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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Os autores foram financiados por subsídios da Fundação Novo Nordisk, a Fundação Lundbeck e o dinamarquês National Research Foundation (DNRF120) através do centro para resposta ao estresse bacteriana e persistência (BASP).

1. coleção da biblioteca do Transposon Nota: O locus DARS2 cromossômico foi clonado para o Tn mini 10-baseado transposon, NKBOR (na pNKBOR) 21, resultando em NKBOR:: DARS2 (pJFM1). pNKBOR pode ser obtido on-line de 22. pNKBOR é um vetor de suicídio baseado em R6K que requer a π de proteínas do iniciador para replicação 23. Plasmídeo pJFM1, portanto, é capaz de replicar em uma cepa d...

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A metodologia utilizada aqui aproveita-se de técnicas de estado-da-arte para responder a uma pergunta difícil em relação a posição ideal de genômica de um elemento genético. A inserção aleatória do elemento genético (mediada pelo transposon) possibilita a coleta fácil e rápida de milhares de clones, que então podem ser feitas para competir uns contra os outros para selecionar para a posição ideal do elemento genético investigada (ou seja, o clone mais apto).
Aqui, o ...

A função de qualquer elemento genético pode ser afetada pela sua localização no genoma. Em bactérias, isto principalmente resulta da interferência pela transcrição dos genes vizinhos, topologia de DNA local, e/ou dosagem de gene associada a replicação. Em particular, os processos de replicação de DNA e segregação são controlados, pelo menos em parte, pelas regiões cromossômicas não-codificantes1e o funcionamento destas regiões depende de localização/contexto genômico. Em e. coli, exemplos são o site dif , necessário para a irmã de resolução do cromossomo2; Sequências KOPS, necessárias para segregação de cromossomo3; e dados, DARS1e DARS2 regiões, necessárias para o controle adequado de replicação cromossômica (abaixo; 4). apresentamos um método permitindo a realocação aleatória, seleção e determinação do contexto genético ideal de qualquer elemento genético determinado, exemplificado aqui o estudo da região não-codificante DARS2 .
Em e. coli, DnaA é a proteína de iniciador responsável pela cadeia de DNA, abrindo a única replicação origemoriquee para o recrutamento do helicase DnaB5,6,7. DnaA pertence a AAA+ (i.e., ATPase associados com diversas atividades) proteínas e pode vincular tanto ATP e ADP com alta semelhante afinidades5. O nível de DnaAATP picos no início8, onde DnaAATP forma um multimer na orique que aciona o DNA abertura frente e verso9. Após a iniciação, orique feita temporariamente indisponível para re-iniciação devido ao sequestro por um mecanismo que envolve a ligação da proteína SeqA a hemimethylated orique10,11. Durante o sequestro, o nível de DnaAATP é reduzido pelo menos dois mecanismos: a inactivação regulamentar de DnaA (RIDA)12,13 e dados-dependente DnaAATP hidrólise (DDAH)14 ,15. Tanto RIDA e DDAH promovem a conversão de DnaAATP DnaAADP. Antes de uma nova rodada de iniciação, DnaAADP é re-ativado para DnaAATP em sequências específicas de DnaA-reativando (DARS): DARS1 e DARS216,17. Os cromossomas dados, DARS1, regiõese DARS2 são não-codificantes e agir em uma maneira semelhante ao acompanhante para modular DnaAATP/DnaAADP interconversão. Nestas regiões, localizadas fora a origem de replicação, permitem a montagem de um complexo de DnaA para qualquer a inactivação (dados; 14) ou ativação (DARS1 e DARS2; 17) de DnaA. Exclusão de DARS2 em uma célula não altera massa dobrando tempo mas resultados em replicação assíncrona iniciação15,16,18. No entanto, DARS2-células deficientes têm uma aptidão custo comparado a uma sua outra forma isogénicas durante ambas as competição de contínuo crescimento em meio rico, ou durante o estabelecimento da colonização do intestino de rato18. Isso indica que mesmo pequenas alterações na concentração de assincronia/origem tem um efeito negativo na aptidão bacteriana. Em e. coli, há uma pressão seletiva para manter a simetria (ou seja, dois braços de replicação de comprimento quase igual) de cromossomo19. Os dados, DARS1e DARS2 regiões têm a mesma distância relativa à orique em todas as e. coli estirpes sequenciadas18, apesar das grandes variações no tamanho do cromossoma.
Aqui, usamos a região de DARS2 de e. coli como exemplo para a identificação da posição cromossômica ideal para sua função. DARS2 foi inserido o transposon NKBOR e o resultante NKBOR::DARS2 transposon posteriormente inserido aleatoriamente no genoma de MG1655 ΔDARS2. Assim, geramos uma coleção de células, cada possuidor DARS2 colocados em um local diferente no cromossomo. Em vitro concorrência experimento, onde todas as células na coleção foram agrupadas e competiam entre si durante o crescimento contínuo em LB para um número estimado de 700 gerações, foi realizado. O resultado da experiência de competição foi monitorado/determinado usando o Southern blot, gene fácil caminhar e sequenciamento do genoma inteiro (WGS; A Figura 1). Clones de ponto de extremidade, resolvidos por gene fácil andar foram caracterizados por citometria de fluxo para avaliar parâmetros do ciclo celular. Em uma análise de fluxo cytometric, tamanho da célula, conteúdo de DNA e sincronia de iniciação podem ser medidos por um grande número de células. Durante citometria de fluxo, um fluxo de células únicas passa um feixe de luz de comprimento de onda apropriado para excitar o DNA manchado, então simultaneamente registado por photomultipliers que coletam a fluorescência emitida, uma medida do conteúdo de DNA, desde o as células são coradas para DNA. A luz difusa é uma medida da massa de célula20.
O experimento concorrência em vitro que apresentamos aqui é usado para abordar questões relacionadas com a importância da posição cromossômica e contexto genômico do elemento genético. O método é imparcial e fácil de usar.

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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>
			<td style="width:164px;">Fisher Scientific</td>
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		<tr height="21">
			<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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		</tr>
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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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		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Rifampicin</td>
			<td style="width:164px;">Serva</td>
			<td style="width:119px;">34514.01</td>
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		<tr height="21">
			<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 style="width:119px;">246964</td>
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			<td height="21" style="width:369px;height:21px;">Apogee A10 instrument</td>
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</table>

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

O ideal posição cromossômica (ões) de um determinado elemento de DNA foi/foram determinada por inserção aleatória mediada por transposon seguida pela seleção de aptidão. Em bactérias, o impacto do contexto genético na função de um elemento genético pode ser difícil de avaliar. Vários mecanismos, incluindo efeitos topológicos, interferência transcricional de genes vizinhos e/ou dosagem de gene associada a replicação, podem afetar a função de um determinado elemento genético. Aqui, descrevemos um método que permite a integração aleatória de um elemento do DNA no cromossomo da Escherichia coli e selecione os locais mais favoráveis, usando uma competição simples crescimento experimentar. O método aproveita-se de um sistema baseado em transposon bem-descrito de inserção aleatória, juntamente com uma seleção de clone(s) os mais aptos, pela vantagem de crescimento, um procedimento que é facilmente ajustável às necessidades experimentais. A natureza da clone(s) mais apto pode ser determinada pelo sequenciamento do inteiro-genoma sobre uma população clonal multi complexo ou pelo gene fácil caminhar para a rápida identificação de clones selecionados. Aqui, a região de DNA não-codificante DARS2, que controla a iniciação da replicação do cromossomo em e. coli, foi usada como um exemplo. A função de DARS2 é conhecida por ser afetado pela dosagem de gene associada a replicação; o mais perto DARS2 Obtém-se a origem da replicação do DNA, torna-se mais ativo. DARS2 foi inserido aleatoriamente o cromossomo de um DARS2-excluído estirpe. Os clones resultantes contendo inserções individuais foram agrupados e competiu contra o outro por centenas de gerações. Finalmente, os clones mais aptos foram caracterizados e encontrados para conter o DARS2 inserido em estreita proximidade com o local original de DARS2 .

Um borrão do Sul foi feito para verificar o que DARS2 foi distribuídas aleatoriamente o cromossomo na biblioteca transposon (t = 0) e que os clones mais aptos que persistem ao longo do tempo. O borrão do Sul foi realizado no DNA extraído do pool do transposon inicial (em t = 0) e cada 100 estimado de 700 gerações de concorrência (Figura 3). Aqui, o DNA celular total de cada time ponto foi digerido com o Pvuenzima de restrição, conhe...

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

Aqui, o poder de uma inserção aleatória de um elemento de DNA não-codificante, mediada por transposon foi usado para resolver a sua posição ideal cromossômica.

Determinação da localização cromossômica ideal para um elemento de DNA em Escherichia coli usando uma nova abordagem mediada por Transposon

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

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