Name: producing gene deletions in escherichia coli by p1 transduction with excisable antibiotic resistance cassettes
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Keio koleksiyonu suşları Ulusal BioResource (Zen, Japonya) projeden elde: E. coli. Dirk Linke (Biosciences bölümü, Oslo Üniversitesi) onun sürekli destek için teşekkür ederiz. Bu eser Norveç genç araştırmacı hibe 249793 (Jack C. Leo) araştırma kurumu tarafından finanse edildi.

1. suşları ve Plazmidlerin
<ol><li>
Bakteri suşları
	<ol>
<li>E. coli suşları BW251135, JW4179 (BW25113 tamA::kan)7, BL21(DE3)20ve BL21ΔABCF21kullanın. Malzemeler tablo daha fazla bilgi için bkz:.</li>
	</ol>
</li>
	<li>
Bacteriophages
	<ol>
<li>Feyc P1vir genel iletim için kullanın. Kloroform birkaç damla ile sıvı bir hisse senedi ol...

<ol>
	<li>Blount, Z. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unexhausted+potential+of+E.+coli.">The unexhausted potential of E. coli.</a> eLife. 4, e05826 (2015).</li><li>Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P., Kushner, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+for+generating+deletions+and+gene+replacements+in+Escherichia+coli.">New method for generating deletions and gene replacements in Escherichia coli.</a> Journal of Bacteriology. 171 (9), 4617-4622 (1989).</li><li>Link, A. J., Phillips, D., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+generating+precise+deletions+and+insertions+in+the+genome+of+wild-type+Escherichia+coli:+application+to+open+reading+frame+characterization.">Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization.</a> Journal of Bacteriology. 179 (20), 6228-6237 (1997).</li><li>Sawitzke, J. A., Thomason, L. C., Costantino, N., Bubunenko, M., Datta, S., Court, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombineering:+in+vivo+genetic+engineering+in+E.+coli,+S.+enterica,+and+beyond.">Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond.</a> Methods in Enzymology. 421, 171-199 (2007).</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> Proceedings of the National Academy of Sciences of the United States of America. 97 (12), 6640-6645 (2000).</li><li>Muyrers, J. P., Zhang, Y., Stewart, A. 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C., Datsenko, K., Wanner, B. L., Mori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+applications+of+systematic+in-frame,+single-gene+knockout+mutant+collection+of+Escherichia+coli+K-12.">The applications of systematic in-frame, single-gene knockout mutant collection of Escherichia coli K-12.</a> Methods in Molecular Biology. 416, 183-194 (2008).</li><li>Chattopadhyay, M. K., Tabor, C. 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C., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunoglobulin-binding+Eib+proteins+from+Escherichia+coli+are+receptors+for+IgG+Fc.">The immunoglobulin-binding Eib proteins from Escherichia coli are receptors for IgG Fc.</a> Molecular Immunology. 46 (8-9), 1860-1866 (2009).</li><li>M&#252;hlenkamp, M., Oberhettinger, P., Leo, J. C., Linke, D., Sch&#252;tz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yersinia+adhesin+A+(YadA)+-+Beauty+&amp;+beast.">Yersinia adhesin A (YadA) - Beauty &amp; beast.</a> International Journal of Medical Microbiology. 305 (2), 252-258 (2015).</li><li>Studier, F. W., Moffatt, B. A. <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+bacteriophage+T7+RNA+polymerase+to+direct+selective+high-level+expression+of+cloned+genes.">Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.</a> Journal of Molecular Biology. 189 (1), 113-130 (1986).</li><li>Meuskens, I., Michalik, M., Chauhan, N., Linke, D., Leo, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strain+collection+for+improved+expression+of+outer+membrane+proteins.">A new strain collection for improved expression of outer membrane proteins.</a> Frontiers in Cellular and Infection Microbiology. 7, 464 (2017).</li><li>Cherepanov, P. P., Wackernagel, 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+disruption+in+Escherichia+coli:+TcR+and+KmR+cassettes+with+the+option+of+Flp-catalyzed+excision+of+the+antibiotic-resistance+determinant.">Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant.</a> Gene. 158 (1), 9-14 (1995).</li><li>Grosskinsky, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+glycine+residue+of+trimeric+autotransporter+domains+plays+a+key+role+in+Yersinia+adhesin+A+autotransport.">A conserved glycine residue of trimeric autotransporter domains plays a key role in Yersinia adhesin A autotransport.</a> Journal of Bacteriology. 189 (24), 9011-9019 (2007).</li><li>Leo, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+E.+coli+IgG-binding+protein+D+suggests+a+general+model+for+bending+and+binding+in+trimeric+autotransporter+adhesins.">The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins.</a> Structure. 19 (7), 1021-1030 (1993).</li><li>Bertani, G. <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+lysogenesis.+I.+The+mode+of+phage+liberation+by+lysogenic+Escherichia+coli.">Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli.</a> Journal of Bacteriology. 62 (3), 293-300 (1951).</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> Journal of Molecular Biology. 166 (4), 557-580 (1983).</li><li>Leo, J. C., Oberhettinger, P., Linke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+outer+membrane+insertion+and+folding+of+multimeric+transmembrane+&#946;-barrel+proteins.">Assessing the outer membrane insertion and folding of multimeric transmembrane &#946;-barrel proteins.</a> Methods in Molecular Biology. , 157-167 (2015).</li><li>Mikula, K. M., Leo, J. C., &#321;yskowski, A., Kedracka-Krok, S., Pirog, A., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translocation+domain+in+trimeric+autotransporter+adhesins+is+necessary+and+sufficient+for+trimerization+and+autotransportation.">The translocation domain in trimeric autotransporter adhesins is necessary and sufficient for trimerization and autotransportation.</a> Journal of Bacteriology. 194 (4), 827-838 (2012).</li><li>Thomason, L. C., Costantino, N., Court, D. L., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E.+coli+genome+manipulation+by+P1+transduction.">E. coli genome manipulation by P1 transduction.</a> Current Protocols in Molecular Biology. , (2007).</li><li>Franklin, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+in+gal+U+gene+of+E.+coli+blocks+phage+P1+infection.">Mutation in gal U gene of E. coli blocks phage P1 infection.</a> Virology. 38 (1), 189-191 (1969).</li><li>Jeong, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequences+of+Escherichia+coli+B+strains+REL606+and+BL21(DE3).">Genome sequences of Escherichia coli B strains REL606 and BL21(DE3).</a> Journal of Molecular Biology. 394 (4), 644-652 (2009).</li><li>Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Sequence+Alignment+during+Homologous+Recombination.">DNA Sequence Alignment during Homologous Recombination.</a> Journal of Biological Chemistry. 291 (22), 11572-11580 (2016).</li><li>Watt, V. M., Ingles, C. J., Urdea, M. S., Rutter, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homology+requirements+for+recombination+in+Escherichia+coli.">Homology requirements for recombination in Escherichia coli.</a> Proceedings of the National Academy of Sciences of the United States of America. 82 (14), 4768-4772 (1985).</li><li>Ho, T. D., Waldor, M. 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Molecular origin of the fragments.</a> Journal of Molecular Biology. 14 (1), 85-109 (1965).</li><li>Murooka, Y., Harada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+the+host+range+of+coliphage+P1+and+gene+transfer+from+enteric+bacteria+to+other+Gam-negative+bacteria.">Expansion of the host range of coliphage P1 and gene transfer from enteric bacteria to other Gam-negative bacteria.</a> Applied and Environmental Microbiology. 38 (4), 754-757 (1979).</li><li>Goldberg, R. B., Bender, R. A., Streicher, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+selection+for+P1-sensitive+mutants+of+enteric+bacteria.">Direct selection for P1-sensitive mutants of enteric bacteria.</a> Journal of Bacteriology. 118 (3), 810-814 (1974).</li><li>O'Connor, K. A., Zusman, D. 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P1 iletim gen silme E. coliüretmek için hızlı, sağlam ve güvenilir bir yöntemdir. Bu burada bir BL21 elde edilen alıcıya Keio donör zorlanma tamA silme mutant transducing tarafından gösterilmiştir. İletim işlemi büyük aşamasında bulunmaktadır lysate transducing üretim, iletim, Kan direnç kaset eksizyon ve knock-out PCR tarafından doğrulanması. Toplamda, süreci yaklaşık 1 hafta alır ve doğrulama için son PCR dışında kullanılmak üzere hiçbir moleküler biyoloji yöntem...

İşlev bir çalışmaya ortak bir ilk yaklaşım knock-out mutagenesis gerçekleştirmek ve elde edilen fenotip dikkat etmektir. Bu da geriye doğru genetik olarak adlandırılır. E. coli bakteri moleküler biyoloji beygir son 70 yıl ya da çok, kendi kültür ve onun amenability genetik manipülasyon1kolaylığı nedeniyle olmuştur. E. coliişaret exchange mutagenesis2,3 ve son zamanlarda, λ kırmızı veya Rac ET sistemleri4,5 kullanarak recombineering da dahil olmak üzere, gen silme üretmek için çeşitli yöntemler geliştirilmiştir , 6.
Yaygın olarak kullanılan bir sistemde, bireysel genlerin kodlama dizileri daha sonra kromozom5,7' den eksize bir antibiyotik direnci kaset değiştirilir. Kodlama dizileri, hangi flippase (FLP) tanıma hedef (FRT) siteleri iki tarafında çevrili örneğin bir sefaloridin (Kan) direnç kaset, değiştirilir. FRT siteleri recombinase siteye özgü rekombinasyon Kan kaset silinmesi için önde gelen FRT siteler arasında aracılık eder FLP tarafından tanınır. Bu şekilde, belirli bir genin kodlama dizisi tam bir silme, yalnızca yaklaşık 100 baz çifti (bp) (resim 1) en az yara dizisi bırakarak elde edilebilir.
Sadece bir on yıl kadar önce sözde Keio koleksiyonu geliştirilmiştir. Bu nerede hemen hemen tüm gerekli olmayan genler ayrı ayrı λ kırmızı rekombinasyon7,8tarafından silindi Standart laboratuvar E. coli K12 zorlanma, temel bakteriyel bir kütüphanedir. Klonlar bu koleksiyon içindeki bir excisable Kan direnç kaset ile yerine bir kodlama dizisi var. Keio koleksiyon birçok uygulamaları9için yararlı bir araç olduğu ispatlanmıştır. Böyle bir uygulama diğer E. coli suşları silme mutantlar yapımıdır. Kan kaset üzerinden verilen silme klon genellikle bacteriophages, P110,11,12,13,14gibi transducing tarafından seferber. Böyle bir yük hazırlanan bir fajının hisse senedi daha sonra alıcı faiz, e.coli suşu bulaştırmak için nerede bir düşük ama güvenilir frekansta Kan kaset içeren bölge alıcı Genom tarafından Homolog rekombinasyon dahil edilebilir kullanılabilir (Şekil 2). Transductants Kan içeren ortamda büyümesi için seçilebilir. Antibiyotik direnci kaset kaldırılmasını isterseniz, bunu, FLP recombinase trans transductant zorlanma sağlanabilir. Ampisilin (Amp) direnç işaret taşıyan FLP içeren plazmid kür sonra Kan ve Amp duyarlı klonlar için ekranlı ve vahşi-türü kodlama dizisi ve Kan kaset doğru eksizyon PCR colony tarafından doğrulandı.
Burada, her bir knock-out yukarıda özetlenen strateji dayalı E. coli zorlanma üreten adımları açıklayan ayrıntılı bir protokol sunulmuştur. Örnek olarak, tamA gen silinmesiyle gösterilmiştir. tamA bir parçası olan taşıma ve montaj modülü (TAM), bazı autotransporter proteinler ve pili15,16,17Biyogenez yer bir dış membran β-varil protein kodlar. Bu knock-out zorlanma sonra iki trimeric autotransporter adhesins (TAAs), Yersinia adhesin YadA ve E. coli immünglobulin (Ig) tamA silinmesi Biyogenez üzerinde etkisini incelemek için kullanılan-TAA EibD bağlama 18,19.

<table>
	<tbody>
		<tr>
			<td>Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli BW25113</td>
			<td>NIG</td>
			<td>ME6092</td>
			<td>Wild-type strain of Keio collection</td>
		</tr>
		<tr>
			<td>E. coli BL21(DE3)</td>
			<td>Merck</td>
			<td>69450-3</td>
			<td>Expression strain</td>
		</tr>
		<tr>
			<td>E. coli BL21DABCF</td>
			<td>Addgene</td>
			<td>102270</td>
			<td>Derived from BL21(DE3)</td>
		</tr>
		<tr>
			<td>E. coli JW4179</td>
			<td>NIG</td>
			<td>JW4179-KC</td>
			<td>tamA deletion mutant</td>
		</tr>
		<tr>
			<td>P1 vir</td>
			<td>NIG</td>
			<td>HR16</td>
			<td>Generally transducing bacteriophage</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCP20</td>
			<td>CGSC</td>
			<td>14177</td>
			<td>conditionally replicating plasmid with FLP</td>
		</tr>
		<tr>
			<td>pASK-IBA2</td>
			<td>IBA GmbH</td>
			<td>2-1301-000</td>
			<td>expression vector</td>
		</tr>
		<tr>
			<td>pEibD10</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>for production of EibD; plasmid available on request</td>
		</tr>
		<tr>
			<td>pET22b+</td>
			<td>Merck</td>
			<td>69744-3</td>
			<td>expression vector</td>
		</tr>
		<tr>
			<td>pIBA2-YadA</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>for production of YadA; plasmid available on request</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>ThermoFisher</td>
			<td>33209</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD Bacto</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Applichem</td>
			<td>A0839</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrotetracycline</td>
			<td>Abcam</td>
			<td>ab145350</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-collagen type I antibody COL-1</td>
			<td>Sigma</td>
			<td>C2456</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine collagen type I</td>
			<td>Sigma</td>
			<td>C9791</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Merck</td>
			<td>102382</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Merck</td>
			<td>102445</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-sodium hydrogen phosphate</td>
			<td>VWR</td>
			<td>28029</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA dye</td>
			<td>Thermo</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA molecular size marker</td>
			<td>New England BioLabs</td>
			<td>N3232S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>New England Biolabs</td>
			<td>N0447</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL HRP substrate</td>
			<td>Advansta</td>
			<td>K-12045</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Applichem</td>
			<td>A2937</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>24388</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse IgG-HRP</td>
			<td>Santa Cruz</td>
			<td>sc-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-rabbit IgG-HRP</td>
			<td>Agrisera</td>
			<td>AS10668</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>30487</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl thiogalactoside</td>
			<td>VWR</td>
			<td>43714</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Applichem</td>
			<td>A1493</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Applichem</td>
			<td>A4972</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>VWR</td>
			<td>25108</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese chloride</td>
			<td>Sigma</td>
			<td>221279</td>
			<td></td>
		</tr>
		<tr>
			<td>N-lauroyl sarcosine</td>
			<td>Sigma</td>
			<td>L9150</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Sigma</td>
			<td>70166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>27808</td>
			<td></td>
		</tr>
		<tr>
			<td>tamA forward primer</td>
			<td>Invitrogen</td>
			<td>N/A</td>
			<td>Sequence 5&#39;-GAAAAAAGGATATTCAGGAGAAAATGTG-3&#39;</td>
		</tr>
		<tr>
			<td>tamA reverse primer</td>
			<td>Invitrogen</td>
			<td>N/A</td>
			<td>Sequence 5&#39;-TCATAATTCTGGCCCCAGACC-3&#39;</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0267</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-sodium citrate</td>
			<td>Merck</td>
			<td>106448</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>VWR</td>
			<td>84610</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Merck</td>
			<td>103753</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis chamber</td>
			<td>Hoefer</td>
			<td>SUB13</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead beater</td>
			<td>Thermo</td>
			<td>FP120A-115</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Kodak</td>
			<td>4000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation cuvettes</td>
			<td>Bio-Rad</td>
			<td>165-2089</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation unit</td>
			<td>Bio-Rad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel imager</td>
			<td>Nippon Genetics</td>
			<td>GP-03LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubating shaker</td>
			<td>Infors HT</td>
			<td>Minitron</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>390-0482</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5415D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave oven</td>
			<td>Samsung</td>
			<td>CM1099A</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Biometra</td>
			<td>Tpersonal</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR strips</td>
			<td>Axygen</td>
			<td>PCR-0208-CP-C</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Hanna Instruments</td>
			<td>HI2211-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>ThermoFisher</td>
			<td>88518</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAG electrophoresis chamber</td>
			<td>ThermoFisher</td>
			<td>A25977</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>B06322</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>GFL</td>
			<td>D3006</td>
			<td></td>
		</tr>
		<tr>
			<td>Wet transfer unit</td>
			<td>Hoefer</td>
			<td>TE22</td>
			<td></td>
		</tr>
	</tbody>
</table>

producing gene deletions in escherichia coli by p1 transduction with excisable antibiotic resistance cassettes

Bakterilerde bilinmeyen bir gen işlevini incelemek için bir ilk yaklaşım bir knock-out bu gen oluşturmaktır. Burada, bakteriyofaj P1 ile jeneralize iletim kullanarak gen silme mutasyonlar bir Escherichia coli zorlanma diğerine aktarmak için sağlam ve hızlı bir iletişim kuralı açıklar. Bu yöntem mutasyon (antibiyotik kaset eklemeleri kullanarak gen kesintileri göre seçilebilirÖrneğin, ) olmasını gerektirir. Bu tür antibiyotik kaset bir donör yük seferber ve hızlı bir şekilde ilgi alıcı bir gerilim girmiştir ve kolayca bir gen silme mutant oluşturmak. Antibiyotik kaset kaset eksizyon izin flippase tanıma siteleri dahil etmek için temiz bir knock-out ile genom bir ~ 100 baz çifti uzun yara sırasına üretmek için siteye özgü bir recombinase tarafından dizayn edilebilir. Protokol autotransporter Biyogenez ilgili bir derleme faktör kodlama tamA gene dışarı knocking göstermek ve bu knock-out Biyogenez üzerinde etkisi ve iki trimeric autotransporter adhesins fonksiyonu test. Gen silme P1 iletim sınırlamaları olsa, kolaylık ve hız onun uygulama gene silme işlemini diğer yöntemleri için cazip bir alternatif olun.

Bir tamA Knock-out BL21ΔABCF, nesil:
Yukarıda özetlenen strateji BL21(DE3), dış membran protein üretimi için en iyi duruma getirilmiş ve BL21ΔABCF21denilen protein üretimi için kullanılan bir Standart laboratuvar yük türetilmiş bir tür üretmek için daha önce kullanılmış. Bu zorlanma dört genler bol dış membran proteinlerinin için kodlama yoksun ve sonuç olarak, daha hetero...

Watch this Scientific Journal Video about Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes at JoVE.com

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Escherichia coli Excisable antibiyotik direnci kasetleri ile P1 iletim tarafından üreten gen silme

Burada bir protokol antibiyotik direnç-kaset silme yapıları silme mutantlar diğer E. coli suşları yapmak için bir temel olarak önceden var olan kullanım için mevcut. Böyle silme mutasyonlar seferber ve büyük bir alıcı yük P1 bakteriyofaj iletim kullanarak odağı karşılık gelen eklenmiş.

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Research

JoVE Journal

Genetics

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Yetiştirme ve Gizle böcek RNA aracılı gen demonte çift sarmallı,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL Haritalama ve CRISPR / Cas9 Düzenlemesi, bir İlaç Direnç Geninin Belirlenmesi için<emToksoplasma gondii</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

En iyi kromozom Location(s) belirlenmesi için yeni bir Transposon-aracılı yaklaşımın kullanarak Escherichia coli DNA öğesinde

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Yerinde mutagenisitesinin CRISPR/Cas9 ve SsODN insan hücrelerinde tarafından katalize nokta mutasyon onarım bir fonksiyonu olarak incelemek için standart bir metodoloji

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Nesil bir gen kesintiye Streptococcus mutans çoğunlukla zorlanma olmadan Gen Klonlama

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Gen ifadesinin artan işlem hacmi ile alı arpa Aleurone katmanlarında ölçme

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plazmid rolü antibiyotik direnci gelişimi test

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Klinik kohort çalışmaları çoğaltılmış Genotipleme ve kütle spektrometresi ile test aday gen

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

CRISPR/Cas9 ribonucleoprotein-boru Elektroporasyon tarafından aracılı hassas gen düzenleme

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Deneysel Evrim Yaklaşımında Plazmid Aracılı Antibiyotik Direncinin Ölçülmesi

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...