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

Nazik integraz klon sağlamak için CM Margaret Smith Destek finansmanı: Genom Kanada / Genom Prairie NSERC Discovery ve Stratejik Proje hibeleri

1. Kültür Üretimi <ol><li> TY 10 (5 g / l tripton, 3 g / l maya ekstresi, 0.44 g / l kalsiyum klorid dihidrat), ve LB 11 (10 g / l tripton, 5 g / L maya ekstresi, 5 g /: steril sıvı ortam hazırlamak sodyum klorid, pH 7). </li><li> SmUW227: 50 mg / ml spectinomycin ile TY medya (S. meliloti LP-suşu: inşaat kodlanabilir, istek üzerine inşaat detayları zorlanma) () 5 ml içine tek bir koloni inoküle. E.: verilen antibiyotik ile ilave aşağıdaki suşlar LB sıvı ortam...

<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>

IMCE tekniği önceden tasarlanmış suşu LP-lokusunda tek bir attB çevrili DNA kaset verimli entegrasyon sağlar. Istenen yapı donör kaset oluşturmak için RFP yerine klonlanmış sonra, tekniğin son derece güçlü hale, müteakip DNA arıtma ve dönüşüm gerektirmez. Bu antibiyotik direnci trans-integrants ve diğer değil faktörlerin oluşturulması nedeniyle belirli olabilmesi için, uygun büyüme kontrolleri dahil olduğunu önemlidir. IMCE yaklaşık% 0.5 v...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Streptomisin </td><td> Bioshop Kanada A.Ş. </td><td> STP101 </td><td></td></tr><tr><td> Spectinomycin </td><td> Bioshop Kanada A.Ş. </td><td> <a href="https://ssl34.alentus.com/bioshopcanada/detail.asp?Pin=SPE201">SPE201</a> </td><td></td></tr><tr><td> Gentamisin </td><td> Bioshop Kanada A.Ş. </td><td> GTA202 </td><td></td></tr><tr><td> Choramphenicol </td><td> Bioshop Kanada A.Ş. </td><td> CLR201 </td><td></td></tr><tr><td> Tetrasiklin </td><td> Bioshop Kanada A.Ş. </td><td> TET701 </td><td></td></tr><tr><td> Kanamisin </td><td> Bioshop Kanada A.Ş. </td><td> KAN201 </td><td></td></tr><tr><td> Bakteriyolojik notu agar </td><td&gt; Bioshop Kanada A.Ş. </td><td> AGR001 </td><td></td></tr><tr><td> Tripton </td><td> Bioshop Kanada A.Ş. </td><td> TRP402 </td><td></td></tr><tr><td> Maya Ekstraktı </td><td> Bioshop Kanada A.Ş. </td><td> YEX401 </td><td></td></tr><tr><td> Sodyum klorür </td><td> Bioshop Kanada A.Ş. </td><td> SOD001 </td><td></td></tr><tr><td> Kalsiyum Klorür </td><td> Bioshop Kanada A.Ş. </td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td> Altın Biyoteknoloji A.Ş. </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli suşu MT616 </td><td> Istek üzerine </td><td></td><td> Ayrıca laboratuar dışında kullanılan </td></tr><tr><td> E. coli suşu pJC2 </td><td> Isteği ile evde, mevcut </td><td></td><td></td></tr><tr><td> E. coli suşu pJH110 </td><td> Reques tarafından evde, mevcutt </td><td></td><td></td></tr><tr><td> SmUW227 gerginlik </td><td> Isteği ile evde, mevcut </td><td></td><td></td></tr></tbody></table>

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

Bakteriyel kromozom stably Mega baz aralığında 1 yabancı DNA korumak için de kullanılabilir. Kromozom entegrasyon gibi plazmid çoğaltma, plazmid istikrar, plazmid uyumsuzluk ve plazmid kopya sayısı varyans gibi konular circumvents. Bu yöntem, Streptomyces faj (Φ) C31 2,3 den site-specific integraz kullanır. AttB ve attP (sırasıyla 34 ve 39 bp) 4: ΦC31 integraz iki spesifik DNA bölgeleri arasında doğrudan bir rekombinasyon katalizler. Bu rekombinasyon istikrarlı ve 5 geri almaz. Bir spectinomycin-direnç geninden, Aada (SPR), ve E. oluşan A &quot;tampon iniş&quot; (LP) sekansı attP siteleri çevrili coli ß-glukuronidaz geni (uidA) Sinorhizobium meliloti, Ochrobactrum anthropi ve bir intergenik bölgede Agrobacterium tumefaciens kromozomlarını entegre edilmiştir, duyuyorumsırasıyla pC odağı ve teta odağı,. S. meliloti Bu protokolde kullanılır. Bir stuffer kırmızı floresan proteini (rfp) geni ve antibiyotik direnç geni kuşatan attB siteleri içeren Mobilizable donör vektörler aynı zamanda inşa edilmiştir. Bu örnekte gentamisin dirençli plazmid pJH110 kullanılır. RFP geni 6 SPH I ve Pst I ile istenen yapı ile ikame edilebilir Alternatif attB siteleri tarafından çevrili sentetik bir yapı gibi pK19mob 7 gibi bir mobilizable vektörüne alt-klonlandı olabilir. ΦC31 integraz geni (pHS62 8 den klonlanmış) ile ifade pRK7813 9 plazmid bir mobilizable geniş bir aralığı üzerinde ana, lac promotör tarafından tahrik edilir. Bir eşleşme tetraparental protokolü LP suşu böylece donör kaset ile LP sırayla değiştirme belirteçler içine donör kaset transfer etmek için kullanılır. Bu hücreler vardır trans-integrants. Trans-integrants% 0.5 arasında, tipik bir verim ile oluşturulanlardır. Trans-integrants tipik olarak 5-bromo-4-kloro-3-indolil-beta-D-glukuronik asit (X-gluc) kullanılarak antibiyotik duyarlılığı veya mavi-beyaz tarama ile taranmıştır olanlar 500-1,000 koloniler içinde bulunurlar. Bu protokol, trans-integrants oluşturma ve izole etmek için çiftleşme ve seçme işlemleri içerir.

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

Önceden hazırlanmış alıcı suşlar olarak adlandırılan iniş takımı suşları, içine faiz yabancı DNA entegre hızlı ve verimli bir yöntem tarif edilmiştir. Metodu ΦC31 integraz konjugasyonu ve ifadesi ile, belirli bir lamine suşun iniş pedi odağına içine bir DNA kasetinin site-specific entegrasyon sağlar.

Siteye özgü Bakteriyel Kromozom Mühendislik: ΦC31 integraz Aracılı Kaset Borsası (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

Akciğer Mühendisliği Prosedürü

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

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

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

İkincil Elektrosprey İyonizasyon Kütle Spektrometresi (Sesi-MS) ile karakterize Bakteriyel uçucu

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

Mikroelektrod Diziler Kullanarak siRNA Bağlanan Hücre Yüksek verim, Siteye özgü Transfeksiyon (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

Kardiyak Doku Mühendisliği kılcal Kuvvetleri Litografi

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

Fibroblast aracılı Fibrin Jel Sıkıştırmayı Gözlemleme ve Ölçme

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

Bir Intein aracılı yapay protein Hidrojel sentezi

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

Binder Bakteriyel Selüloz kullanmak Sağlam Doğal Elyaf preform üretim

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

Mikrobiyolojik Kalsit Yağış Bağlı tarafından Aracılı<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

Bir ıntrinsic Vaskülarizasyon tarafından Doku Mühendisliği<em&gt; İn Vivo</em&gt; Doku Mühendisliği Odası

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

Parazit Mikro / Nanoparçacık Hücre Mühendisliği mikroakışkan Tampon Değişim

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