Name: producing gene deletions in escherichia coli by p1 transduction with excisable antibiotic resistance cassettes
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Keio samling stammar erhölls från det nationella BioResource-projektet (NIG, Japan): E. coli. Vi tackar Dirk Linke (Institutionen för biovetenskaper, universitetet i Oslo) för hans fortsatta stöd. Detta arbete finansierades av Vetenskapsrådet Norge ung forskare bidrag 249793 (till Jack C. Leo).

1. stammar och plasmider
<ol><li>
Bakteriestammar
	<ol>
<li>Använd det E. coli stammar BW251135, JW4179 (BW25113 tamA::kan)7, BL21(DE3)20och BL21ΔABCF21. Se Tabell för material för ytterligare information.</li>
	</ol>
</li>
	<li>
Bakteriofager
	<ol>
<li>Använd phage P1vir för den allmänna transduktion. Lagra phage som en flytande lager med n...

<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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques:+Recombinogenic+engineering--new+options+for+cloning+and+manipulating+DNA.">Techniques: Recombinogenic engineering--new options for cloning and manipulating DNA.</a> Trends in Biochemical Sciences. 26 (5), 325-331 (2001).</li><li>Baba, T., 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=Construction+of+Escherichia+coli+K-12+in-frame,+single-gene+knockout+mutants:+the+Keio+collection.">Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection.</a> Molecular Systems Biology. 2, (2006).</li><li>Yamamoto, N., 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=Update+on+the+Keio+collection+of+Escherichia+coli+single-gene+deletion+mutants.">Update on the Keio collection of Escherichia coli single-gene deletion mutants.</a> Molecular Systems Biology. 5, 335 (2009).</li><li>Baba, T., Huan, H. -. 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. W., Tabor, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyamines+are+not+required+for+aerobic+growth+of+Escherichia+coli:+preparation+of+a+strain+with+deletions+in+all+of+the+genes+for+polyamine+biosynthesis.">Polyamines are not required for aerobic growth of Escherichia coli: preparation of a strain with deletions in all of the genes for polyamine biosynthesis.</a> Journal of Bacteriology. 191 (17), 5549-5552 (2009).</li><li>Xie, X., Wong, W. W., Tang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+simvastatin+bioconversion+in+Escherichia+coli+by+deletion+of+bioH.">Improving simvastatin bioconversion in Escherichia coli by deletion of bioH.</a> Metabolic Engineering. 9 (4), 379-386 (2007).</li><li>Maeda, T., Sanchez-Torres, V., Wood, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+hydrogenase+3+is+a+reversible+enzyme+possessing+hydrogen+uptake+and+synthesis+activities.">Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities.</a> Applied Microbiology and Biotechnology. 76 (5), 1035-1042 (2007).</li><li>Meehan, B. M., Landeta, C., Boyd, D., Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+disulfide+bond+formation+pathway+is+essential+for+anaerobic+growth+of+Escherichia+coli.">The disulfide bond formation pathway is essential for anaerobic growth of Escherichia coli.</a> Journal of Bacteriology. 199 (16), (2017).</li><li>Niba, E. T. E., Naka, Y., Nagase, M., Mori, H., Kitakawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+approach+to+identify+the+genes+involved+in+biofilm+formation+in+E.+coli.">A genome-wide approach to identify the genes involved in biofilm formation in E. coli.</a> DNA Research. 14 (6), 237-246 (2008).</li><li>Heinz, E., 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=Conserved+features+in+the+structure,+mechanism,+and+biogenesis+of+the+inverse+autotransporter+protein+family.">Conserved features in the structure, mechanism, and biogenesis of the inverse autotransporter protein family.</a> Genome Biology and Evolution. 8 (6), 1690-1705 (2016).</li><li>Selkrig, J., 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=Discovery+of+an+archetypal+protein+transport+system+in+bacterial+outer+membranes.">Discovery of an archetypal protein transport system in bacterial outer membranes.</a> Nature Structural &amp; Molecular Biology. 19 (5), 506-510 (2012).</li><li>Stubenrauch, 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=Effective+assembly+of+fimbriae+in+Escherichia+coli+depends+on+the+translocation+assembly+module+nanomachine.">Effective assembly of fimbriae in Escherichia coli depends on the translocation assembly module nanomachine.</a> Nature Microbiology. 1 (7), 16064 (2016).</li><li>Leo, J. 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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterohemorrhagic+Escherichia+coli+O157:H7+gal+mutants+are+sensitive+to+bacteriophage+P1+and+defective+in+intestinal+colonization.">Enterohemorrhagic Escherichia coli O157:H7 gal mutants are sensitive to bacteriophage P1 and defective in intestinal colonization.</a> Infection and Immunity. 75 (4), 1661-1666 (2006).</li><li>Ikeda, H., Tomizawa, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transducing+fragments+in+generalized+transduction+by+phage+P1.+I.+Molecular+origin+of+the+fragments.">Transducing fragments in generalized transduction by phage P1. I. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coliphage+P1-mediated+transduction+of+cloned+DNA+from+Escherichia+coli+to+Myxococcus+xanthus:+use+for+complementation+and+recombinational+analyses.">Coliphage P1-mediated transduction of cloned DNA from Escherichia coli to Myxococcus xanthus: use for complementation and recombinational analyses.</a> Journal of Bacteriology. 155 (1), 317-329 (1983).</li></ol>

P1 transduktion är en snabb, robust och tillförlitlig metod för att generera gen borttagningar i E. coli. Detta framgår här av transducing en tamA radering mutant från en Keio givare stam till en BL21-derived mottagare. De stora stegen i signaltransduktion processen är produktionen av den transducing lysate, transduktion själv, excision av Kan motstånd kassetten och kontrollen av den knock-out med PCR. Totalt, processen tar cirka 1 vecka och kräver ingen molekylärbiologiska metoder användas,...

En vanlig första metod att studera funktionen av en gen är att utföra knock-out mutagenes och observera den resulterande fenotypen. Detta kallas också omvänd genetik. Bakterien E. coli har varit arbetshästen i molekylärbiologi för de senaste 70 åren eller så, på grund av förenklar dess odling och dess föredragandens genmanipulation1. Flera metoder har utvecklats för att producera gen borttagningar i E. coli, inklusive markör exchange mutagenes2,3 och, mer nyligen, recombineering använder den λ röd eller Rac ET system4,5 , 6.
I ett allmänt använt system ersättas kodande sekvenser av enskilda gener med en antibiotikaresistens kassett som senare kan vara censurerade från kromosom5,7. De kodande sekvenserna byts ut, till exempel genom en kanamycin (Kan) motstånd kassett, som flankeras av flippase (FLP) erkännande mål (FRT) platser på vardera sidan. FRT platserna identifieras av recombinase FLP, som medlar platsspecifika rekombination mellan FRT platser leder till borttagningen av Kan kassett. På detta sätt kan en fullständig radering av en given genens kodande sekvens uppnås, lämnar efter sig endast en minimal ärr sekvens av ungefärligt 100 baspar (bp) (figur 1).
Drygt ett decennium sedan utvecklades den så kallade Keio-samlingen. Detta är en bakteriell bibliotek baserat på en standard laboratorium E. coli K12 stammen, där nästan alla icke-väsentliga gener ströks individuellt genom λ röd rekombination7,8. Klonerna inom denna samling varje har en kodande sekvens ersatts med en punktskattepliktiga Kan motstånd kassett. Keio samlingen har visat sig vara ett användbart verktyg för många applikationer9. En sådan ansökan är produktionen av radering mutanter i andra E. coli -stammar. Kan kassetten från en given radering klon kan mobiliseras av allmänt transducing bakteriofager, såsom P110,11,12,13,14. En fag lager beredd från sådan en stam kan sedan användas till infektera en mottagare E. coli stam av intresse, där en låg men pålitlig frekvens Kan kassett-innehållande regionen kan införlivas i mottagarens genomet av homolog rekombination (Figur 2). Transductants kan väljas för tillväxten på Kan-med mediet. Efter detta, om avlägsnandet av antibiotikaresistens kassetten önskas, kan den FLP recombinase levereras till transductant stam i trans. Efter härdning av FLP-innehållande plasmid, som bär en ampicillin (Amp) motstånd markör, Kan och Amp-känsliga kloner screenas för, och den rätta excision av vildtyp kodande sekvens och kassetten Kan verifieras av kolonin PCR.
Här presenteras ett detaljerat protokoll, som beskriver varje steg i att producera en knock-out E. coli stam baserat på den strategi som beskrivs ovan. Som ett exempel demonstreras en borttagning av tamA genen. tamA kodar en yttre membranprotein i β-fat, som är en del av Transport och montering modul (TAM), som är involverat i biogenes av vissa autotransporter proteiner och pili15,16,17. Denna knock-out stam användes sedan för att undersöka effekten av tamA borttagningen på biogenes av två trimeric autotransporter adhesiner (TAAs), den Yersinia adhesin YadA och den E. coli immunglobulin (Ig)-bindande TAA EibD 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

En första metod att studera funktionen av en okänd gen hos bakterier är att skapa en knock-out av denna gen. Här beskriver vi en robust och snabb protokoll för att överföra genmutationer borttagning från en Escherichia coli stam till en annan med hjälp av generaliserad transduktion med bacteriophage P1. Denna metod kräver att mutationen kan väljas (t.ex. baserat på genen störningar med antibiotika kassett infogningar). Sådana antibiotika kassetter kan mobiliseras från en donator stam och införas till en mottagare stam av intresse för snabbt och generera enkelt en gen radering mutant. Antibiotikum kassetten kan utformas att inkludera flippase erkännande webbplatser som tillåter excision av kassetten genom en platsspecifik recombinase att producera en ren knock-out med endast en ~ 100-base-par-långa ärr sekvens i genomet. Vi visar protokollet genom att slå ut den tamA -gen som kodar en församling faktor inblandad i autotransporter biogenes och testa effekten av denna knock-out på biogenes och funktion av två trimeric autotransporter adhesiner. Även gen radering av P1 transduktion har sina begränsningar, gör den lätthet och snabbhet av dess genomförande det ett attraktivt alternativ till andra metoder för genterapi radering.

Generation av en tamA Knock-out av BL21ΔABCF:
Den strategi som beskrivs ovan har tidigare använts för att producera en härledda stam av BL21(DE3), en standard laboratorium stam används för produktion av proteiner, som är optimerade för yttre membran proteinproduktion och kallas BL21ΔABCF21. Denna stam saknar fyra gener som kodar för riklig yttre membranproteiner och, följaktligen är kunn...

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

Producerar genen borttagningar i Escherichia coli av P1 transduktion med punktskattepliktiga antibiotikaresistens kassetter

Här presenterar vi ett protokoll för användning av befintlig antibiotika resistens-kassett radering konstruktioner som underlag för att göra strykningen mutanter i andra E. coli -stammar. Sådan radering mutationer kan mobiliseras och infogas i det motsvarande locus av en mottagare stam med P1 bacteriophage transduktion.

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 ...

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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

Uppfödning och dubbelsträngade RNA-medierad Gene Knockdown i Hide Beetle,<em&gt; Rävänger</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-kartläggning och CRISPR / Cas9 redigering för att identifiera en läkemedelsresistensgen i<em&gt; Toxoplasma 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

Bestämning av optimala kromosomala belägenheten för ett DNA-Element i Escherichia coli använder en ny metod för Transposon-medierad

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

En standardmetod att undersöka Hotellets mutagenicitet som en funktion av punktmutation reparation katalyseras av CRISPR/Cas9 och SsODN i mänskliga celler

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

Generation av en gen-stört Streptococcus mutans stam utan genen kloning

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

Mäta genuttryck i bombarderade korn Aleurone lager med ökad genomströmning

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

Testning Multicopy plasmider roll i utvecklingen av antibiotikaresistens

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

Kandidat Gene tester i klinisk kohortstudier med multiplexade genotypning och masspektrometri

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-medierad exakt gen redigering av Tube Electroporation

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

Kvantifiering av plasmid-medierad antibiotikaresistens i en experimentell evolutions metod

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