Name: site-specific bacterial chromosome engineering: &#934;c31 integrase mediated cassette exchange (imce)
Uploaded: 2012-03-16T13:00:00
Description: Watch this Scientific Journal Video about Site-specific Bacterial Chromosome Engineering: ÃŽÂ¦C31 Integrase Mediated Cassette Exchange IMCE at JoVE.com

Margaret CM Smith vänligt ge integras klonen Finansiering stöd från: Genome Canada / Genome Prairie NSERC Discovery och strategiska projektbidrag

1. Produktion av kultur <ol><li> Framställa sterila flytande media: TY 10 (5 g / I trypton, 3 g / I jästextrakt, 0,44 g / liter kalciumklorid dehydrat) och LB-11 (10 g / I trypton, 5 g / I jästextrakt, 5 g / natriumklorid, pH 7). </li><li> Inokulera från en enda koloni: SmUW227 (S. meliloti LP-stam: konstruktion kommer att beskrivas på annat håll, stamkonstruktion information tillgänglig på begäran) () i 5 ml TY media med 50 pg / ml spektinomycin. Ympa följande stammar i 5 ml m...

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Den IMCE Tekniken möjliggör effektiv integrering av ett enda flankerad attB DNA-kassett till LP-platsen för en tidigare konstruerad stam. När den önskade konstruktionen klonas i stället för RFP för att skapa givaren kassetten kräver tekniken inte efterföljande DNA rening och transformation, vilket gör det mycket robust. Det är viktigt att lämpliga tillväxt kontroller ingår, för att vara säker på att antibiotikaresistens beror på skapandet av trans-integranter och inte andra faktorer....

<table border="1"><tbody><tr><td> Namnet på reagens </td><td> Företaget </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Streptomycin </td><td> Bioshop Canada Inc. </td><td> STP101 </td><td></td></tr><tr><td> Spektinomycin </td><td> Bioshop Canada Inc. </td><td> <a href="https://ssl34.alentus.com/bioshopcanada/detail.asp?Pin=SPE201">SPE201</a> </td><td></td></tr><tr><td> Gentamicin </td><td> Bioshop Canada Inc. </td><td> GTA202 </td><td></td></tr><tr><td> Choramphenicol </td><td> Bioshop Canada Inc. </td><td> CLR201 </td><td></td></tr><tr><td> Tetracyklin </td><td> Bioshop Canada Inc. </td><td> TET701 </td><td></td></tr><tr><td> Kanamycin </td><td> Bioshop Canada Inc. </td><td> KAN201 </td><td></td></tr><tr><td> Bakteriologisk kvalitet agar </td><td&gt; Bioshop Canada Inc. </td><td> AGR001 </td><td></td></tr><tr><td> Trypton </td><td> Bioshop Canada Inc. </td><td> TRP402 </td><td></td></tr><tr><td> Jästextrakt </td><td> Bioshop Canada Inc. </td><td> YEX401 </td><td></td></tr><tr><td> Natriumklorid </td><td> Bioshop Canada Inc. </td><td> SOD001 </td><td></td></tr><tr><td> Kalciumklorid </td><td> Bioshop Canada Inc. </td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td> Gold Biotechnology Inc. </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli-stam MT616 </td><td> Tillgänglig på begäran </td><td></td><td> Används även utanför vårt laboratorium </td></tr><tr><td> E. coli-stam pJC2 </td><td> I hus, finns på begäran </td><td></td><td></td></tr><tr><td> E. coli-stam pJH110 </td><td> I hus, tillgänglig genom de krävst </td><td></td><td></td></tr><tr><td> SmUW227 stam </td><td> I hus, finns på begäran </td><td></td><td></td></tr></tbody></table>

site-specific bacterial chromosome engineering: &#934;c31 integrase mediated cassette exchange (imce)

Den bakteriella kromosomen kan användas för att stabilt hålla främmande DNA i mega-basen intervallet 1. Integrering i kromosomen kringgår frågor som plasmidreplikation, plasmidstabilitet, plasmid inkompatibilitet och plasmidkopieantalet varians. Denna metod används platsspecifika integras från Streptomyces fag (Φ) C31 2,3. Den ΦC31 integras katalyserar en direkt rekombination mellan två specifika DNA webbplatser: attB och attP (34 och 39 bp respektive) 4. Denna rekombination är stabil och inte återgå 5. En &quot;landning dyna&quot; (LP)-sekvens bestående av en spektinomycin-resistensgen, aadA (SPR), och E. coli ß-glukuronidasgenen (uidA) flankerad av ATTP ställen har integrerats i kromosomerna i Sinorhizobium meliloti, Ochrobactrum anthropi och Agrobacterium tumefaciens i en intergenregion, AMpC-lokuset och den tetA-lokuset, respektive. S. meliloti används i detta protokoll. Mobiliserbara donator vektorer innehållande attB-ställen som flankerar en stuffer röd fluorescerande protein (RFP)-genen och en gen för antibiotikaresistens har också konstruerats. I detta exempel är gentamicin resistenta plasmiden pJH110 används. RFP-genen 6 kan ersättas med en önskad konstruktion med användning av Sphl-och Pstl Alternativt en syntetisk konstruktion flankerad av attB ställen kan vara sub-klonades in i en mobiliserbar vektor såsom pK19mob 7. Uttrycket av ΦC31 integras-genen (klonad från pHS62 8) drives av lac-promotorn, på en mobiliserbar brett värdområde plasmiden pRK7813 9. En tetraparental parning protokoll används för att överföra givaren kassetten in i LP-stam därigenom ersätta markörer i LP-sekvensen med givaren kassetten. Dessa celler är trans-integranter. Trans-integranter är utformade med en typisk effektivitet av 0,5%. Trans-integranter normalt återfinns inom de första 500-1,000 kolonier screenades genom antibiotikakänslighet eller blå-vit screening med användning av 5-brom-4-klor-3-indolyl-beta-D-glukuronsyra (X-gluc). Detta protokoll innehåller parning och urvalsförfaranden för att skapa och isolera trans-integranter.

Watch this Scientific Journal Video about Site-specific Bacterial Chromosome Engineering: ÃŽÂ¦C31 Integrase Mediated Cassette Exchange IMCE at JoVE.com

Site-specific Bacterial Chromosome Engineering: ÃƒÅ½Ã‚Â¦C31 Integrase Mediated Cassette Exchange IMCE

En snabb och effektiv metod för att integrera främmande DNA av intresse i färdiga acceptor stammar, sk landning pad stammar, beskrivs. Metoden tillåter platsspecifik integration av ett DNA-kassett in i den konstruerade landningsplats lokus för en given stam, genom konjugering och uttryck av ΦC31 integras.

Platsspecifik bakteriekromosomen Engineering: ΦC31 Integras Mediated kassett Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

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Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

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