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

Per CM Margaret Smith per aver gentilmente fornire il clone integrasi Un sostegno finanziario a partire da: Genome Canada / Genome Prairie NSERC Discovery e borse di studio progetti strategici

1. Produzione di Cultura <ol><li> Preparare liquidi sterili: TY 10 (5 g / l triptone, 3 g / l di estratto di lievito, 0,44 g / l di cloruro di calcio diidrato), e LB 11 (10 g / l triptone, 5 g / l di estratto di lievito, 5 g / cloruro di sodio, pH 7). </li><li> Inoculare da una singola colonia: SmUW227 (S. meliloti LP-ceppo: verranno descritte altrove, ceppo dettagli costruttivi disponibili su richiesta) () in 5 ml di TY media con 50 ug / ml spectinomicina. Inoculare le seguenti ceppi in...

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La tecnica IMCE consente l&#39;integrazione efficace di una singola cassetta DNA attB affiancato nella LP-locus di un ceppo precedentemente progettato. Una volta che il costrutto desiderato viene clonato al posto di RFP per creare la cassetta donatore, la tecnica non richiede successiva purificazione del DNA e trasformazione, che rende molto robusto. È fondamentale che controlli crescita appropriati sono inclusi, per essere certi della resistenza agli antibiotici è dovuta alla creazione di trans-inte...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Streptomicina </td><td> Bioshop Canada Inc. </td><td> STP101 </td><td></td></tr><tr><td> Spectinomicina </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> Gentamicina </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> Kanamicina </td><td> Bioshop Canada Inc. </td><td> KAN201 </td><td></td></tr><tr><td> Batteriologica agar grade </td><td&gt; Bioshop Canada Inc. </td><td> AGR001 </td><td></td></tr><tr><td> Triptone </td><td> Bioshop Canada Inc. </td><td> TRP402 </td><td></td></tr><tr><td> Estratto di lievito </td><td> Bioshop Canada Inc. </td><td> YEX401 </td><td></td></tr><tr><td> Cloruro di sodio </td><td> Bioshop Canada Inc. </td><td> SOD001 </td><td></td></tr><tr><td> Cloruro di calcio </td><td> Bioshop Canada Inc. </td><td> CCL444 </td><td></td></tr><tr><td> X-Gluc </td><td> Oro Biotechnology Inc. </td><td> G1281C1 </td><td></td></tr><tr><td> MT616 ceppo E. coli </td><td> Disponibile su richiesta </td><td></td><td> Utilizzato anche al di fuori del nostro laboratorio </td></tr><tr><td> PJC2 ceppo E. coli </td><td> In casa, disponibile su richiesta </td><td></td><td></td></tr><tr><td> PJH110 ceppo E. coli </td><td> In casa, a disposizione dal richt </td><td></td><td></td></tr><tr><td> SmUW227 ceppo </td><td> In casa, disponibile su richiesta </td><td></td><td></td></tr></tbody></table>

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

Il cromosoma batterico può essere utilizzato per mantenere stabilmente il DNA estraneo nel mega-base gamma 1. Integrazione nel cromosoma elude questioni quali la replicazione del plasmide, la stabilità plasmide, incompatibilità plasmide, e il plasmide varianza numero di copie. Questo metodo utilizza il site-specific integrasi dal fago Streptomyces (Φ) C31 2,3. Il ΦC31 integrasi catalizza una ricombinazione diretto tra due specifici siti del DNA: ATTB e attP (34 e 39 bp, rispettivamente) 4. Tale ricombinazione è stabile e non ripristinare 5. Una &quot;pista&quot; (LP) costituito da una sequenza spectinomicina gene di resistenza, aadA (SPR), e la E. coli ß-glucuronidasi gene (uidA) affiancato da siti attP è stato integrato nei cromosomi di Sinorhizobium meliloti, Ochrobactrum anthropi e tumefaciens in una regione intergenica, l&#39;ampC locus, e il locus teta, rispettivamente. S. meliloti viene utilizzato in questo protocollo. Vettori contenenti siti donatori mobilizzabile attB fiancheggiano una stuffer rosso proteina fluorescente (RFP) gene e un gene di resistenza agli antibiotici sono stati costruiti. In questo esempio la gentamicina pJH110 resistente plasmide viene utilizzato. Il gene RFP 6 può essere sostituito con un costrutto desiderata con Sph I e Pst I. In alternativa, un costrutto sintetico fiancheggiata da siti attB possono essere subclonato in un vettore mobilizzabile come pK19mob 7. L&#39;espressione del gene ΦC31 integrasi (clonato da pHS62 8) è guidata dal promotore lac, su una vasta gamma mobilizzabile ospite plasmide pRK7813 9. Un protocollo accoppiamento tetraparental viene utilizzato per trasferire la cassetta nel ceppo donatore LP sostituendo i marcatori nella sequenza LP con la cassetta donatore. Queste cellule sono trans-integrants. Trans-integrants sono formate con una efficienza tipica di 0,5%. Trans-integrants trovano tipicamente entro i primi 500-1.000 colonie schermati dalla sensibilità agli antibiotici o blu-nero screening utilizzando 5-bromo-4-cloro-3-indolil-beta-D-glucuronico (X-Gluc). Questo protocollo contiene le procedure di accoppiamento e di selezione per la creazione e isolare trans integranti.

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

Un metodo rapido ed efficace per integrare DNA estraneo di interesse in pre-fatti ceppi accettori, chiamati ceppi pista di atterraggio, è descritta. Il metodo permette integrazione sito-specifica di una cassetta DNA nel locus ingegnerizzato atterraggio di un dato ceppo, tramite coniugazione e l&#39;espressione del ΦC31 integrasi.

Site-specific Ingegneria batterica Cromosoma: ΦC31 integrasi 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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Bioengineering

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

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