTGAACAAGACCTGGGCCACCATGCACTGCGGGACCAG1320IMENVQGLNKTWATMHCGTSCCCCGGCGGGCCCTGCAACGAGACCAGCGGCATCGGCAATTTGACCGCCTGCCCGAACAC1380PGGPCNETSGIGNLTACPNTCACCTGCCATTCGGGCTTGCACACCTACACCATGGAGTGGGACCGCTCGGTCAGCCCCGA1440TCHSGLHTYTMEWDRSVSPEGGCGATCCGCTTCTCCGTCGACGGCGTCACCTACCAGACGGTCACGGCGAATCATATGGA1500AIRFSVDGVTYQTVTANHMDCGCGGTCACCTGGACCAACGCCACGAACCACGGTTTCTTCGTTATCCTGAACGTGGCGAT1560AVTWTNATNHGFFVILNVAMGGGCGGCGGCTTCCCGGGTGCCTTCGGCGGCGGCCCGACCGGCGCCACCGAGCCCGGCCA1620GGGFPGAFGGGPTGATEPGHCCCCATGGTCGTCGACTACGTCCAGGTGCTCCAGTCCTCGGGCGGCGGGGGCGGCGGGGG1680PMVVDYVQVLQSSGGGGGGGCGGGGGCACCACTCCCCCGCCGACCGGCGACCGGGACGCGTACGGTCAGATCCAGGCGGA1740GGTTPPPTGDRDAYGQIQAEGTCGTACGACGGCCAGTCCGGTGTCGCCACCGAGACCACCACGGACACCGGCGGCGGACA1800SYDGQSGVATETTTDTGGGQGGACATGGGCTACCTCGCCAATGGCGACTGGGCGCTGTACAAGGGCGTGAACTTCGGCTC1860DMGYLANGDWALYKGVNFGSGACACCGGCCACCCAGTTCTACGGTCGGGTGGCGAGCGGCGCGGGCGGCGGGGTCAGCGG1920TPATQFYGRVASGAGGGVSGCCTGGTGGAGGTGCGGCTGGACAGCCGTACGAACGCGCCGATCGGGAGCTTCGCGGTGGG1980LVEVRLDSRTNAPIGSFAVGCGACACCGGGGGCTGGCAGTCCTGGCGGACGGTTCCGGCCAACATCGGGTCCGTCACCGG2040DTGGWQSWRTVPANIGSVTGCACGCACGATGTGTACCTCACGTTCAGCAGCGGGCAGCCGGCCGACTTCGTGAACGTCAA2100THDVYLTFSSGQPADFVNVNCTGGTTCGACTTCGGGCACTGATCCGCCGCGGGTACCGGGGTAGCGTTCACAGCATGTCG2160WFDFGH*PartialalignmentoftheS.Sioyaensisenzymewithotherb-1,3-glucanaseofglycosylhydrolasesfamily1657WSQVFLDDFDGAAGSSVNTANWQFDTGTSYPGGAGNWG--TGEVESMTSSTSNVSLDGNGDLLITPRRDASGNWTSGRSsio57LLWSDEFDGAAGSAPNPAVWNHETGA------HGWG--NAELQNYTASRANSALDGQGNLVITARRE-------GDOxan46WRLIWHDEFEG---SEVNKEYWTFEKGNGIAYGIPGWG--NGELEYYAE---NNTYIVNGTLVIEARKEIITDPNEGTPfur216WQLVWSQEFDD---GVIDPNVWNFEIGNGHAKGTPGWG--NAELEYYTD---KNAFVENGCLVIEARKEQVSD-EYGTTnea425LIWQDEFN---GTTLDTSKWNYETGYYLNNDPATWGWGNAELQHYTNS-TQNVYVQDGKLNIKAMNDSKSFPQDPNBcir30WELVWSDEFD--YNGLPDPAKWDYDVGG------HGWG--NQELQYYTRARIENARVGGGVLIIEARRE-----SYEGRmar135IETTRTDFQPPAGGKLR------VEARLQMPNVTGDAAAGYWPAFWMLGAPFRGNYQNWPGVGELDIMENV-QGLNKTSsio117--GSYTSARMTTQGKYQPQYGRI-EARIQIPR-----GQGIWPAFWMLGG--SFPGTPWPSSGEIDIMENVGFEPHRVOxan115--FLYTSSRLKTEGKVEFSPPVVVEARIKLPK-----GKGLWPAFWMLGS--NIREVGWPNCGEIDIMEFLGHEPRTIPfur284--YDYTSARITTEGKFEIKYGKI-EIRAKLPK-----GKGIWPALWMLGN--NIGEVGWPTCGEIDIMEMLGHDTRTVTnea496RYAQYSSGKINTKDKLSLKYGR-VDFRAKLPT-----GDGVWPALWMLPKDSVYGT--WAASGEIDVMEARGRLPGSVBcir92R--EYTSARLVTRGKASWTYGRF-EIRARLPS-----GRGTWPAIWMLPDRQTYGSAYWPDNGEIDIMEHVGFNPDVVRmar205WATMHCGTSPGG-PCNETSGIGNLTACPNTTCHSGLHTYTMEWDRSVSPEAIRFSVDGVTYQTVTANH---MDAVTWTSsio184HGTVHGPGYSG----GSGITGMYQHPQGWS-FADTFHTFAVDWK----PGEITWFVDGQQFHRVTRAS-VGANA--W-Oxan183HGTVHGPGYSG----SKGITRAYTLPEGVPDFTEDFHVFGIVWY----PDKIKWYVDGTFYHEVTKEQ-VEAMGYEW-Pfur352LRTAHGPGYSG----GASIGVAYHLPEEVPDFSEDFHVFSIEWD----ENEVEWYVDGQLYHVLSKDE-LAELGLEW-Tnea566SGTIH---FGGQWPVNQSSGGDYHFPEG-QTFANDYHVYSVVWE----EDNIKWYVDGKFFYKVTNQQWYSTAAPNNPBcir162HGTVHTKAYNHL--LGTQRGGSIRVPTARTDF----HVYAIEWT----PEEIRWFVDDSLYYRFPNER-LTNPEADWRRmar378NAT-NHGFFVILNVAMGGSsio249--VFDQPFFLILNVAVGGOxan250--VFDKPFYIILNLAVGGPfur393--VFDHPFFLILNVAMGGTnea636NAPFDEPFYLIMNLAVGGBcir228HWPFDQPFHLIMNIAV--Rmar