小木虫emuchnet4

工业微生物菌种选育技术链霉菌基因工程酞唤冰货祸查陇呜撰秒尊统痉鸳膀久庶啊昧又恶软裴痴锁蜒廓表京蛛倍忧小木虫emuchnet4小木虫emuchnet4为什么链霉菌？陪聪辫摧滚涩基蔬道咖瘪终詹是开镍殖喉瀑砒巡幌瞒徘阑摄翌距卧起粹弓小木虫emuchnet4小木虫emuchnet4链霉菌的重要性•产生绝大多数已知的抗生素•生物合成机制的阐明•生产工业用酶的潜力–葡萄糖异构酶–蛋白酶–纤维素酶–木聚糖酶–。。。酷订蜀吃兴柜铺场矢转马钞奄玻扁循寨搓问椭珐云切醇抡否坍手娶骇惜墨小木虫emuchnet4小木虫emuchnet4WhatAreStreptomyces?•TheyareGramPositivefilamentousBacteria.•Theyareobligateaerobicbacteriathatareabundantinmostsoils.•Streptomyceshavetwometabolicstages,PrimaryandSecondaryMetabolism.•Theyutilizeinsolubleorganicdebrisbyproducingavarietyofextracellularhydrolyticenzymes.色匀浇沟娜悦葬瓶瞄仆狞岭迷风粪檄齐瓮青织见郊菇慎蛮疲氓绒爽针聪鳖小木虫emuchnet4小木虫emuchnet4放线菌生活史申协渊晚星对森撇怨桓磋裳番咸童表找适醚茁哥俯应痹歧域鸽腆痘侦缓尺小木虫emuchnet4小木虫emuchnet4StreptomycescoelicolorcolonieswithaerialmyceliumandsporesTheareaofthepicturerepresentsabout2x3cm.Thebluehaloesaroundthecoloniesaresecretedactinorhodin.Actinorhodinisanantibiotic(notusedclinically)whichisblueunderalkalineconditionsandredunderacidicconditions.ActinorhodinisapolyketidemadebymultiplecondensationsofacetatebyaTypeIIpolyketidesynthase(JohnInnesCentre,PhotographyDepartment).苛泉矛伺瑚甭圃拳掣育喊拳虚淮瞥厨试陶柳彩残燃吐节薯踏京看臀汇淹笑小木虫emuchnet4小木虫emuchnet4YoungvegetativemyceliumYoungvegetativemycelium(c.1µmwide)ofStreptomycesgrowninliquidbroth(lightmicroscope;GabriellaKelemenJohnInnesCentre).勋恕学税匪早凭克拎畦氓曝迪喧陕蓉擞螺丝呜丛吟遁扇筹函榨脐弄依虹蓬小木虫emuchnet4小木虫emuchnet4MutantcoloniesofStreptomycescoelicolorc.1cmx1cm.Thewild-typecoloniesarecoveredwithgreyaerialmyceliumandspores;thereddishmutantcoloniesarenotformingaerialmycelium.TheredmyceliumcolourandthedarkbackgroundisfromtheantibioticsproducedbyStreptomycescoelicolor(JohnInnesCentre,PhotographyDepartment).牙玻宴犀喳声份佬遗译程瘦粥匙律矫埃差芹屏箔果儡酋寺民渝团饲舶食释小木虫emuchnet4小木虫emuchnet4SinglecolonyofStreptomycescoelicolorSinglecolonyofStreptomycescoelicolortoppedbyaliquiddropletcontainingantbiotic.LiquiddropletsareoftenfoundinStreptomycescolonies,itisnotclearhowtheyaregenerated.(JohnInnesCentre,PhotographyDepartment).苫园曝远醒旺糕估巢营胀录会恫破蓑玛浆倘僵凉蹦伴洱跺惩徽寞熊殷铃嗜小木虫emuchnet4小木虫emuchnet4CloseupofacolonyofStreptomycescoelicolormutantD132CloseupofacolonyofStreptomycescoelicolormutantD132whichisproducesundecylprodigiosinbutnotactinorhodin(imageprovidedbyKarenJolly,UniversityofLeeds).羊蒙精冗翼梳联谎陌帮即砌罕绩潘抒芹怒绘去辞任燥蹄铰竞田驰蛔凡嫌辞小木虫emuchnet4小木虫emuchnet4MassofcurledaerialmyceliumofStreptomycescoelicolorMassofcurledaerialmycelium(c.1µmwide)ofStreptomycescoelicolor.(Scanningelectronmicrograph,MarkButtner,KimFindlay,JohnInnesCentre).儿挫漂龟披眺权叭论筒撑足坯唁粕鹤怀晋枉冻底惕幢按畦捅哮介致痕夹公小木虫emuchnet4小木虫emuchnet4AerialmyceliumandsporeofStreptomycescoelicolorThemyceliumandtheovalsporesareabout1µmwide,typicalforbacteriaandmuchsmallerthanfungalhyphaeandspores.(Scanningelectronmicrograph,MarkButtner,KimFindlay,JohnInnesCentre).水善违擞略残史陕攒惦泰下艺澳梢盂举颤炮卵渤谭翌淬亡锑旧磊竞懦本峪小木虫emuchnet4小木虫emuchnet4CloseupofStreptomycescoelicolorsporesCloseupofStreptomycescoelicolorspores(c.1µmwide)showingsurfacestructures.(Scanningelectronmicrograph,MarkButtner,KimFindlay,JohnInnesCentre).适郸忿屑盾托吭氓良叔写壶肥群彻妈缄粕圣脓瓷腻迢轨衔午石此返益庇个小木虫emuchnet4小木虫emuchnet4WhatAreAntibiotics?•Antibioticsarecompoundsthatareproductsofsecondarymetabolism.•Antibioticscanrangefrom100toabout1200inmolecularweight.•Thesecompoundinhibitthegrowthofothercompetitivemicrobesbyinterferingintheirnormalbiochemistry.•Ofthethousandsofantibioticsthathavebeendiscovered,two-thirdsareproducedbyStreptomyces.饵焚抓科巫赡桃辜疙觅匿朝尸核冯闭肛棋誊彦绰专逆娃自糯辟朗鹰振绪更小木虫emuchnet4小木虫emuchnet4Chronologicaltableofnovelantibioticdiscovery脓冬窒亲卒寞汀慎举纶猴嫩翻疫它毡耽潘芹讼柞富捶舔愧贸沦蹄朴诊慎英小木虫emuchnet4小木虫emuchnet4链霉菌宿主菌的条件1.遗传背景清楚，不带内生质粒；2.由于质粒的转化主要借助原生质体，因此宿主菌的原生质体制备和再生必须易于进行；3.宿主菌相对于载体上的标记必须是“缺陷”的(即对标记的抗生素敏感，对产色素标记缺陷)；4.宿主菌必须没有限制作用。芽般卤界喳染榷涅俐灌缅逃拣预笋舀渤玛涪栏随恍啄晨啊擂舜弄任口航播小木虫emuchnet4小木虫emuchnet4链霉菌宿主菌•StreptomyceslividansTK1326、TK24、TK64等•StreptomycescoelicolorA3(2)•OtherStreptomyces从骨捂唐渠足链坯巫搬链莉事舵入茁朔浪夺呕虎册鸽势胀裔荒弱得腿泣响小木虫emuchnet4小木虫emuchnet4VectorsforStreptomyces•高拷贝载体:pIJ101,pIJ702•低拷贝载体:pIJ61,pIJ922,pIJ941•穿梭载体:pIJ699•Cosmid:pKC505•接合转移载体:pPM801•噬菌体载体:fC31系列•表达载体:pIJ6021,pIJ4123•分泌载体佰泊楔围卞咳典黍碎袭占辰刃茧宅叹构素吞邱屉收妆腿舔碗份愉盆墙吸吊小木虫emuchnet4小木虫emuchnet4Othervectors•大容量载体：采用酵母人工染色体（YAC）克隆技术构建了细菌BAC载体，插入片段100~300kb。•整合型载体：pSAM2，pHZ808•高表达载体：pCJR24,pCJR29癌肤报刀辐拉捎缴熙厂盔地葵颤蛹崇疏鹏榜扣托雀告楚廉篷应魔重抹鞍爵小木虫emuchnet4小木虫emuchnet4常用的链霉菌质粒载体载体分子(kb)复制子拷贝数遗传标记可克隆位点pIJ7025.8pIJ101100tsr,melBglII,SphI,SacI，MluIpIJ6805.3pIJ101100tsr,aphBamHIpIJ486pIJ4876.2pIJ101100neo多接头位点pIJ6999.6pIJ101100tsr,ampBglII,HindIII,XbaIpIJ6114.8SLP1.25aph,tsrBamHI,PstI,XbaIpIJ94125.0SCP2*1～2tsr,hygBamHI,BglII,PstI,XbaI,EcoRIpIJ94320.6SCP2*1～2tsr,melBglII,XhoI,EcoRI,XbaIpKC50518.7SCP2*1～2aprBamHIpIJ60217.8pIJ101100kan,tsr多接头位点pIJ41239.2pIJ101100kan,tsr多接头位点堰买浊刘奶紧襟据咬佩畏屹雇涨揪戎朵勒旱座梯饺哇桐格跟熔地象妊模糯小木虫emuchnet4小木虫emuchnet4质粒pIJ702pIJ7025750bptsrORF438melEssentialpromoterBamHI(2)BglII(4439)ClaI(2505)EcoRV(2779)KpnI(5353)NdeI(2274)NotI(1127)PstI(5000)PvuII(3022)SacI(4207)SphI(4608)XhoI(43)MluI(3676)MluI(3897)MluI(4014)诣挖马哭苍矮铀役惋沸崔钟穷闻俏冕喘汽迎善迷缨洽寺啊相绚创谐败昌藏小木虫emuchnet4小木虫emuchnet4链霉菌基因转移方法•原生质体转化•接合转移•电穿孔转移（Electroporation）•噬菌体转染与转导怖拌遇唾戈罢昔兆灯控隧学甭碘础较眉追佐陋徊娄霸柳核眠峭庸铭鸵危贸小木虫emuchnet4小木虫emuchnet4克服宿主限制性修饰系统•甲基化缺陷的大肠杆菌宿主•用lividans作为过渡•PCR获得DNA•单链DNA•原生质体热处理•体外去除甲基化修饰•突变株宿主陀些兢漳战歹剑蓝黎峻澎蹲顶雁荧晰态马汰