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

For at Margaret CM Smith for venlig at give integrase klon Økonomisk støtte fra: Genome Canada / Genome Prairie NSERC Discovery og strategiske projekt tilskud

1. Produktion af Kultur <ol><li> Fremstille sterile flydende medier: TY 10 (5 g / l trypton, 3 g / l gærekstrakt, 0,44 g / l calciumchlorid dehydrat) og LB 11 (10 g / l trypton, 5 g / l gærekstrakt, 5 g / natriumchlorid, pH 7). </li><li> Podes fra en enkelt koloni: SmUW227 (S. meliloti LP-stammen: konstruktion vil blive beskrevet andetsteds, stamme konstruktionsdetaljer til rådighed efter anmodning) () i 5 ml TY medier med 50 pg / ml spectinomycin. Inokulere følgende stammer i 5 ml L...

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Den IMCE teknik muliggør effektiv integration af en enkelt attB flankeres DNA&#39;et kassetten i LP-locus af en tidligere udviklet stamme. Når den ønskede konstruktion klones i stedet for RFP at skabe donor kassetten, er teknikken ikke kræver efterfølgende DNA-oprensning og transformation, hvilket gør det meget robust. Det er vigtigt, at passende vækst kontroller er indbefattet, for at være sikker på antibiotikaresistens skyldes indførelsen af ​​trans-integranter og ikke andre faktorer. <...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Streptomycin </td><td> Bioshop Canada Inc. </td><td> STP101 </td><td></td></tr><tr><td> Spectinomycin </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> Gærekstrakt </td><td> Bioshop Canada Inc. </td><td> YEX401 </td><td></td></tr><tr><td> Natriumchlorid </td><td> Bioshop Canada Inc. </td><td> SOD001 </td><td></td></tr><tr><td> Calciumchlorid </td><td> Bioshop Canada Inc. </td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td> Guld Biotechnology Inc. </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli MT616-stamme </td><td> Til rådighed efter anmodning </td><td></td><td> Kan også bruges uden for vores laboratorium </td></tr><tr><td> E. coli pJC2 stamme </td><td> I huset, er tilgængeligt ved forespørgsel </td><td></td><td></td></tr><tr><td> E. coli pJH110-stamme </td><td> I huset, til rådighed af request </td><td></td><td></td></tr><tr><td> SmUW227 stammen </td><td> I huset, er tilgængeligt ved forespørgsel </td><td></td><td></td></tr></tbody></table>

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

Det bakterielle kromosom, kan anvendes til stabilt at opretholde fremmed DNA i mega-base mellem 1. Integration i kromosomet omgår emner såsom plasmidreplikation, plasmidstabilitet, plasmid uforenelighed, og antallet af plasmidkopier varians. Denne metode anvender den stedspecifikke integrase fra Streptomyces fagen (Φ) C31 2,3. Den ΦC31 integrase katalyserer en direkte rekombination mellem to specifikke DNA steder: attB og attP (34 og 39 bp, henholdsvis) 4. Denne rekombination er stabilt og ikke vende tilbage 5. En &quot;landing pad&quot; (LP) sekvensen bestående af en spectinomycin-resistens gen, Aada (SPR) og E. coli ß-glucuronidase-genet (uidA) flankeret af attP sites er blevet integreret i kromosomerne i Sinorhizobium meliloti, Ochrobactrum anthropi og Agrobacterium tumefaciens i en intergenisk region, ampC locus, og den tetA locus, hhv. S. meliloti anvendes i denne protokol. Mobiliserbar donor vektorer indeholdende attB-sites flankerer en stuffer rødt fluorescerende protein (RFP) gen og en antibiotisk resistens-gen er også blevet konstrueret. I dette eksempel gentamicin resistent plasmid pJH110 anvendes. RFP gen 6 kan erstattes med en ønsket konstruktion med Sph I og Pst I. Alternativt kan en syntetisk konstruktion flankeret af attB-sites kan være sub-klonet ind i en mobiliserbar vektor, såsom pK19mob 7. Ekspressionen af ΦC31 integrase-genet (klonet fra pHS62 8) drives af lac-promotoren på en mobiliserbar bredt værtsspektrum plasmid pRK7813 9. En tetraparental parring protokol anvendes til at overføre donor kassetten i LP-stammen derved erstatte markører i LP-sekvensen med donor kassetten. Disse celler er trans-integranter. Trans-integranter er udformet med en typisk effektivitet på 0,5%. Trans-integranter er typisk inden for de første 500-1000 kolonier screenet ved antibiotisk følsomhed eller blå-hvid-screening ved anvendelse af 5-brom-4-chlor-3-indolyl-beta-D-glucuronsyre (X-gluc). Denne protokol indeholder de parring og udvælgelse procedurer for at skabe og isolere 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 hurtig og effektiv metode til at integrere fremmed DNA af interesse i præfremstillede acceptor stammer, betegnet landingsplads stammer, er beskrevet. Fremgangsmåden tillader stedspecifik integration af et DNA-kassette i det konstruerede landingsplads locus af en given stamme, gennem konjugation og ekspression af det ΦC31 integrase.

Site-specifik bakteriekromosomet 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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