Name: transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination
Uploaded: 2012-10-08T12:00:00
Description: Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com

Jeg vil gerne anerkende Olga de Souza Silva til teknisk bistand. Dette arbejde blev støttet af den schweiziske National Science Foundation (SNSF) Grant 31003A_127029.

The TransFLP metoden (fig. 1) er eksemplificeret ved tre forskellige metoder: I), at to nabostillede gener, der koder virulensdeterminanter af V. cholerae (f.eks ΔctxAB), II) fjernelse af en patogenitetstest ø (f.eks ΔVPI-1), og III) integration af en T7 RNA-polymerase-afhængige promotorsekvensen opstrøms for et gen-of-interesse, som efterfølgende giver sit kunstige ekspression i den pågældende stamme baggrund (fx T7-tfoX) (figur 2). <p class="jove...

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The TransFLP fremgangsmåden beskrevet ovenfor og andre steder 5, er blevet ekstensivt anvendt i vores laboratorium. Den form for genetisk manipulation, der er mulig omfatter bl.a.: deletion af enkelte gener og gen-clustere, deletion af genomiske øer (fx VPI-1), insertion af sekvenser opstrøms for et gen af interesse (fx promotorsekvenser), og indsættelse af sekvenser ved slutningen af et specifikt gen (f.eks kodende for affinitetsmærker). En import ...

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transflp &#x2014; a method to genetically modify vibrio cholerae based on natural transformation and flp-recombination

Der findes flere metoder til at manipulere bakterielle kromosomer 1-3. De fleste af disse protokoller er afhængige af indsættelsen af konditionelt replikative plasmider (f.eks huser pir-afhængige eller temperatur-sensitive replicons 1,2). Disse plasmider integreret i bakterielle kromosomer på basis af homologi-medieret rekombination. Sådanne insertionelle mutanter ofte anvendes direkte i forsøgsopstillingen. Alternativt kan selektion for plasmidet excision efterfulgt af tabet udføres, som for gramnegative bakterier ofte afhængig af kontra-selekterbare levansucraseenzymet kodet af sacB-genet 4. The excision kan enten gendanne før insertion genotype eller medfører en udveksling mellem kromosomet og plasmidkodet kopi af det modificerede gen. En ulempe ved denne teknik er, at det er tidskrævende. Plasmidet, klones først, det kræver horisontal overførsel til V. cholerae (mest N<html> otably ved parring med et E. coli donor stamme) eller kunstige omdannelse af dette produkt, og udskæring af plasmidet er tilfældig og kan enten genoprette den oprindelige genotype eller skabe den ønskede modifikation, hvis der ikke positiv selektion udøves. Her præsenteres en metode til hurtig manipulation af V. cholerae kromosom (er) 5 (fig. 1). Denne TransFLP metode er baseret på den nyligt opdaget chitin-medieret induktion af naturlig kompetence i denne organisme 6 og anden repræsentant af slægten Vibrio, såsom V. fischeri 7. Naturlig kompetence tillader optagelse af frit DNA, herunder PCR-dannede DNA-fragmenter. Når taget op, DNA rekombinerer med kromosomet følge af tilstedeværelsen af mindst 250-500 bp flankerende homologe region 8. Herunder en selektionsmarkør i-mellem disse flankerende regioner gør det nemt at detektion af hyppigt forekommende transformanter. t &quot;&gt; Denne metode kan anvendes til forskellige genetiske manipulationer af V. cholerae og eventuelt også andre naturligt kompetente bakterier. Vi giver tre hidtil ukendte eksempler på hvad der kan opnås ved denne fremgangsmåde ud over vores tidligere offentliggjorte undersøgelse enkelt gen deletioner og tilsætning af affinitets-tag sekvenser 5. Adskillige optimering trin vedrørende indledende protokol af chitin-induceret naturlig transformation 6 er indarbejdet i denne TransFLP protokol. Disse omfatter blandt andet at erstatte krabbeskallen fragmenter ved kommercielt tilgængelig chitin flager 8 donation af PCR -afledt DNA som transformerende materiale 9, og tilsætningen af FLP-rekombinationssitene målsteder (FRT) 5. FRT sites tillader sted-rettet udskæring af selektionsmarkøren medieret af Flp-rekombinasen 10.

Repræsentative resultater af de tre eksempler er vist i figurerne 4-6. Den første strategi til at slette de tilstødende gener ctxA og ctxB. Sammen de koder for den store virulensfaktor V. cholerae, koleratoksin. Vi designede oligonucleotider til TransFLP metoden som beskrevet ovenfor og ifølge figur 3. Den parentale stamme, blev den mellemliggende stamme, der huser den FRT-flankeret antibiotikaresistens kassetten på den oprindelige DNA-locus af ctxAB<...

Watch this Scientific Journal Video about TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination at JoVE.com 

TransFLP â€” A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination (Video) | JoVE 

En hurtig metode til at modificere genomet af<em&gt; V. cholerae</em&gt; Er beskrevet. Disse ændringer omfatter sletning af enkelte gener, gen-clustere og genomiske øområder samt integration af korte sekvenser (f.eks promotorelementer eller affinitet-tag-sekvenser). Fremgangsmåden er baseret på det naturlige transformation og FLP-rekombination.

TransFLP - en metode til at gensplejse<em&gt; Vibrio cholerae</em&gt; Baseret på Natural Transformation og FLP-rekombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

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TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

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