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

Om Margaret CM Smith voor vriendelijk het verstrekken van de integrase kloon Financiering steun van: Genome Canada / Genome Prairie NSERC Discovery en strategische Projectsubsidies

1. Productie van Cultuur <ol><li> Bereid steriele vloeibare media: TY 10 (5 g / l trypton, 3 g / l gistextract, 0,44 g / l calciumchloride dehydrateren) en LB 11 (10 g / l trypton, 5 g / l gistextract, 5 g / natriumchloride, pH 7). </li><li> Inoculeer uit een kolonie: SmUW227 (S. meliloti LP-stam: de bouw zal elders worden beschreven, stam bouw details zijn beschikbaar op aanvraag) () in 5 ml TY media met 50 ug / ml spectinomycine. Inoculeer de volgende stammen in 5 ml LB vloeibare media...

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De IMCE techniek maakt het mogelijk voor de efficiënte integratie van een enkele attB geflankeerd DNA-cassette in de LP-locus van een eerder ontworpen stam. Zodra de gewenste construct wordt gekloond in plaats van RFP aan de donor cassette te creëren, is de techniek niet nodig latere DNA-zuivering en transformatie, waardoor het zeer robuust. Het is cruciaal dat goede groei controles zijn opgenomen, om zeker te zijn van de resistentie tegen antibiotica is het gevolg van het ontstaan ​​van trans-in...

<table border="1"><tbody><tr><td> Naam van het serum </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Reacties </td></tr><tr><td> Streptomycine </td><td> Bioshop Canada Inc </td><td> STP101 </td><td></td></tr><tr><td> Spectinomycine </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> Gentamicine </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> Tetracycline </td><td> Bioshop Canada Inc </td><td> TET701 </td><td></td></tr><tr><td> Kanamycine </td><td> Bioshop Canada Inc </td><td> KAN201 </td><td></td></tr><tr><td> Bacteriologische kwaliteit 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> Gist Extract </td><td> Bioshop Canada Inc </td><td> YEX401 </td><td></td></tr><tr><td> Natriumchloride </td><td> Bioshop Canada Inc </td><td> SOD001 </td><td></td></tr><tr><td> Calcium Chloride </td><td> Bioshop Canada Inc </td><td> CCL444 </td><td></td></tr><tr><td> X-Gluc </td><td> Goud Biotechnology Inc </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli stam MT616 </td><td> Beschikbaar op aanvraag </td><td></td><td> Wordt ook gebruikt buiten ons lab </td></tr><tr><td> E. coli stam pJC2 </td><td> In huis, op aanvraag beschikbaar </td><td></td><td></td></tr><tr><td> E. coli stam pJH110 </td><td> In het huis, beschikbaar gesteld door request </td><td></td><td></td></tr><tr><td> SmUW227 stam </td><td> In huis, op aanvraag beschikbaar </td><td></td><td></td></tr></tbody></table>

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

De bacteriële chromosoom kan worden gebruikt om vreemd DNA stabiel te houden in de mega-base bereik 1. De integratie in het chromosoom omzeilt onderwerpen als plasmide replicatie, plasmide stabiliteit, plasmide onverenigbaarheid, en plasmide aantal kopieën variantie. Deze methode maakt gebruik van de site-specific integrase van de Streptomyces faag (Φ) C31 2,3. De ΦC31 integrase katalyseert een directe recombinatie tussen twee specifieke DNA-sites: attB en attP (34 en 39 bp, respectievelijk) 4. Dit recombinatie stabiel is en niet terug 5. Een &quot;landingsplaats&quot; (LP) sequentie bestaande uit een spectinomycine-resistentiegen, aada (SPR) en E. coli ß-glucuronidase gen (uidA) geflankeerd door attP plaatsen is geïntegreerd in de chromosomen van Sinorhizobium meliloti, Ochrobactrum anthropi en Agrobacterium tumefaciens in een intergene regio, de ampc locus en de TETA locus respectievelijk. S. meliloti wordt in dit protocol. Mobiliseerbare vectoren die donor attB flankeren een stuffer rood fluorescent eiwit (RFP) gen en een antibioticumresistentiegen zijn aangelegd. In dit voorbeeld gentamicine resistente plasmide pJH110 gebruikt. De RFP gen 6 kan worden vervangen door een gewenste construct met Sphl en Pst I. Als alternatief kan een synthetische construct geflankeerd door attB sites kunnen sub-gekloneerd is in een mobiliseerbare vector, zoals pK19mob 7. De expressie van het ΦC31 integrase gen (gekloneerd pHS62 8) wordt aangedreven door de lac-promotor, een mobiliseerbare breed gastheerbereik plasmide pRK7813 9. Een tetraparental koppelen protocol wordt gebruikt om de donor cassette over in de LP stam en vervangt de merkers met de LP sequentie met de donor cassette. Deze cellen zijn trans-integranten. Trans-integranten gevormd met een typische rendement van 0,5%. Trans-integranten zijn kenmerkend in de eerste 500-1000 kolonies gescreend met antibiotica gevoeligheid of blauw wit screenen met 5-bromo-4-chloor-3-indolyl-ß-D-glucuronzuur (X-Gluc). Dit protocol bevat de paren en de selectieprocedures voor het maken en isoleren van de trans-integranten.

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

Een snelle en efficiënte methode om vreemd DNA van belang te integreren in pre-en-klare acceptor stammen, aangeduid als landingsplaats stammen, beschreven. De methode maakt het mogelijk site-specifieke integratie van een DNA-cassette in de gemanipuleerde landingsplaats locus van een bepaalde stam, door middel van conjugatie en de expressie van de ΦC31 integrase.

Site-specifieke bacteriële chromosoom Engineering: ΦC31 Integrase Mediated Cassette 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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