以細菌為平台發展新的大片段DNA複製技術

在細菌的研究中，我們會透過一個自殺式質體幫助我們將細菌染色體上的基因刪除。

... 在我的研究裡，我將這段跳出的環狀DNA透過細菌接合的方式送到大腸桿菌內保存並進行 ... 資料載入處理中... 跳到主要內容 臺灣博碩士論文加值系統 ::: 網站導覽| 首頁| 關於本站| 聯絡我們| 國圖首頁| 常見問題| 操作說明 English |FB專頁 |Mobile 免費會員 登入| 註冊 功能切換導覽列 (165.22.106.144)您好！臺灣時間：2022/09/0417:09 字體大小：       ::: 詳目顯示 recordfocus 第1筆/ 共1筆  /1頁 論文基本資料 摘要 外文摘要 目次 參考文獻 紙本論文 論文連結 QRCode 本論文永久網址: 複製永久網址Twitter研究生:謝堉文研究生(外文):Yu-WenHsieh論文名稱:以細菌為平台發展新的大片段DNA複製技術論文名稱(外文):DevelopmentofanovelinvivotargetcloningsystemforlongDNAfragmentinbacteria指導教授:鄧景浩、橋本昌征指導教授(外文):Ching-HaoTeng、MasayukiHashimoto學位類別:碩士校院名稱:國立成功大學系所名稱:分子醫學研究所學門:醫藥衛生學門學類:醫學學類論文種類:學術論文論文出版年:2016畢業學年度:104語文別:英文論文頁數:59中文關鍵詞:活體克隆技術、自殺式質體、基因島外文關鍵詞:invivocloning、suicidevector、genomicisland相關次數: 被引用:0點閱:165評分:下載:0書目收藏:0 隨著科技發展，現在我們可以透過高通量定序的技術定序細菌的基因體。

透過比較與分析，我們可以找到一些外來的基因序列存在於細菌的基因體中，而這些序列通常是來自細菌間水平傳播，並且被定義為基因島。

這些基因島的長度通常都大於五萬對鹼基序列。

聚合酶連鎖反應通常被應用在研究一個目標基因，但基因島的序列長度遠超過聚合酶連鎖反應所能放大的範圍。

因此，在我的研究中，我將發展一個新的系統能夠複製像基因島這般大的長片斷鹼基序列。

在細菌的研究中，我們會透過一個自殺式質體幫助我們將細菌染色體上的基因刪除。

這個自殺式質體包含欲刪除目標序列的上下游片段以及一個抗生素標記。

當我們將這個自殺式質體導入細菌中，透過兩次的同源重組，我們可以將目標基因置換成抗生素標記以達成刪除基因的目的。

其中，在第二次的同源重組裡，一段帶有目標序列的環狀DNA會從染色體跳出。

但這個帶有目標序列的環狀DNA並不能在細菌中被保存，因此會消失在細菌中。

在我的研究裡，我將這段跳出的環狀DNA透過細菌接合的方式送到大腸桿菌內保存並進行其他操作。

在我的研究中，我建構一個自殺式質體和一隻能夠保存此自殺式質體的大腸桿菌。

為了證明我們系統的可行性，我利用了這個系統複製了一段在沙門氏菌中長度約四萬對鹼基序列的基因島SPI-2及一段在細菌性葉斑病菌中長度為七萬對鹼基序列的非核醣體胜肽合成酵素。

在實驗中，我們先將自殺式質體送入細菌中，透過第一次同源重組，這個自殺式質體會潛入細菌染色體。

之後，我們利用細菌接合將發生第二次同源重組所跳出並帶有目標序列的質體送到大腸桿菌中。

在實驗結果中，我們得到一些含有目標DNA質體的細菌，並透過聚合酶連鎖反應進行確認。

此外，將含有基因島SPI-2的質體送入SPI-2突變的沙門氏菌能夠成功回補SPI-2的功能，這個結果顯示通過此複製技術所複製出的DNA片段是有功能的。

綜合以上實驗結果，我們發展了一個新的活體大片段複製技術，且這個技術只需要一個自殺式質體且已知最少能夠用來複製長七萬對鹼基序列的DNA片段。

High-throughputsequencingtechnologieshavemadeitpossibletostudybacteriathroughanalyzingtheirgenomesequences.Byanalyzingbacterialgenomes,wecandiscoverforeigngenomicregionsinthebacteria,andtheymighthorizontallytransferfromotherbacteria,whicharedefinedasgenomicislands.Thelengthsofthegenomicislandsareoftenlongerthan50kb.PCRenablestargetcloning,however,itisbeyondthelimitationofPCRtoamplifysuchalongDNAfragment.Here,weaimtodevelopaneasymethodtoclonealargeDNAfragmentininvivo,whichisnecessarytohandlethegenomicislands.Inbacteria,asuicideplasmidisgenerallyusedforgenereplacementtodeletearegionfromthechromosome.Thesuicideplasmidcontainsanupstreamregionoftargetregiontobecloned,anantibioticmarker,andadownstreamregionofthat.Throughthedouble-crossingoverprocess,theantibioticmarkeroriginallylocatedontheplasmidwillbereplacedwiththeregiontobedeletedonthechromosomeviahomologousrecombination.Thepopped-outcircularDNAiscarryingthetargetedregion,butisnon-replicableinthebacterialcell.Then,wesendthepopped-outcircularDNAtoE.colibyconjugationtorescueitasaplasmidcontainingtargetregion.Finally,theplasmidcanbemaintainedinE.coliforfurthermanipulation.Fortheaim,IconstructedasuicideplasmidvectorandanE.colistraintomaintaintheplasmid.Tovalidatethisinvivocloningmethod,wetargeteda40kbgenomicislandSPI-2encodingtypeIIIsecretionmachineryfromSalmonellaentericaserovarTyphimuriumanda70kbfragmentencodingnon-ribosomalpeptidesynthaseinPseudomonascichorii.Briefly,thesuicideplasmidwasintegratedintotargetregionthroughhomologousrecombination,andthen,conjugationwascarriedouttorescuethepoppedoutplasmidtoE.coli.Astheresult,weobtainedseveralE.colicoloniesharboringthelongtargetedDNAfragment.PCRanalysisshowedthattherescuedplasmidcontainsthetargetregion.ComplementationofΔSPI-2mutantofSalmonellastrainbyusingtheplasmidcloningSPI-2regionshowedtheclonedSPI-2wasfunctional.Takentogether,theeasyinvivocloningsystembyusingsinglesuicideplasmidwasdevelopedwhichallowedtocloneatleast70kbDNAfragment. 中文摘要IAbstractII誌謝IVContentVIntroduction11.1CloningofalongDNAfragment11.2ReplicationcontrolofRK2plasmid21.3Suicidevector41.4Bacterialconjugation51.5Cloningtargets5Specificaims8Materialsandmethods92.1Bacterialstrainsandculturecondition92.2Cellculture92.3PCRforvectorconstruction92.4Ligation102.5PCRwithTaqpolymerase102.6Plasmidtransformation102.7TransformationwithlinearizedDNAfragmentbyredrecombination112.8Constructionofcloningvector,pYW3112.9Bacterialconjugation122.10Gentamicinprotectionassaytodeterminebacterialsurvivalwithinmacrophage122.11Kado-Liumethod132.12P1phagetransduction132.13Statisticalanalysis14Results153.1ConstructionofpYW3,avectorforcaptureplasmid153.2CloningtheSPI-2regionfromSalmonellawiththecapturevector163.3CloningtheSPI-2regionfromSalmonellawiththenewcapturevector183.4CloningtheNRPSregionfromPseudomonascichoriibythenewvector193.5Complementationassaywiththepopped-outplasmid20Discussion22Acknowledgment25FiguresandTables26Reference51 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 國圖紙本論文 連結至畢業學校之論文網頁點我開啟連結註:此連結為研究生畢業學校所提供，不一定有電子全文可供下載，若連結有誤，請點選上方之〝勘誤回報〞功能，我們會盡快修正，謝謝！ 推文 網路書籤 推薦 評分 引用網址 轉寄                                                                                                                                                                                                                    top 相關論文 相關期刊 熱門點閱論文 無相關論文   無相關期刊   1. 探討能幫助E.coli攝鐵的抗藥接合質體 2. 蛋白酶Prc在腸道外致病性大腸桿菌致病機轉的角色 3. 以莢托馬尾藻進行褐藻多醣之萃取、純化及生物活性探討 4. 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