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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Die Autoren wurden durch Zuschüsse aus der Novo Nordisk Stiftung, die Lundbeck-Stiftung und der dänischen National Research Foundation (DNRF120) durch das Zentrum für bakterielle Stressantwort und Persistenz (BASP) finanziert.

1. Sammlung der Bibliothek Transposon Hinweis: die chromosomale Locus DARS2 wurde in die Mini Tn 10 geklont-basierte Transposon, NKBOR (auf pNKBOR) 21, was zu NKBOR:: DARS2 (pJFM1). pNKBOR erhalten Sie Online- 22. pNKBOR ist ein R6K-basierte Selbstmord-Vektor, der Initiator Protein π für Replikation 23 erfordert. Plasmid pJFM1 ist daher in der Lage, in einer E. Coli-Stamm (z. B.<...

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Die hier verwendete Methodik nutzt State-of-the-Art Techniken, eine schwierige Frage in Bezug auf die optimale genomische Position eines genetischen Elements zu beantworten. Die zufällige Aufnahme des genetischen Elements (vermittelt durch das Transposon) ermöglicht die schnelle und einfache Sammlung von Tausenden von Klone, die dann vorgenommen werden kann, wählen für die optimale Position des untersuchten genetischen Elements gegeneinander antreten (d.h. der fitteste Klon).
Hier...

Durch seine Lage im Genom kann die Funktion eines genetischen Elements beeinträchtigt werden. In Bakterien ergibt sich somit vor allem vor Störungen durch die Transkription des benachbarten Gene, lokalen DNA Topologie und/oder Replikation-assoziierten Gen Dosierung. Insbesondere die Prozesse der DNA-Replikation und Segregation gesteuert werden, mindestens im Teil, durch nicht-kodierenden chromosomalen Regionen1und die ordnungsgemäße Funktion dieser Regionen hängt genomische Position/Kontext. In e. coli, Beispiele sind die Dif -Standort, erforderlich für Schwester Chromosom Auflösung2; KOPS-Sequenzen, erforderlich für Chromosom Abtrennung3; Daten, DARS1und DARS2 Regionen, für die ordnungsgemäße chromosomale Replikation Steuerung (unten; ( 4). präsentieren wir Ihnen eine Methode, für die zufällige Umzug, Auswahl und Bestimmung der optimalen genetischen Kontext eines bestimmten genetischen Elements, hier veranschaulicht durch das Studium der DARS2 nicht-kodierende Region.
In E. Coli, DnaA ist der Initiator Protein verantwortlich für DNA-Strang Eröffnung um einzelne Replikation HerkunftoriC, und für die Einstellung von der Helikase DnaB5,6,7. DnaA gehört zu den AAA+ (d. h. ATPasen verbunden mit vielfältigen Aktivitäten) Proteine und ATP und ADP zu binden mit ähnlich hohen Affinitäten5. Das Niveau der DnaAATP peaks bei Einleitung8, wo DnaAATP bildet ein Multimer auf oriC auslöst, die DNA duplex Öffnung9. Nach der Einleitung oriC erfolgt durch einen Mechanismus mit der Bindung des Proteins SeqA Hemimethylated vorübergehend nicht verfügbar für Wiederaufnahme wegen Sequestrierung oriC10,11. Während der Sequestrierung, sinkt das Niveau der DnaAATP durch mindestens zwei Mechanismen: die regulatorischen Inaktivierung der DnaA (RIDA)12,13 und Daten-abhängige DnaAATP Hydrolyse (Gefässwiderstand)14 ,15. RIDA und Gefässwiderstand fördern die Umwandlung von DnaAATP zuADPDnaA. Vor einer neuen Runde der Initiation, DnaAADP wird wieder aktiviert, DnaAATP bei bestimmten DnaA-Reaktivierung Sequenzen (DARS): DARS1 und DARS216,17. Die chromosomalen Daten, DARS1und DARS2 Regionen sind nicht-kodierende und Handeln in einem Chaperon-Manier zu modulieren, DnaAATP/DnaAADP fußenden. Diese Regionen außerhalb der Ursprung der Replikation, ermöglichen der Montage von einem komplexen DnaA für entweder die Inaktivierung (Daten, 14) oder Aktivierung (DARS1 und DARS2; 17) der DnaA. DARS2 in einer Zelle löschen, ändert nicht Masse Verdoppelung Zeit aber Ergebnisse asynchroner Replikation Einleitung15,16,18. Jedoch DARS2-mangelhafte Zellen haben eine Fitness Kosten im Vergleich zu einer ansonsten isogenen Wildtyp, während beide kontinuierliches Wachstum Wettbewerb im Reich Medium oder die Einrichtung der Besiedlung im Darm Maus18. Dies bedeutet, dass bereits geringe Veränderungen Asynchronie/Herkunft Konzentration wirkt sich negativ auf die bakterielle Fitness haben. In E. Coligibt es einen Selektionsdruck auf Chromosom Symmetrie (d. h. zwei fast gleich lang Replikation Arme)19pflegen. Daten, DARS1und DARS2 Regionen haben den gleichen relativen Abstand zu oriC in alle E. Coli Stämme18, trotz großen Schwankungen in der Größe von Chromosom sequenziert.
Hier verwenden wir die DARS2 Region von E. Coli als Beispiel für die Identifizierung von der chromosomalen Position(en) optimal für seine Funktion. DARS2 wurde in das Transposon NKBOR und die daraus resultierende NKBOR eingefügt::DARS2 Transposon anschließend nach dem Zufallsprinzip eingefügt in das Genom der MG1655 ΔDARS2. Wir erzeugt somit eine Ansammlung von Zellen, jede besitzen DARS2 platziert an einem anderen Ort auf dem Chromosom. Ein in-vitro- Wettbewerb Experiment, wo alle Zellen in der Auflistung wurden gebündelt und während des kontinuierlichen Wachstums in LB für eine geschätzte 700 Generationen gegeneinander angetreten, wurde durchgeführt. Das Ergebnis des Experiments Wettbewerb wurde überwacht/ermittelt mithilfe von südlicher Fleck, leicht gen zu Fuß und Sequenzierung des gesamten Genoms (WGS; ( Abbildung 1). Endpunkt-Klone zu Fuss leicht gen gelöst waren gekennzeichnet durch Durchflusszytometrie, Zellzyklus Parameter zu bewerten. In einer durchflusszytometrischen Analyse können die Zellengröße, DNA-Gehalt und Einleitung Synchronität für eine große Anzahl von Zellen gemessen werden. Während der Durchflusszytometrie, ein Strom von Einzelzellen übergibt einen Lichtstrahl der entsprechenden Wellenlänge die gefärbte DNA begeistern die dann gleichzeitig durch Photomultiplier, die sammeln die emittierte Fluoreszenz, ein gewisses Maß an DNA-Gehalt, registriert ist sofern die Zellen sind für DNA gefärbt. Das vorwärts-verstreut Licht ist ein Maß für die Zelle Masse20.
Die in-vitro- Wettbewerb Experiment, das wir hier vorstellen wird verwendet, um Fragen in Bezug auf die Bedeutung der chromosomalen Position und genomische Kontext des genetischen Elements. Die Methode ist neutral und einfach zu bedienen.

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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>
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			<td height="21" style="width:369px;height:21px;">Rifampicin</td>
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			<td height="21" style="width:369px;height:21px;">Mithramycin</td>
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			<td style="width:164px;">Apogee</td>
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		</tr>
	</tbody>
</table>

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

Die optimale chromosomalen Position(en) eines bestimmten DNA-Elements war/waren bestimmt durch Transposon-vermittelten zufällige Einblendung gefolgt von Fitness-Auswahl. In Bakterien kann die Auswirkungen der genetischen Kontext auf die Funktion eines genetischen Elements schwer abzuschätzen sein. Mehrere Mechanismen, einschließlich topologische Effekte, transkriptionelle Interferenz von benachbarten Gene und/oder Replikation-assoziierten Gen Dosierung, können die Funktion eines bestimmten genetischen Elements beeinflussen. Hier beschreiben wir eine Methode, die erlaubt die zufällige Integration eines DNA-Elements in das Chromosom von Escherichia coli und wählen Sie die günstigsten Standorten mit einem einfachen Wachstum Wettbewerb zu experimentieren. Die Methode nutzt eine gut beschriebene Transposon-basiertes System der zufällige Insertion, gepaart mit einer Auswahl an der stärkeren Clone(s) von Wachstum nutzen, ein Verfahren, das auf experimentelle Bedürfnisse leicht einstellbar ist. Die Art der fitteste Clone(s) kann durch ganze Genomsequenzierung auf eine komplexe Multi-klonale Population oder einfach zu Fuß für die schnelle Identifizierung von ausgewählten Klonen gen bestimmt werden. Hier war die nicht-kodierenden DNA-Region DARS2, die die Initiation der Replikation der Chromosomen in E. Colikontrolliert, als Beispiel verwendet. Die Funktion des DARS2 ist bekannt durch Replikation-assoziierten Gen Dosierung beeinträchtigt werden; der engere DARS2 ruft nach der Herkunft der DNA-Replikation, desto aktiver wird es. DARS2 wurde nach dem Zufallsprinzip in das Chromosom ein DARS2eingefügt-Belastung gelöscht. Die daraus resultierende Klone mit einzelnen Einfügungen wurden gebündelt und für Hunderte von Generationen gegeneinander konkurrierten. Schließlich wurden die fittesten Klone gekennzeichnet und festgestellt, dass DARS2 eingefügt in unmittelbarer Nähe zu den Originalschauplätzen der DARS2 enthalten.

Ein südlicher Fleck wurde getan, um sicherzustellen, dass DARS2 das Chromosom in der Transposon-Bibliothek nach dem Zufallsprinzip verteilt wurde (t = 0) und, die die stärksten Klonen würde im Laufe der Zeit beibehalten. Der Southern Blot erfolgte auf DNA extrahiert aus dem anfänglichen Transposon-Pool (bei t = 0) und alle geschätzten 100 aus 700 Generationen des Wettbewerbs (Abbildung 3). Hier war die totale zelluläre DNA aus jeden Zeitpunkt v...

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

Hier war die Macht der ein Transposon-vermittelten zufällige einführen eines nicht-kodierende DNA-Elements verwendet, um seine optimale chromosomalen Position zu lösen.

Bestimmung der optimalen chromosomalen Speicherorte für eine DNA-Element in Escherichia coli mit einem neuartigen Transposon-vermittelte Ansatz

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.

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Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

Verwendung von gefrorenem Gewebe im Kometentest zur Bewertung von DNA-Schäden

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...