酵母单杂

酵母单杂1、启动子构在载体pLacZi上；2、基因构在载体pB42AD上；接头引物LACZ-F：5`-ATCTGTCGACCTCGAG-3`酶切XhoILACZ-R：5`-GAGCACATGCCTCGAG-3`PB42AD-F：5`-TGCCTCTCCCGAATTC-3`酶切EcoRIPB42AD-R：5`-CGAGTCGGCCGAATTC-3`测序引物Pb42AD-F：ACCAGCCTCTTGCTGAGTGGAGATGCCPb42AD-R：GAAGTGTCAACAACGTATCTAPLacZ2U-F：GAATTCGAGCTCGGTACCCPLacZ2U-R：GGACCTAATGTATAAGGAAAG3、菌株：EGY48；4、转化步骤参照一般的酵母转化，共转到2缺平板上；5、显色培养基：X-gal(20mg/mlinDMF)将5-溴-4-氯-3-吲哚-β-半乳糖苷(X-GAL；#8060-1)溶解在二甲基甲酰胺(DMF)中，–20°C避光保存。10XBUsaltsDissolvethefollowingcomponentsin1L(total)ofH2O:70gNa2HPO4•7H2O30gNaH2PO4调pH为7，高压灭菌，室温保存。SD/Gal/Raf培养基的配制（包含X-gal和BUsalts，并且灭菌完降到55度以下再加）FinalconcentrationToprepare1Lofmediumβ-Galactose2%50mlof40%stock20gRaffinose1%25mlof40%stock10g10XBUsalts1X100mlof10XstockX-Gal80mg/L4mlof20mg/mlYNB6.7gAgar20gSD-URA-TRP0.72g倒平板，室温下凝固。倒置平板，用塑料套筒套住，黑暗保存。在4°C下最多至两个月。注意事项：A、当加入BUsalts和X-Gal时培养基温度不得超过55°C，如果超过55°CBUsalts会沉淀，X-Gal会降解。B、半乳糖必需是高纯度的，并且葡萄糖含量要小于0.01%；C、调节培养基pH到7，因为这个值接近β-半乳糖苷酶反应的最适pH，能够为β-gal反应提供必需的磷酸盐。Figure1.Mapandmultiplecloningsite(MCS)ofpLacZi.pLacZiisayeastintegrationandreportervectorforusewiththeMATCHMAKEROne-HybridSystem.ThisplasmidcontainsthebacteriallacZgenedownstreamoftheminimalpromoteroftheyeastiso-1-cytochromeCgene(PCYC1).TargetelementscanbeinsertedintotheMCSupstreamofthePCYC1-lacZreporter.Withoutactivationfromacis-regulatoryelement,lacZexpressionisverylowwhenthevectorisintegratedintotheyeastgenome.TheyeastURA3geneisusedasaselectablemarkerforintegrationintothenonfunctionaluralocusoftheYM4271hoststrainafterlinearizingthevectorattheNcoIorApaIsite.pLacZicannotreplicateautonomouslyinyeast.Thisplasmidcontainstheampicillinresistancegene(Amp)andtheColE1originforselectionandpropagationinE.coli.UniquerestrictionsitesareinboldFigure2.pB42ADplasmidmapandMCSsequence.(pB42ADwasoriginallypublishedaspJG4-5inGyurisetal.,1993.)pB42ADexpressescDNAsorothercodingsequencesinsertedintotheuniqueECORIandXhoIsitesastranslationalfusionstoacassetteconsistingoftheSV40nuclearlocalizationsequence,the88residueB42acidicactivator(AD),andthehemagglutininepitopetag.FusionproteinexpressionisunderthecontroloftheGAL1induciblepromoter.MaybepropagatedandselectedforinE.coliandyeast.TheTRP1transformationmarkerisusedforselectioninyeast.Uniquesitesareinbold.ApB42ADinsertsequencingprimerisincludedintheMATCHMAKERLexATwo-HybridSystemforsequencinginserts.