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

Para Margaret CM Smith por gentilmente fornecer o clone integrase Apoio financeiro de: Genoma Canadá / Genoma Prairie NSERC Discovery e subsídios projeto estratégico

1. Produção de Cultura <ol><li> Preparar meios líquidos estéreis: TY 10 (5 g / l de triptona, extracto de levedura 3 g / l, 0,44 g / l de cloreto de cálcio hidratado), e LB 11 (10 g / l de triptona, 5 g de extracto de levedura / l, 5 g / cloreto de sódio, pH 7). </li><li> Inocular a partir de uma única colônia: SmUW227 (S. meliloti LP-deformação: a construção será descrito em outro lugar, a estirpe detalhes de construção disponíveis mediante solicitação) () em 5 ml de T...

<ol>
	<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>

A técnica IMCE permite a eficiente integração de uma cassete de ADN único attB flanqueada no LP-locus de uma estirpe previamente manipulada. Uma vez que a construção desejada é clonado no lugar de rfp para criar a cassete de dador, a técnica não requer purificação de ADN e transformação subsequente, o que torna muito robusta. É crítico que controla o crescimento adequados estão incluídos, para ser determinada a resistência a antibióticos é devido à criação de trans-integrantes e ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Estreptomicina </td><td> Bioshop Canada Inc. </td><td> STP101 </td><td></td></tr><tr><td> Espectinomicina </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> Tetraciclina </td><td> Bioshop Canada Inc. </td><td> TET701 </td><td></td></tr><tr><td> Canamicina </td><td> Bioshop Canada Inc. </td><td> KAN201 </td><td></td></tr><tr><td> Agar grau bacteriológica </td><td&gt; Bioshop Canada Inc. </td><td> AGR001 </td><td></td></tr><tr><td> Triptona </td><td> Bioshop Canada Inc. </td><td> TRP402 </td><td></td></tr><tr><td> Extracto de levedura </td><td> Bioshop Canada Inc. </td><td> YEX401 </td><td></td></tr><tr><td> Cloreto de Sódio </td><td> Bioshop Canada Inc. </td><td> SOD001 </td><td></td></tr><tr><td> Cloreto de cálcio </td><td> Bioshop Canada Inc. </td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td> Ouro Biotecnologia Inc. </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli estirpe MT616 </td><td> Disponível em cima do pedido </td><td></td><td> Também é usado fora do nosso laboratório </td></tr><tr><td> E. coli estirpe pJC2 </td><td> Em casa, disponíveis mediante solicitação </td><td></td><td></td></tr><tr><td> E. coli estirpe pJH110 </td><td> Em casa, disponível por Request </td><td></td><td></td></tr><tr><td> SmUW227 tensão </td><td> Em casa, disponíveis mediante solicitação </td><td></td><td></td></tr></tbody></table>

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

O cromossoma bacteriano pode ser utilizado para manter de forma estável de ADN estranho na gama mega-base 1. Integração no cromossoma contorna questões tais como a replicação do plasmídeo, a estabilidade do plasmídeo, a incompatibilidade do plasmídeo, e variância número plasmídeo de cópia. Este método utiliza a integrase site-specific a partir do fago de Streptomyces (Φ) C31 2,3. A integrase ΦC31 catalisa uma recombinação direta entre dois sítios de DNA específicas: attB e attP (34 e 39 pb, respectivamente) 4. Esta recombinação é estável e não revertem 5. A &quot;pista&quot; sequência (LP), constituído por um gene de resistência à espectinomicina, aadA (SPR), ea E. gene coli ß-glucuronidase (uidA) ladeado por sites attP foi integrado nos cromossomos de Sinorhizobium meliloti, anthropi Ochrobactrum e Agrobacterium tumefaciens em uma região intergênica, a ampC locus, eo locus Teta, respectivamente. S. meliloti é usado neste protocolo. Vectores de dadores contendo locais attB mobilizável que flanqueiam um stuffer vermelho fluorescente proteína do gene (SDP) e um gene de resistência a antibiótico também têm sido construída. Neste exemplo, o pJH110 resistente gentamicina plasmídeo é usado. O gene rfp 6 pode ser substituída com uma construção desejada utilizando Sph I e Pst I. Alternativamente um construto sintético flanqueado por sítios attB podem ser sub-clonado num vector mobilizável, tais como pK19mob 7. A expressão do gene da integrase ΦC31 (clonado a partir de pHS62 8) é conduzida pelo promotor lac, em uma ampla gama de hospedeiros mobilizável plasmídeo pRK7813 9. Um protocolo de acasalamento tetraparental é usado para transferir a cassete de doador na estirpe LP substituindo assim os marcadores na sequência de LP com a cassete de dador. Estas células são trans-integrantes. Trans-integrantes são formados com uma eficiência típica de 0,5%. Trans-integrantes são tipicamente encontrados nos primeiros 500-1,000 colónias filtradas através da sensibilidade aos antibióticos ou azul-branco rastreio utilizando 5-bromo-4-cloro-3-indolil-beta-D-glucurónico ácido (X-gluc). Este protocolo contém os procedimentos de acasalamento e de seleção para criar e isolar trans-integrantes.

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

Um método rápido e eficiente para integrar DNA estranho de juros em cepas pré-fabricados receptoras, chamadas de tensões almofada de aterragem, é descrito. O método permite site-specific integração de uma cassete de ADN no locus engenharia aterragem almofada de uma estirpe dada, por meio de conjugação e expressão da integrase ΦC31.

Site-specific Engenharia cromossomo bacteriano: 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 ...

Research

JoVE Journal

Bioengineering

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

Procedure for Lung Engineering

Procedimento para a Engenharia de pulmão

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Bioengenharia

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Caracterizando Voláteis bacteriana secundária usando ionização Espectrometria de Massa (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Alta eficiência, Transfecção site-specific de células aderentes com siRNA Usando matrizes de microeletrodos (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Capillary Force Lithography for Cardiac Tissue Engineering

Capilar Força Litografia de Engenharia tecido cardíaco

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Observando e quantificando compactação de gel de fibrina mediada por fibroblasto

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

Síntese de uma proteína artificial intein mediada Hidrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

Fabricação de fibras naturais robustos Preformas Utilizando bacteriana Celulose como Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Microbiologicamente induzida Calcita Precipitation Mediada por<em&gt; Sporosarcina pasteurii</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Microfluídico troca de tampão para Micro / nanopartículas celular Engenharia sem interferências

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...