친절 integrase의 클론을 제공하는 마가렛 CM 스미스에게 부터 지원 자금 : 게놈 캐나다 / 게놈 프레리 NSERC 발견과 전략적 사업 보조금

1. 문화 생산 <ol><li> 타이 10 (5g / L tryptone, 3g / L 효모 추출물, 0.44 g / L 칼슘 염화물 탈수) 및 LB 11 (10g / L tryptone, 5g / L 효모 추출물, 5 G / : 무균 액체 매체를 준비합니다 나트륨 염화물, 산도 7). </li><li> SmUW227 : 50 μg / ML의 spectinomycin와 타이의 미디어 (S. meliloti 천자 - 변형 : 건설 다른 곳에서 설명한 것이다는 요청시 사용 가능한 건축 세부 변형) () 5에 ML 단일 콜로니 접종. E. : 주어진 항생제와 보완 다음과 같은 변종 LB 액체 매체의 5 ML로를 접종 대장균은 DH5α 12 10 μg / ML 테트라 사이클린과 pJC2, 플라스미드 integrase 표현식을 포함하는, E. 대장균 10 μg / ML의 chloramphenicol과 MT616 13 mobilizer, 및 E. 대장균 DH5α 5 μg / ML의 gentamicin과 pJH110, 플라스미드 기증자 카세트를 포함하는. </li><li> 품어 E. 37 AT &amp; 하룻밤 대장균의 변종있길; 지속적인 진동과 C #. 품어 S. 떨고있는 이틀 동안 30 ° C에서 meliloti 변형. 그것은 S.를 얻을 수 있습니다 큰 inoculum (포화 문화 1:500 subculture)와 하룻밤 meliloti 문화. </li></ol> 2. 문화의 준비 및 혼합 <ol><li> 30 초 동안 17000 XG에서 원심 분리하여 세포 펠렛를 수집하고 1 ML 멸균 0.85 % NaCl에 resuspending하여 각 변형에 대한 문화의 1.5 ML 씻으십시오, 반복을 한 번. </li><li> 멸균 0.85 % NaCl을 100 μl의 Resuspend 펠릿. </li><li> 개별적으로 일반 타이 한천 플레이트의 각 변형에 대한 형편없는 문화 10 μl을 더럽힐. 이들 명소는 제어 명소입니다. </li><li> E.을 제외한 각 변형의 40 μl를 추가하십시오 대장균 DH5α 12 멸균 튜브, 믹스, 그리고 평범한 타이 한천 플레이트에이 혼합물의 장소 120 μl에 pJC2를 포함. 이 지점이 노 integrase 부정적인 컨트롤입니다. </li><li> 멸균 t 각 변형의 40 μl를 추가하십시오ube, 믹스, 그리고 장소 평범한 타이 한천 플레이트에이 혼합물 160 μl. 이 지점이 IMCE 짝짓기 장소입니다. </li><li> 장소는 층류 후드에서 건조하도록 허용합니다. </li><li> 인감 플레이트 30 ° C에서 하룻밤 사이에서 품어. </li></ol> 3. 횡단 integrants의 분리 <ol><li> 타이 한천 플레이트를 향해 행진 제어 명소와 IMCE 장소는 200 μg / ML의 스트렙토 마이신 (SmUW227가 스트렙토 마이신 14 높은 수준의 저항을 부여 돌연변이를 운반하는 Rm1021에 기초) 30 μg / ML gentamicin과 보충. </li><li> IMCE 효율의 계산의 경우 약 resuspend ¼ 500 μl 멸균 0.85 % NaCl에서 IMCE 짝짓기 장소로. 10 -7로 10 배 시리얼 dilutions하십시오. 플레이트 dilutions 10 -2 횡단 integrants위한 선택 200 μg / ML의 스트렙토 마이신과 30 μg / ML gentamicin와 보충 타이 한천 플레이트에 -5 ~ 10. 플레이트 dilutions 10 -3 through 횡단 integrants하고 잠재받는 사람, 즉 총받는 사람 모두를 위해 선택 200 μg / ML의 스트렙토 마이신과 보완 타이 한천 플레이트 10 -7. </li><li> markerless 횡단 integrants의 절연 위해 멸균 0.85 % NaCl 500 μl에 약 IMCE 짝짓기 장소의 4 분의 1을 resuspend. 10 -6으로 10 배 시리얼 dilutions하십시오. 200 μg / ML의 스트렙토 마이신 200 μg / ML X-gluc과 보완 네 타이 한천 플레이트 10 -6 ~ 10 -4 플레이트 dilutions. </li><li> 3 일간 30 ° C에서 번호판을 품어. </li><li> 녹색 표시등 (525 nm의) 아래와 빨간색 필터 (&gt; 610 nm의) 15 조회수 RFP 횡단 integrants. </li><li> 트랜스 integrants의 CFU 총받는 CFU에 비해​​ 퍼센트 IMCE 효율을 계산합니다. 트랜스 integrants의 약 절반이 민​​감한 그들이 흰색과 spectinomycin 만드는 진정한 카세트 교환을받은 것입니다. </li><li> markerless 횡단 integrants (W를 찾으려면여기 기증자 벡터가 하얀 식민지 (오랜 잠복기, 상온에서 1-2 여분의 일, X-gluc 통해 식민지의 차별을 향상시킬 것입위한 화면) pJH110에서처럼 GM R을 포함하지 않으며, 이것은 S.에 필요 meliloti SmUW227), 항생제 및 100 μg / ML의 spectinomycin를 포함하는 타이 한천 플레이트없이 타이 한천 플레이트에서 심사하여 식민지의 spectinomycin 감도를 확인합니다. </li></ol> 4. 대표 결과 타이에서 부화 3 일간은 스트렙토 마이신과 보완 및 gentamicin 후 제어 줄무늬가 더 성장을 할 필요가 거의 없습니다. IMCE의 행진은 헤드 탄력과 그림 2에서 본 두 번째 행진,에 많은 식민지에 합류 성장이 있어야합니다. 총받는 사람에게 횡단 integrants의 백분율로 표시 횡단 통합의 효율성은 0.5 %의 범위에 있어야합니다. 약 횡단 integrants의 절반은 spectinomycin 민감한과 흰색이 될 것입니다그들은 진정한 카세트 교환을받은 보여주는. 녹색 표시등 (525 nm의) 아래와 빨간색 필터 (&gt; 610 nm의) 15을 통해 볼 때 pJH110에서 테스터 RFP 기증자 카세트를 포함하는 횡단 integrants는 뚜렷한 RFP의 형광을 표시해야합니다. <img src="/files/ftp_upload/3698/3698fig1.jpg" alt="그림 1"/> . 그림 1 활용 혼합물 일러스트 : pRK600에서 전송 유전자의 표현에 의해 주었는 plasmids의 모든 세포에서 세포로 무작위로 전송됩니다. IMCE, integrase (INT) 헬퍼 플라스미드 pJC2 및 기증자 플라스미드 pJH110에 필요한 두 plasmids의 LP - 변형의 인수를 통해 혼합물의 횡단 integrants의 생성,이 전송 결과입니다. 플라스미드 아닌 복제 기증자 (pJH110)에서 기증자 카세트 (LP-마커의 손실의 결과로, 염색체의 LP-카세트의 마커로 ΦC31 integrase 활동을 통해 교환되는 SPR과 uidA) 및 그 결과 횡단 필요 불가결의 기증자 카세트 (RFP와 GM R)의 유지. <img src="/files/ftp_upload/3698/3698fig2.jpg" alt="그림 2"/> 그림 2. :. 한천 표면에 건조 세포 섞어 보여주는 비 선택적 타이 플레이트의 번식 장소 플레이트 B : 위로부터 스트렙토 마이신-gentamicin-X-gluc 플레이트에 streaked 번식 장소는 S.으로, 시계 방향으로 IMCE을 더 integrase 제어를 떠난 적이 meliloti, E. 대장균은 DH5α pJC2 컨트롤을 포함하는, E. 대장균은 pJH110 제어, E를 포함하는 DH5a 대장균 MT616 제어 및 S. . meliloti UW227 컨트롤 C :. 타이 스트렙토 마이신-X-gluc 한천 D의 짝짓기 현장 resuspension 10 -2 희석 : 타이 스트렙토 마이신-gentamicin-X-gluc 한천 (파란색 식민지가 단일 recombinants 위치에서 짝짓기 현장 resuspension 10 -2 희석, 하얀 식민지 사실받은. 카세트 교환) E : 형광의 부족을 보여주는 타이 스트렙토 마이신-X-gluc 한천의 짝짓기 현장 resuspension 10 -2 희석 F :. 두 가지 수준을 보여주는 타이 스트렙토 마이신-gentamicin-X-gluc 한천의 짝짓기 현장 resuspension 10 -2 희석 형광 (밝은 식민지가 청색 식민지에 해당하며 읽기 비록 식민지 사실 - 카세트 교환없이에서의 LAC업자로부터 읽기를 통해서만 그 즉시 발기인으로 RFP를 포함 겪은 것에 벡터 시퀀스에서 발기인으로 인해 아마도 높은 RFP 표현을 가지고 백터는 어느 결석입니다.).

<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>

관심의 어떠한 충돌 선언 없습니다.

IMCE 기술은 이전에 설계 변형율의 LP-현장으로 단일 attB 어귀의 DNA 카세트의 효율적인 통합을 허용합니다. 원하는 구조가 기증자 카세트를 만드는 RFP 자리에 복제되면 기술은 매우 강력하고, 이후 DNA를 정제 및 변환을 필요로하지 않습니다. 그것은 항생제 저항이 횡단 integrants 및 기타 아니라 요소의 생성에 의한 특정 말하면, 적절한 성장 컨트롤이 포함되어있는 것이 중요합니다. ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 스트렙토 마이신 </td><td> Bioshop 캐나다 주식 회사 </td><td> STP101 </td><td></td></tr><tr><td> Spectinomycin </td><td> Bioshop 캐나다 주식 회사 </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 캐나다 주식 회사 </td><td> GTA202 </td><td></td></tr><tr><td> Choramphenicol </td><td> Bioshop 캐나다 주식 회사 </td><td> CLR201 </td><td></td></tr><tr><td> 테트라 싸이클린 </td><td> Bioshop 캐나다 주식 회사 </td><td> TET701 </td><td></td></tr><tr><td> Kanamycin </td><td> Bioshop 캐나다 주식 회사 </td><td> KAN201 </td><td></td></tr><tr><td> 세균 학년 한천 </td><td&gt; Bioshop 캐나다 주식 회사 </td><td> AGR001 </td><td></td></tr><tr><td> Tryptone </td><td> Bioshop 캐나다 주식 회사 </td><td> TRP402 </td><td></td></tr><tr><td> 효모 엑기스 </td><td> Bioshop 캐나다 주식 회사 </td><td> YEX401 </td><td></td></tr><tr><td> 나트륨 염화물 </td><td> Bioshop 캐나다 주식 회사 </td><td> SOD001 </td><td></td></tr><tr><td> 염화칼슘 </td><td> Bioshop 캐나다 주식 회사 </td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td> 골드 바이오 주식 회사 </td><td> G1281C1 </td><td></td></tr><tr><td> E. 대장균 MT616 변형 </td><td> 요청시 사용 가능 </td><td></td><td> 또한 저희 실험실 밖에서 사용되는 </td></tr><tr><td> E. 대장균 pJC2 변형 </td><td> 요청에 의해 집안에서 사용 가능한 </td><td></td><td></td></tr><tr><td> E. 대장균 pJH110 변형 </td><td> reques에 의한 하우스에서 사용 가능한티 </td><td></td><td></td></tr><tr><td> SmUW227 변형 </td><td> 요청에 의해 집안에서 사용 가능한 </td><td></td><td></td></tr></tbody></table>

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

박테리아 염색체가 안정적으로 메가베이스 범위의 1에 외국의 DNA를 유지하는 데 사용할 수 있습니다. 염색체에 통합은 플라스미드 복제, 플라스미드 안정성, 플라스미드 호환성 및 플라스미드 사본 번호 분산 등의 문제를 circumvents. 이 방법은 Streptomyces 파지 (Φ) C31 2,3에서 특정 사이트 integrase를 사용합니다. attB 및 attP (각각 34 39 BP) 4 : ΦC31 integrase 두 개의 특정 유전자 사이트 간의 직접적인 재조합을 catalyzes. 이러한 재조합은 안정하고 5 되돌릴 수 없습니다. spectinomycin - 저항 유전자, aadA (SpR), 그리고 E. 구성된 &quot;방문 패드&quot;(LP) 시퀀스 attP 사이트의 어귀 대장균 SS-glucuronidase 유전자가 (uidA) Sinorhizobium meliloti, Ochrobactrum anthropi 및 intergenic 지역의 Agrobacterium tumefaciens의의 염색체에 통합되었고, 오전각각 PC 현장, 그리고 tetA 현장. S. meliloti이 프로토콜에서 사용됩니다. stuffer 적색 형광 단백질 (RFP) 유전자와 항생제 내성 유전자 측면을 노릴 attB 사이트가있는 Mobilizable 기증자 벡터도 구축되었습니다. 이 예제에서는 gentamicin 내성 플라스미드 pJH110이 사용됩니다. RFP 유전자 6 SPH I 및 PST 나를 사용하여 원하는 구조로 대체될 수 있습니다 또는 attB 사이트의 어귀 합성 구조는 pK19mob 7과 mobilizable 벡터에 서브 복제있을 수 있습니다. ΦC31 integrase 유전자 (pHS62 8 일부터 복제)의 표현은 pRK7813 9 플라스미드 mobilizable 광범위한 호스트 범위에 LAC 프로 모터에 의해 구동됩니다. tetraparental 짝짓기 프로토콜은 LP 변형이 됨으로써 기증자 카세트와 LP 시퀀스에서 마커를 교체로 기증자 카세트를 전송하는 데 사용됩니다. 이러한 세포는 트란입니다S-integrants. 횡단 integrants은 0.5 %의 전형적인 효율을 형성하고 있습니다. 횡단 integrants는 일반적으로 5 - 브로모 -4 - 클로로 -3 - 인돌릴 - 베타-D-glucuronic 이상해진 (X-gluc)을 사용하여 항생제 감수성이나 청색 - 흰색 심사에 의해 상영 첫 500-1,000 식민지 내에 찾을 수 있습니다. 이 프로토콜은 횡단 integrants를 생성하고 분리를위한 짝짓기 및 선정 절차가 포함되어 있습니다.

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: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

미리 만든 수용체의 직전 되나 상륙 패드 종자에 관심있는 외국의 DNA를 통합하는 신속하고 효율적인 방법이 설명되어 있습니다. 방법은 ΦC31 integrase의 활용과 표현을 통해 주어진 변형율의 공학 상륙 패드 현장에 DNA 카세트의 특정 사이트 통합을 허용합니다.

사이트 특정 박테리아 염색체 공학 : ΦC31 Integrase 중재 카세트 거래소 (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 ...

site-specific-bacterial-chromosome-engineering-c31-integrase-mediated

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Bioengineering

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

15.5K 조회수.  University of Waterloo. 미리 만든 수용체의 직전 되나 상륙 패드 종자에 관심있는 외국의 DNA를 통합하는 신속하고 효율적인 방법이 설명되어 있습니다. 방법은 ΦC31 integrase의 활용과 표현을 통해 주어진 변형율의 공학 상륙 패드 현장에 DNA 카세트의 특정 사이트 통합을 허용합니다.

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

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

룽 공학을위한 절차

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

연구

생체공학

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

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

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

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

보조 전기 분무 이온화 질량 분석법 (SESI - MS)를 사용하여 특성화 세균 휘발성 물질

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)

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 silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

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

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

Microelectrode 배열을 사용하여 siRNA와 자기편 세포의 높은 효율, 특정 사이트 Transfection (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

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

심장 조직 공학을위한 모세관 력 리소그래피

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

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 reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

섬유아세포 매개 피브린 겔 다짐 관찰 및 정량화

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 ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as 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 and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

바인더로 박테리아 셀룰로오스를 활용하여 강력한 천연 섬유 프리폼 제조

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

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

미생물 학적으로 방해석 강수량 유도에 의해 중재<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

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

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

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

에 내장 혈관 신생에 의하여 조직 공학<em&gt; 생체</em&gt; 조직 공학 상공 회의소

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

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

간섭이없는 마이크로 / 나노 입자의 세포 공학을위한 미세 유체 버퍼 교환

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

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

세포 배양 응용 프로그램에 대한 히드로 겔의 빛 매개 형성 및 패터닝

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...