Name: determination of the optimal chromosomal location(s) for a dna element in escherichia coli using a novel transposon-mediated approach
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Description: Watch this Scientific Journal Video about Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach at JoVE.com

Forfatterne var finansieret af tilskud fra Novo Nordisk Fonden, Lundbeckfonden og den danske National Research Foundation (DNRF120) via Center for bakteriel stressrespons og persistens (BASP).

1. samling af biblioteket Transposon NOTE: den kromosomale DARS2 locus er klonet i den mini Tn 10-baseret transposon, NKBOR (efter pNKBOR) 21, hvilket resulterer i NKBOR:: DARS2 (pJFM1). pNKBOR kan fås online 22. pNKBOR er en R6K-baseret selvmord vektor, der kræver, at initiatoren protein π for replikering 23. Plasmidet pJFM1 er derfor i stand til at replikere i en E. coli-stamme (...

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	<li>Frimodt-Moller, J., Charbon, G., Lobner-Olesen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+bacterial+chromosome+replication+by+non-coding+regions+outside+the+origin.">Control of bacterial chromosome replication by non-coding regions outside the origin.</a> Curr Genet. , (2016).</li><li>Sherratt, D. 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=Recombination+and+chromosome+segregation.">Recombination and chromosome segregation.</a> Philos Trans R Soc Lond B Biol Sci. 359 (1441), 61-69 (2004).</li><li>Bigot, S., 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=DNA+motifs+that+control+E.+coli+chromosome+segregation+by+orienting+the+FtsK+translocase.">DNA motifs that control E. coli chromosome segregation by orienting the FtsK translocase.</a> EMBO J. 24 (21), 3770-3780 (2005).</li><li>Frimodt-Moller, J., Charbon, G., Krogfelt, K. A., Lobner-Olesen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Replication+Control+Is+Linked+to+Genomic+Positioning+of+Control+Regions+in+Escherichia+coli.">DNA Replication Control Is Linked to Genomic Positioning of Control Regions in Escherichia coli.</a> PLoS Genet. 12 (9), 1006286 (2016).</li><li>Kornberg, A., Baker, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DNA Replication, Second Edition. , (1992).</li><li>Kaguni, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+initiation+at+the+Escherichia+coli+chromosomal+origin.">Replication initiation at the Escherichia coli chromosomal origin.</a> Curr Opin Chem Biol. 15 (5), 606-613 (2011).</li><li>Leonard, A. C., Grimwade, 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=Regulation+of+DnaA+assembly+and+activity:+taking+directions+from+the+genome.">Regulation of DnaA assembly and activity: taking directions from the genome.</a> Annu Rev Microbiol. 65, 19-35 (2011).</li><li>Kurokawa, K., Nishida, S., Emoto, A., Sekimizu, K., Katayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+cycle-coordinated+change+of+the+adenine+nucleotide-bound+forms+of+DnaA+protein+in+Escherichia+coli.">Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli.</a> EMBO J. 18 (23), 6642-6652 (1999).</li><li>Sekimizu, K., Bramhill, D., Kornberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+activates+dnaA+protein+in+initiating+replication+of+plasmids+bearing+the+origin+of+the+E.+coli+chromosome.">ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome.</a> Cell. 50 (2), 259-265 (1987).</li><li>Brendler, T., Austin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+SeqA+protein+to+DNA+requires+interaction+between+two+or+more+complexes+bound+to+separate+hemimethylated+GATC+sequences.">Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences.</a> EMBO J. 18 (8), 2304-2310 (1999).</li><li>Lu, M., Campbell, J. L., Boye, E., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SeqA:+a+negative+modulator+of+replication+initiation+in+E.+coli.">SeqA: a negative modulator of replication initiation in E. coli.</a> Cell. 77 (3), 413-426 (1994).</li><li>Katayama, T., Kubota, T., Kurokawa, K., Crooke, E., Sekimizu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+initiator+function+of+DnaA+protein+is+negatively+regulated+by+the+sliding+clamp+of+the+E.+coli+chromosomal+replicase.">The initiator function of DnaA protein is negatively regulated by the sliding clamp of the E. coli chromosomal replicase.</a> Cell. 94 (1), 61-71 (1998).</li><li>Kato, J., Katayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hda,+a+novel+DnaA-related+protein,+regulates+the+replication+cycle+in+Escherichia+coli.">Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli.</a> EMBO J. 20 (15), 4253-4262 (2001).</li><li>Kasho, K., Katayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DnaA+binding+locus+datA+promotes+DnaA-ATP+hydrolysis+to+enable+cell+cycle-coordinated+replication+initiation.">DnaA binding locus datA promotes DnaA-ATP hydrolysis to enable cell cycle-coordinated replication initiation.</a> Proc Natl Acad Sci U S A. 110 (3), 936-941 (2013).</li><li>Kitagawa, R., Ozaki, T., Moriya, S., Ogawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+control+of+replication+initiation+by+a+novel+chromosomal+locus+exhibiting+exceptional+affinity+for+Escherichia+coli+DnaA+protein.">Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein.</a> Genes Dev. 12 (19), 3032-3043 (1998).</li><li>Fujimitsu, K., Senriuchi, T., Katayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+genomic+sequences+of+E.+coli+promote+replicational+initiation+by+directly+reactivating+ADP-DnaA.">Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA.</a> Genes Dev. 23 (10), 1221-1233 (2009).</li><li>Kasho, K., Fujimitsu, K., Matoba, T., Oshima, T., Katayama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timely+binding+of+IHF+and+Fis+to+DARS2+regulates+ATP-DnaA+production+and+replication+initiation.">Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation.</a> Nucleic Acids Res. 42 (21), 13134-13149 (2014).</li><li>Frimodt-Moller, J., Charbon, G., Krogfelt, K. A., Lobner-Olesen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+regions+for+chromosome+replication+are+conserved+with+respect+to+sequence+and+location+among+Escherichia+coli+strains.">Control regions for chromosome replication are conserved with respect to sequence and location among Escherichia coli strains.</a> Front Microbiol. 6, 1011 (2015).</li><li>Bergthorsson, U., Ochman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+chromosome+length+variation+in+natural+isolates+of+Escherichia+coli.">Distribution of chromosome length variation in natural isolates of Escherichia coli.</a> Mol Biol Evol. 15 (1), 6-16 (1998).</li><li>Boye, E., Steen, H. B., Skarstad, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+bacteria:+a+promising+tool+in+experimental+and+clinical+microbiology.">Flow cytometry of bacteria: a promising tool in experimental and clinical microbiology.</a> J Gen Microbiol. 129 (4), 973-980 (1983).</li><li>Rossignol, M., Basset, A., Espeli, O., Boccard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKBOR,+a+mini-Tn10-based+transposon+for+random+insertion+in+the+chromosome+of+Gram-negative+bacteria+and+the+rapid+recovery+of+sequences+flanking+the+insertion+sites+in+Escherichia+coli.">NKBOR, a mini-Tn10-based transposon for random insertion in the chromosome of Gram-negative bacteria and the rapid recovery of sequences flanking the insertion sites in Escherichia coli.</a> Res Microbiol. 152 (5), 481-485 (2001).</li><li>Shafferman, A., Helinski, 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=Structural+properties+of+the+beta+origin+of+replication+of+plasmid+R6K.">Structural properties of the beta origin of replication of plasmid R6K.</a> J Biol Chem. 258 (7), 4083-4090 (1983).</li><li>Gonzales, M. F., Brooks, T., Pukatzki, S. U., Provenzano, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+protocol+for+preparation+of+electrocompetent+Escherichia+coli+and+Vibrio+cholerae.">Rapid protocol for preparation of electrocompetent Escherichia coli and Vibrio cholerae.</a> J Vis Exp. (80), (2013).</li><li>Smith, 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=Random+primed+labeling+of+DNA.">Random primed labeling of DNA.</a> Methods Mol Biol. 18, 445-447 (1993).</li><li>Lobner-Olesen, A., von Freiesleben, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+replication+incompatibility+in+Dam+methyltransferase+deficient+Escherichia+coli+cells.">Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells.</a> EMBO J. 15 (21), 5999-6008 (1996).</li><li>Harrison, R. W., Miller, J. C., D'Souza, M. J., Kampo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+gene+walking.">Easy gene walking.</a> Biotechniques. 22 (4), 650-653 (1997).</li><li>Langmead, B., Salzberg, 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=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nat Methods. 9 (4), 357-359 (2012).</li><li>Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+local+alignment+search+tool.">Basic local alignment search tool.</a> J Mol Biol. 215 (3), 403-410 (1990).</li><li>Stepankiw, N., Kaidow, A., Boye, E., Bates, 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+right+half+of+the+Escherichia+coli+replication+origin+is+not+essential+for+viability,+but+facilitates+multi-forked+replication.">The right half of the Escherichia coli replication origin is not essential for viability, but facilitates multi-forked replication.</a> Mol Microbiol. 74 (2), 467-479 (2009).</li><li>Lobner-Olesen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+minichromosomes+in+individual+Escherichia+coli+cells:+implications+for+replication+control.">Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control.</a> EMBO J. 18 (6), 1712-1721 (1999).</li><li>Lobner-Olesen, A., Skarstad, K., Hansen, F. G., von Meyenburg, K., Boye, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DnaA+protein+determines+the+initiation+mass+of+Escherichia+coli+K-12.">The DnaA protein determines the initiation mass of Escherichia coli K-12.</a> Cell. 57 (5), 881-889 (1989).</li><li>Carver, T., Thomson, N., Bleasby, A., Berriman, M., Parkhill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNAPlotter:+circular+and+linear+interactive+genome+visualization.">DNAPlotter: circular and linear interactive genome visualization.</a> Bioinformatics. 25 (1), 119-120 (2009).</li><li>Eckert, S. 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=Retrospective+application+of+transposon-directed+insertion+site+sequencing+to+a+library+of+signature-tagged+mini-Tn5Km2+mutants+of+Escherichia+coli+O157:H7+screened+in+cattle.">Retrospective application of transposon-directed insertion site sequencing to a library of signature-tagged mini-Tn5Km2 mutants of Escherichia coli O157:H7 screened in cattle.</a> J Bacteriol. 193 (7), 1771-1776 (2011).</li><li>van Opijnen, T., Bodi, K. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn-seq:+high-throughput+parallel+sequencing+for+fitness+and+genetic+interaction+studies+in+microorganisms.">Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms.</a> Nat Methods. 6 (10), 767-772 (2009).</li><li>van Opijnen, T., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+insertion+sequencing:+a+new+tool+for+systems-level+analysis+of+microorganisms.">Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.</a> Nat Rev Microbiol. 11 (7), 435-442 (2013).</li><li>Jeong, H., Lee, S. J., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+Adaptive+Laboratory+Evolution+of+Microorganisms+Using+a+Chemostat.">Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat.</a> J Vis Exp. (115), (2016).</li><li>Bryant, J. A., Sellars, L. E., Busby, S. J., Lee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+position+effects+on+gene+expression+in+Escherichia+coli+K-12.">Chromosome position effects on gene expression in Escherichia coli K-12.</a> Nucleic Acids Res. 42 (18), 11383-11392 (2014).</li><li>Blattner, F. R., 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+complete+genome+sequence+of+Escherichia+coli+K-12.">The complete genome sequence of Escherichia coli K-12.</a> Science. 277 (5331), 1453-1462 (1997).</li></ol>

Den metode, der anvendes her udnytter state-of-the-art teknikker til at besvare vanskelige spørgsmål vedrørende den optimale genomiske holdning af et genetisk element. Den tilfældige indsættelse af det genetiske element (medieret af transposon) giver mulighed for hurtig og nem indsamling af tusindvis af kloner, som derefter kan bringes til at konkurrere mod hinanden for at vælge den optimale stilling af de undersøgte genetiske element (dvs. de stærkeste klon).
Her, DARS2 <...

Funktionen af et genetisk element kan blive påvirket af dens placering i genomet. I bakterier resulterer hovedsagelig fra indblanding af transkription af nærliggende gener, lokale DNA topologi, og/eller Replikerings-associerede gen dosering. Især processerne af DNA-replikation og segregation styres i det mindste i en del, af ikke-kodende kromosomale områder1, og den korrekte funktion af disse regioner afhænger genomisk placering/kontekst. I E.coli, eksempler er webstedet dif , kræves til søster kromosom opløsning2; KOPS sekvenser, kræves til kromosom segregation3; og datA, DARS1og DARS2 regioner, nødvendige for korrekt kromosom replikation kontrol (nedenfor; 4). vi præsentere en metode giver mulighed for tilfældige udflytning, udvælgelse og bestemmelse af optimal genetiske forbindelse med nogen given genetiske element, her eksemplificeret ved studiet af DARS2 ikke-kodende region.
I E. coli, DnaA er initiativtager protein ansvarlige for DNA streng åbning på enkelt replikation oprindelseoriCsamt ansættelse af helicase DnaB5,6,7. DnaA hører til AAA+ (dvs. ATPases forbundet med forskellige aktiviteter) proteiner og kan binde ATP og ADP med lignende høj samfølelse5. Niveauet af DnaAATP toppe på indledningen8, hvor DnaAATP danner en multimer på oriC , der udløser DNA duplex åbning9. Efter indledningen, oriC er lavet midlertidigt utilgængelig for fornyet indledning som følge af binding af en mekanisme, der involverer bindingen af SeqA proteinet hemimethylated oriC10,11. Under kulstofbindingen, den DnaAATP reduceres af mindst to mekanismer: regulerende inaktivering af DnaA (RIDA)12,13 og datA-afhængige DnaAATP hydrolyse (DDAH)14 ,15. Både RIDA og DDAH fremme omdannelsen af DnaAATP til DnaAADP. Før en ny runde af indledningen, DnaAADP er aktiveret igen til DnaAATP på specifikke genaktivering af DnaA sekvenser (DARS): DARS1 og DARS216,17. Den kromosomale datA, DARS1og DARS2 regioner er ikke-kodende og handle på en anstandsdame-lignende måde til at modulere DnaAATP/DnaAADP energifremtid. Disse regioner, beliggende uden for oprindelsen af replikering, aktiverer forsamlingen af en kompleks DnaA for enten inaktivering (datA; 14) eller aktivisering (DARS1 og DARS2; 17) af DnaA. Sletning af DARS2 i en celle ændres ikke masse fordobling tid men resultater i asynkron replikering indledning15,16,18. Dog DARS2-mangelfuld celler har en fitness omkostninger i forhold til en ellers isogene vildtype under begge kontinuerlig vækst konkurrence i rig medium eller under etableringen af kolonisering i mus tarmen18. Dette indikerer, at selv mindre ændringer i asynkroniske/oprindelse koncentration har en negativ effekt på bakteriel fitness. I E. colier der en selektiv pres for at opretholde kromosom symmetri (dvs. to næsten lige lange replikering arme)19. De datA, DARS1og DARS2 regioner har den samme relative afstand til oriC i alle E. coli stammer sekventeret18, trods store variationer i kromosom størrelse.
Her bruger vi DARS2 regionen af E. coli som et eksempel til identifikation af den kromosomale stilling(er) optimalt for dens funktion. DARS2 blev indsat i NKBOR transposon, og den deraf følgende NKBOR::DARS2 transposon efterfølgende indsat tilfældigt i genomet af MG1655 ΔDARS2. Vi derfor genereret en samling af celler, hver besidder DARS2 placeret på en anden placering på kromosom. En in vitro- konkurrence eksperiment, hvor alle celler i samlingen blev samlet og konkurrerede mod hinanden under kontinuerlig vækst i LB for en anslået 700 generationer, blev udført. Resultatet af konkurrencen eksperiment blev overvåget/bestemmes ved hjælp af det sydlige skamplet, let gen gåture og hele-genome sequencing (WGS; Figur 1). End-point kloner løst fods let gen var karakteriseret ved flowcytometri at evaluere cellecyklus parametre. I en flow cytometric analyse, kan Cellestørrelse, DNA indhold og indledning synchrony måles for et stort antal celler. Under flowcytometri, en strøm af enkelt celler passerer en lysstråle af passende bølgelængde at ophidse den farvede DNA, som er så samtidig registreret af Fotomultiplikatorer, der indsamler de udsendte fluorescens, en foranstaltning af DNA indhold, forudsat at celler er farvet for DNA. Den frem-spredt lys er et mål for celle masse20.
Til i vitro konkurrence eksperiment præsenterer vi her bruges til at behandle spørgsmål vedrørende betydningen af den kromosomale holdning og genomisk forbindelse med det genetiske element. Metoden er fordomsfri og nem at bruge.

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			<td height="20" style="width:369px;height:20px;">Autoclaved Mili-Q water</td>
			<td style="width:164px;"></td>
			<td style="width:119px;">None</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Electroporation Cuvettes, 0.1 cm</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">P41050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Bio-Rad MicroPulser Electroporation System</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;">165-2100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">LB Broth</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12780029&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">LB Agar, powder (Lennox L agar)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22700025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Glycerol</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Fisherbrand&#160;Plastic Petri Dishes</td>
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			<td height="21" style="width:369px;height:21px;">dATP, [&#945;-32P]- 3000Ci/mmol 10mCi/ml, 250 &#181;Ci</td>
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			<td height="21" style="width:369px;height:21px;">GenElute &#160;Gel Extraction Kit</td>
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			<td height="21" style="width:369px;height:21px;">GenElute &#160;PCR Clean-Up Kit</td>
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			<td height="21" style="width:369px;height:21px;">T100&#160; Thermal Cycler</td>
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			<td height="21" style="width:369px;height:21px;">SmartSpec Plus Spectrophotometer</td>
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			<td height="21" style="width:369px;height:21px;">Rifampicin</td>
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			<td style="width:119px;">34514.01</td>
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			<td height="21" style="width:369px;height:21px;">Cephalexin</td>
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			<td height="21" style="width:369px;height:21px;">Mithramycin</td>
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			<td>29803.02</td>
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			<td height="21" style="width:369px;height:21px;">Magnesium chloride hexahydrate</td>
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			<td height="21" style="width:369px;height:21px;">Apogee A10 instrument</td>
			<td style="width:164px;">Apogee</td>
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	</tbody>
</table>

determination of the optimal chromosomal location(s) for a dna element in escherichia coli using a novel transposon-mediated approach

Den optimale kromosomale stilling(er) i en given DNA element var/blev bestemt af transposon-medieret tilfældige indsættelse efterfulgt af fitness udvalg. I bakterier, kan konsekvenserne af den genetiske forbindelse på funktionen af et genetisk element være vanskeligt at vurdere. Flere mekanismer, herunder topologisk effekter, transcriptional interferens fra nærliggende gener, og/eller Replikerings-associerede gen dosering, kan påvirke funktionen af en given genetiske element. Her, beskriver vi en metode, der tillader tilfældig integration af et DNA element i kromosom af Escherichia coli og vælge de mest gunstige placeringer ved hjælp af en simpel vækst konkurrence eksperimentere. Metoden udnytter en velbeskrevne transposon-baseret system for tilfældige indsættelse, kombineret med et udvalg af de stærkeste clone(s) af vækst fordel, en procedure, der kan nemt justeres til eksperimentelle behov. Arten af den stærkestes ret clone(s) kan bestemmes ved hele-genome sequencing på en kompleks multi klonede befolkning eller ved let gen gå til hurtig identifikation af udvalgte kloner. Her, blev regionen med ikke-kodende DNA DARS2, som styrer indledningen af kromosom replikation i E. coli, brugt som et eksempel. Funktionen af DARS2 er kendt for at være påvirket af replikering-associerede gen dosering; den tættere DARS2 får til oprindelsen af DNA-replikation, jo mere aktiv det bliver. DARS2 var tilfældigt indsat i kromosom af en DARS2-slettet stamme. De resulterende kloner indeholder enkelte indsætninger blev samlet og konkurrerede mod hinanden for hundredvis af generationer. Endelig, de stærkestes ret kloner var karakteriseret og fundet for at indeholde DARS2 indsat i umiddelbar nærhed af den oprindelige DARS2 placering.

En sydlige skamplet blev gjort for at kontrollere, at DARS2 var fordelt tilfældigt i kromosom i biblioteket transposon (t = 0), og at de stærkeste kloner ville der stadig over tid. Den sydlige skamplet blev udført på DNA ekstraheret fra den oprindelige transposon pool (ved t = 0) og hver anslået 100 ud af 700 generationer af konkurrence (figur 3). Her, den samlede cellulære DNA fra hvert tidspunkt var fordøjet med Pvubegrænsning enzy...

Watch this Scientific Journal Video about Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach at JoVE.com

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

Her, blev en transposon-medieret tilfældige indsættelse af en ikke-kodende DNA element magt brugt til at løse sin optimale kromosomale holdning.

Bestemmelse af Optimal kromosomale placering for en DNA Element i Escherichia coli ved hjælp af en roman Transposon-medieret tilgang

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

The overall goal of this experiment is to determine the optimal chromosomal position of any given DNA element using transposon-mediated random insertion, competition experiments and whole genome sequencing. This method can help answer key questions in the DNA replication field such as the importance of a chromosomal location of a genetic element. The advantage of this technique is that is couples transposon-based random insertion with the selection of the fittest clone by growth advantage.Here, it's simplified by the E.coli DARS2 region. Though this method can provide insight into the chromosomal organization of Escherichia coli, it can also be applied to other bacteria. After preparing electrocompetant cells such as MG1655 delta DARS2 according to the text protocol, add one microgram of pNKBOR DARS2 to 40 microliters of cells and transfer the mixture to a pre-chilled sterile 0.2-centimeter gap cuvette.Insert the cuvette into the electroporation chamber. Then, electroporate the cells at 18 kilovolts, 500 ohm and 25 microfarad. The time constant should be about five milliseconds and no arcing should occur.Quickly recover the bacterial suspension by re-suspending it in one milliliter of pre-warmed LB broth and transfer to a 15-milliliter test tube. Incubate the cells under aerated growth conditions at 37 degrees Celsius for 30 minutes without antibiotic selection. Then, plate the bacteria onto the LB agar plates supplemented with 50 micrograms per milliliter of kanamycin and incubate the plates at 37 degrees Celsius overnight.The following day, count the colonies from the electroporation. One colony is considered equal to one chromosomal transposon insertion. Add one milliliter of LB broth to each plate and wash off all the colonies.Reuse the same one milliliter of LB broth to increase the bacterial concentration. Then, pool all the colonies in the same 50-milliliter tube. Vortex the tube to freeze the starting material and mix one milliliter of cell suspension with one milliliter of 50%glycerol on ice.Transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen, transfer them to a negative 80-degree-Celsius freezer. Thaw the transposon library from negative 80 degrees Celsius on ice.Mix by pipetting, then transfer 100 microliters of the transposon library to 10 milliliters of LB in a 15-milliliter test tube. Grow the cells aerated with continuous shaking at 37 degrees Celsius for eight hours to stationary phase. Adjust the parameters to different growth conditions as desired.To propagate the bacterial population at approximately every 10 generations, transfer 10 microliters of the previously stationary phase into 10 milliliters of fresh pre-warmed medium and grow the culture for another eight hours to stationary phase. After each 100 generations of competition, save five one-milliliter samples and store them in negative 80 degrees Celsius. Prepare the probe for the Southern blot against a one-kilobyte section of the NKBOR by PCR amplification using specific primers as listed here.Run the samples using the following program. Label the resultant PCR fragment with P32-labeled dATP using the random primer system according to Smith and prepare total cellular DNA according to the text protocol. Digest the total cellular DNA with Pvul which cuts NKBOR containing the region of interest outside of a region covered by the probe.Perform a Southern blot using 0.7%agarose gel according to Loebner-Olsen and von Freiesleben. To use easy gene walking to identify DARS2 insertion sites from single clones, after 700 estimated generations of competition, isolate genomic DNA for the PCR template by first spreading bacteria on an LB agar plate and incubate at 37 degrees Celsius overnight. Inoculate LB broth with single colonies and grow them overnight at 37 degrees Celsius.Then, transfer 20 microliters into 200 microliters of autoclaved distilled water. Vortex the colonies into solution, then heat the mixture at 100 degrees Celsius for 10 minutes. Centrifuge the samples at maximum speed for five minutes, then save the cell lysate at negative 20 degrees Celsius for future use.Next, design three nested primers to anneal within the transposon of choice as shown here. Use 200 nanograms of genomic DNA, nested primer one and a random primer to prepare a 20-microliter PCR reaction. Then, run the samples using the following program.For the second amplification, use one microliter of the first reaction as the template and use nested primer two and the same random primer. Carry out a third reaction in a similar manner using nested primer three, the same random primer and one microliter from the second reaction as the template. Run the products from the final PCR reaction on a 1.5%agarose gel and statin with ethidium bromide.Cut out a band in the range of 100 to 800 base pairs and isolate the DNA using any commercial DNA gel extracting kit according to the manufacturer's directions. To carry out flow cytometry, balance each bacterial culture by maintaining it in the exponential growth phase for at least 10 generations. Measure the OD600 and ensure that it never exceeds 0.3.To carry out rifampicin run-out per the text protocol, transfer one milliliter of culture to a 15-milliliter tube containing 30 microliters of RIF-CEF. Incubate the tube at 37 degrees Celsius with a shaking for a minimum of four hours. For the EXP sample, transfer one milliliter of exponentially growing culture to a 1.5 milliliter tube on ice.Then, harvest the cells by centrifugation at 15, 000 times G and four degrees Celsius for five minutes. Re-suspend the pellet in 100 microliters of ice-cold 10 millimolar tris pH 7.5 and add one milliliter of ice-cold 77%ethanol. Store the samples at four degrees Celsius until use.Stain the cells by harvesting 100 to 300 microliters of fixed cells by centrifugation at 15, 000 times G for 15 minutes. Remove the supernatant and use 130 microliters of DNA staining solution to re-suspend the pellet. Then, incubate the samples on ice and in the dark for 10 minutes.Finally, use flow cytometry to determine the origin per cell, the relative cell mass and the relative DNA content. Following the extraction of DNA from the initial transposon pool and every estimated 100 out of 700 generations of competition, the DNA was cut with Pvul which is known to cut transposon NKBOR DARS2 once in a region not covered by the probe and a Southern blot was performed. As shown here, the initial DARS2 pool lacked distinct bands which shows that DARS2 was inserted randomly throughout the chromosome.Over time, a pattern emerged where the initial large pool of DARS2 clones developed into only one or a few persisting DARS2 clones. Whole genome sequencing was used to identify DARS2 insertion sites from the start pool. And after 400, 500, and 700 generations of competition in a representative subset of insertion sequenced, 98%of all DARS2 insertions were found close to the wild type DARS2 chromosomal location.And 2%were found elsewhere on the chromosome. DARS2-deficient cells were previously shown to initiate replication in a synchrony and to have a decrease in origin concentration relative to wild-type cells. Here, the presence of a DARS2 element in the terminus does not restore synchrony or the cellular origin content to wild-type levels while the selected DARS2 clones IR and RPPH do initiate replication in synchrony.After watching this video, you should have a good understanding of how to determine the optimal chromosomal position of a given DNA element. This is done by transposon-based random insertions followed by competition experiments and finally whole genome sequencing.

Research

JoVE Journal

Genetics

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

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