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: Î¦C31 Integrase Mediated Cassette Exchange IMCE at JoVE.com

Hvis Margaret CM Smith for vennlig å gi integrase klone Finansiering støtte fra: Genome Canada / Genome Prairie NSERC Discovery og strategisk prosjekt tilskudd

1. Produksjon av Kultur <ol><li> Forbered sterile flytende medier: TY 10 (5 g / l tryptone, 3 g / l gjærekstrakt, 0,44 g / l kalsiumklorid dehydrere), og LB 11 (10 g / l tryptone, 5 g / l gjærekstrakt, 5 g / natriumklorid, pH 7). </li><li> Inokuler fra en enkelt koloni: SmUW227 (S. meliloti LP-stamme: konstruksjonen vil bli beskrevet andre steder, strain konstruksjonsdetaljer tilgjengelige på forespørsel) () til 5 ml TY media med 50 mikrogram / ​​ml spektinomycin. Inokuler følge...

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	<li>Itaya, M., Tsuge, K., Koizumi, M., Fujita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+two+genomes+in+one+cell:+Stable+cloning+of+the+Synechocystis+PCC6803+genome+in+the+Bacillus+subtilis+168+genome.">Combining two genomes in one cell: Stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 15971-15976 (2005).</li><li>Kushtoss, S., Rao, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+Integration+Function+of+the+Streptomycete+Bacteriophage+FC31.">Analysis of the Integration Function of the Streptomycete Bacteriophage FC31.</a> J. Mol. Biol. 222, 897-908 (1991).</li><li>Brown, W. R., Lee, N. C., Xu, Z., Smith, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine+recombinases+as+tools+for+genome+engineering.">Serine recombinases as tools for genome engineering.</a> Methods. 53, 372-379 (2011).</li><li>Groth, A. C., Olivares, E. C., Thyagarajan, B., Calos, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phage+integrase+directs+efficient+site-specific+integration+in+human+cells.">A phage integrase directs efficient site-specific integration in human cells.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 5995-6000 (2000).</li><li>Rowley, P. A., Smith, M. C., Younger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+motif+in+the+C-terminal+domain+of+FC31+integrase+controls+the+directionality+of+recombination.">A motif in the C-terminal domain of FC31 integrase controls the directionality of recombination.</a> Nuc. Acid. Res. 36, 3879-3891 (2008).</li><li>Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monomeric+red+fluorescent+protein.">A monomeric red fluorescent protein.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 7877-7882 (2002).</li><li>Schafer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+mobilizable+multi-purpose+cloning+vectors+derived+from+the+Escherichia+coli+plasmids+pK18+and+pK19:+selection+of+defined+deletions+in+the+chromosome+of+Corynebacterium+glutumicum.">Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutumicum.</a> Gene. 145, 69-73 (1994).</li><li>Thorpe, H. M., Smith, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+site-specific+integration+of+bacteriophage+DNA+catalyzed+by+a+recombinase+of+the+resolvase&#47;invertase+family.">In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase&#47;invertase family.</a> Proc. Natl. Acad. Sci. 95, 5505-5510 (1998).</li><li>Jones, J. D., Gutterson, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+mobilizable+cosmid+vector,+pRK7813,+and+its+use+in+a+rapid+method+for+marker+exchange+in+Pseudomonas+fluorescens+strain+HV37a.">An efficient mobilizable cosmid vector, pRK7813, and its use in a rapid method for marker exchange in Pseudomonas fluorescens strain HV37a.</a> Gene. 61, 299-306 (1987).</li><li>Beringer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R+Factor+transfer+in+Rhizobium+leguminosarum.">R Factor transfer in Rhizobium leguminosarum.</a> J. Gen. Microbiol. 84, 188-198 (1974).</li><li>Lennox, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+linked+genetic+characters+of+the+host+by+bacteriophage+P1.">Transduction of linked genetic characters of the host by bacteriophage P1.</a> Virology. 1, 190-206 (1955).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transformation+of+Escherichia+coli+with+plasmids.">Studies on transformation of Escherichia coli with plasmids.</a> J. Mol. Biol. 166, 557-580 (1983).</li><li>Charles, T. C., Finan, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+map+of+Rhizobium+meliloti+megaplasmid+pRmeSU47b.">Genetic map of Rhizobium meliloti megaplasmid pRmeSU47b.</a> J. Bacteriol. 172, 2469-2476 (1990).</li><li>Leigh, J. A., Signer, E. R., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exopolysaccharide-deficient+mutants+of+Rhizobium+meliloti+that+form+ineffective+nodules.">Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 6231-6235 (1985).</li><li>Heil, J. R., Nordeste, R. F., Charles, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescence+theatre:+a+cost-effective+device+using+theatre+gels+for+fluorescent+protein+and+dye+screening.">The fluorescence theatre: a cost-effective device using theatre gels for fluorescent protein and dye screening.</a> Can. J. Microbiol. 57, 339-342 (2011).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Lesic, B., Rahme, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+lambda+Red+recombinase+system+to+rapidly+generate+mutants+in+Pseudomonas+aeruginosa.">Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa.</a> BMC Mol. Biol.. 9, 20-20 (2008).</li><li>Choi, K. -. H., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mini-Tn7+insertion+in+bacteria+with+single+attTn7+sites:+example+Pseudomonas+aeruginosa.">mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa.</a> Nat. Protocols. 1, 153-161 (2006).</li><li>Thomason, L. C., Calendar, R., Ow, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+insertion+and+replacement+in+Schizosaccharomyces+pombe+mediated+by+the+Streptomyces+bacteriophage+FC31+site-specific+recombination+system.">Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage FC31 site-specific recombination system.</a> Molecular Genetics and Genomics. 265, 1031-1038 (2001).</li><li>Katzen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway+recombinational+cloning:+a+biological+operating+system.">Gateway recombinational cloning: a biological operating system.</a> Expert Opin. Drug Discovery. , 571-586 (2007).</li><li>Charles, T. C., Doty, S. L., Nester, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+Agrobacterium+strains+by+electroporation+of+genomic+DNA+and+its+utility+in+analysis+of+chromosomal+virulence+mutations.">Construction of Agrobacterium strains by electroporation of genomic DNA and its utility in analysis of chromosomal virulence mutations.</a> Appl. Environ. Microbiol. 60, 4192-4194 (1994).</li></ol>

Den IMCE teknikken gir mulighet for effektiv integrering av en enkelt attB flankert DNA kassetten inn i LP-locus av en tidligere utviklet belastning. Når ønsket konstruktet er klonet i stedet for RFP for å lage donor kassetten, ikke teknikken ikke krever etterfølgende DNA rensing og transformasjon, som gjør det svært robust. Det er viktig at egnede vekst kontroller er inkludert, for å være sikker på at antibiotikaresistens skyldes etableringen av trans-integrants og ikke andre faktorer. <p...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </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> Tetracycline </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> Tryptone </td><td> Bioshop Canada Inc. </td><td> TRP402 </td><td></td></tr><tr><td> Gjærekstrakt </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> Kalsiumklorid </td><td> Bioshop Canada Inc. </td><td> CCL444 </td><td></td></tr><tr><td> X-Gluc </td><td> Gull Bioteknologi Inc. </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli MT616 belastning </td><td> Tilgjengelig på forespørsel </td><td></td><td> Brukes også utenfor vår lab </td></tr><tr><td> E. coli pJC2 belastning </td><td> I huset, som er tilgjengelig på forespørsel </td><td></td><td></td></tr><tr><td> E. coli pJH110 belastning </td><td> I huset, tilgjengelig ved request </td><td></td><td></td></tr><tr><td> SmUW227 belastning </td><td> I huset, som er tilgjengelig på forespørsel </td><td></td><td></td></tr></tbody></table>

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

Bakteriell kromosom kan brukes til stabilt opprettholde fremmed DNA i mega-basen rekkevidde en. Integrering i kromosomet omgår spørsmål som plasmid replikasjon, plasmid stabilitet, plasmid inkompatibilitet, og plasmid kopi nummer varians. Denne metoden bruker stedsspesifikk integrase fra Streptomyces phage (Φ) C31 2,3. Den ΦC31 integrase katalyserer en direkte rekombinasjon mellom to spesifikke DNA sider: attB og attP (34 og 39 bp, henholdsvis) 4. Dette rekombinasjon er stabil og ikke gå tilbake ikke fem. En &quot;landing pad&quot; (LP) sekvens bestående av en spektinomycin-motstand genet, aadA (SPR), og E. coli ß-glucuronidasesystemer genet (uidA) flankert av attP nettsteder har blitt integrert inn i kromosomene i Sinorhizobium meliloti, Ochrobactrum anthropi, og Agrobacterium tumefaciens i en intergenic regionen, ampC locus, og tetA locus, henholdsvis. S. meliloti brukes i denne protokollen. Mobilizable donor vektorer som inneholder attB nettsteder flankerer en stuffer rød fluorescerende protein (RFP) genet og en antibiotikaresistens genet har også blitt konstruert. I dette eksempelet gentamicin motstandsdyktig plasmidet pJH110 brukes. Den RFP genet 6 kan erstattes med en ønsket konstruksjon med sph jeg og PST I. Alternativt kan en syntetisk konstruksjon flankert av attB nettsteder kan være sub-klonet inn i en mobilizable vektor som pK19mob 7. Uttrykket av ΦC31 integrase genet (klonet fra pHS62 8) er drevet av lac arrangøren, på en mobilizable bredt vertsområde plasmid pRK7813 9. En tetraparental parring protokollen brukes til å overføre donor kassetten inn i LP belastningen dermed erstatte markørene i LP sekvens med donor kassett. Disse cellene er trans-integrants. Trans-integrants er dannet med en typisk virkningsgrad på 0,5%. Trans-integrants finnes vanligvis i løpet av de første 500-1000 koloniene filtrert av antibiotika følsomhet eller blå-hvite screening ved hjelp av 5-Bromo-4-klor-3-indolyl-beta-D-glukuronsyre (X-Gluc). Denne protokollen inneholder sammenføyningsflatene og utvalg prosedyrer for å lage og isolere trans-integrants.

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

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

En rask og effektiv metode for å integrere fremmed DNA av interesse i pre-laget akseptor stammer, betegnes Matte stammer, er beskrevet. Metoden gjør at stedsspesifikk integrering av en DNA kassetten inn i konstruert landing puten locus av en gitt belastning, gjennom konjugering og uttrykk for ΦC31 integrase.

Stedsspesifikk Bakteriell Chromosome 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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