This study was financially supported by the Korean Ministry of Science, ICT and Future Planning (Intelligent Synthetic Biology Center program 2012M3A6A8054887). P. Kim was supported by a fellowship from the Catholic University of Korea (2015).

1. Preparação Equipamento <ol><li> Obter um frasco quimiostato (150-250 ml) ou num balão de Erlenmeyer (250 ml) contendo uma abertura de entrada e uma porta de saída. Ligue as portas com tubo de silicone que permite taxas de fluxo de 10-100 ml / hr. Opcionalmente, usar um respiradouro de ar, uma porta de saída de ar, e portas de entrada e saída de água de temperatura controlada. </li><li> Obter um dispositivo adequado para o frasco quimiostato que fornece para agitação e controlo da temperatura (ou usar um incubador com agitação rotativa). </li><li> Obter duas bombas peristálticas de modo a fornecer meio fresco e recolher a cultura. </li><li> Obter um frasco reservatório (10-20 L) contendo um orifício de saída médio e uma porta de entrada de ar. </li><li> Obter tubo de silicone adequado para a taxa de diluição (isto é, identificação de 0,8 milímetros, a gama de fluxo 0,06-36 mL / min; L / S 13 tubos). </li></ol> 2. Médio Preparação e Esterilização <ol><li> meio inicial <ol><li> Dissolve-se 0,3 g de glucose, 0,08 g de NH 4Cl, 0,05 g de NaCl, 0,75 g de Na 2 HPO 4 · 2H 2 O, e 0,3 g de KH 2 PO 4 em 90 ml de água destilada (DW) em um frasco de quimiostato. </li><li> Selar o frasco quemostato junto com a tubulação usando grampos. Não selar a abertura de ar. </li><li> Esteriliza-se o frasco quimiostato numa autoclave a 121 ° C durante 15 min. Após a esterilização, armazenar o frasco quimiostato à temperatura ambiente. </li><li> Dissolve-se 0,02 g de MgSO 4 · 7H 2 O, 0,01 g de CaCl2, e 0,1 mg de tiamina em 10 ml de DW (solução A). </li><li> Filtrar a solução A, utilizando uma seringa e um filtro de seringa pré-esterilizados (um filtro de 0,45 um de poro). </li><li> Adicionar a solução A filtrados para o frasco quemostato. </li></ol></li><li> Média Tensão <ol><li> Dissolve-se 30 g de glucose, 8 g de NH 4 Cl, 5 g de NaCl, 75 g de Na 2 HPO 4 · 2H 2 O, 30 g de KH 2 PO 4, e 300 g de succinato dissódico hexa-hidratado (Na 2 · · succinato 6H &lt;sub&gt; 2 O; o estressor utilizado neste experimento) em 9,9 L DW em um frasco reservatório. </li><li> Selar o frasco reservatório junto com a tubulação usando grampos. Não selar a abertura de ar. </li><li> Esteriliza-se o frasco reservatório em autoclave a 121 ° C durante 15 min. Após a esterilização, armazenar o frasco à temperatura ambiente. </li><li> Dissolve-se 2 g de MgSO 4 7H 2 O ·, 1 g de CaCl2, e 10 mg de tiamina em 100 ml de DW (solução A). </li><li> Filtrar a solução A com uma seringa e um filtro de seringa pré-esterilizados (um filtro de 0,45 um de poro). </li><li> Adicionar a solução A filtrados para o frasco reservatório. </li><li> Assepticamente conectar o tubo de silicone esterilizados para o frasco reservatório e anexar as bombas peristálticas. </li></ol></li><li> Médio-Alto estresse <ol><li> Prepare o meio como na secção 2.2, mas com uma maior concentração de estressor (ou seja, 3-5 g / L maior na adaptação succinato). Nota: Este protocolo é para adaptação a um estresse thno pode ser entregue através do meio. No caso de factores de stress físicos, tais como a temperatura, agitação, ou iluminação, o cultivo deve ser concebido em conformidade. </li></ol></li></ol> 3. cultivo inicial <ol><li> Inocular uma única colônia de tipo selvagem E. coli em um tubo de ensaio de 15 mL contendo 4 mL de meio inicial. </li><li> Incubar o tubo de ensaio num incubador com agitação durante 12 h a 37 ° C e 220 rpm. </li><li> Transfira assepticamente 1 ml de pré-cultura para o frasco quemostato. </li><li> Incubar o frasco quimiostato, fornecendo para arejamento (ar de 50 ml / min) e agitação (200 rpm), a 37 ° C durante 6 h. </li></ol> Adaptação 4. Estresse <ol><li> Assepticamente ligue a extremidade do tubo de silicone das bombas para o frasco quemostato. </li><li> Inicie a bomba de saída (10 ml / h ou superior) e recolher a cultura. Nota: A cultura deve estar na fase exponencial, tipicamente 4-8 horas após o cultivo inicial. </li><li> CHeck a densidade óptica (600 nm) da cultura a partir do tubo de saída. </li><li> Iniciar a bomba de entrada (10 ml / h, correspondendo a uma taxa de diluição de 0,1 hr -1). </li><li> Verifique a densidade óptica da cultura a 600 nm da tomada da tubagem a cada 24 h. </li><li> Operar o quimiostato durante 96 horas (volume 9,6 vezes) ou mais. Se a densidade óptica é estável, trocar o reservatório que contém o meio de alta tensão. Se a densidade óptica é inferior a 0,2, parar a bomba de entrada alimenta durante 6 h. Reinicie a bomba de entrada e verifique se a densidade óptica é superior a 0,2. </li><li> Aumentar gradualmente a concentração do estressor alterando a um reservatório que contém uma concentração mais elevada estressor. </li><li> Retirar amostras da cultura adaptada quando ela atinge um marco (por exemplo, uma estirpe adaptada a 100 g / L de stress succinato), e armazenamento para posterior análise genómica. </li><li> Para o armazenamento da amostra, misturar a amostra de cultura (0,5 ml) com um soluti esterilizado glicerol 80%na (0,5 ml) e armazená-lo a -80 ° C. Nota: Se o microorganismo adquire a capacidade de degradar o estressor durante o processo de ALE, a concentração estressor no frasco de fermentação não é a mesma que no reservatório fresco. </li></ol> 5.-colônia Único isolamento da estirpe adaptada-Stress <ol><li> Preparar o meio da placa de agar (1,6% de agar) contendo a mesma estressor e na mesma concentração do meio. </li><li> Placa da cultura de saída (0,1 ml) a partir do quimiostato, e incuba-se a 37 ° C durante 16 horas. </li><li> Escolha colónias isoladas da placa utilizando um palito esterilizado e inocular-los em 15 ml de tubos de ensaio que contêm o mesmo estressor e na mesma concentração forma como no quimiostato, e incuba-se durante 6 h. </li><li> Transferir 1 ml do caldo de cultura num balão de Erlenmeyer de 250 mL contendo 50 mL de meio. Colheita de 0,5 ml do caldo de cultura a cada um de RH, e medir a DO a 600 nm. Compare a taxa de crescimento do strai adaptadaN ao da estirpe de tipo selvagem dado o estressor. </li></ol>

<ol>
	<li>Rando, O. J., Verstrepen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timescales+of+genetic+and+epigenetic+inheritance.">Timescales of genetic and epigenetic inheritance.</a> Cell. 128, 655-668 (2007).</li><li>Kim, H. 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=Short-term+differential+adaptation+to+anaerobic+stress+via+genomic+mutations+by+Escherichia+coli+strains+K-12+and+B+lacking+alcohol+dehydrogenase.">Short-term differential adaptation to anaerobic stress via genomic mutations by Escherichia coli strains K-12 and B lacking alcohol dehydrogenase.</a> Front Microbiol. 5, 476 (2014).</li><li>Mendizabal, I., Keller, T. E., Zeng, J., Yi, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+and+evolution.">Epigenetics and evolution.</a> Integr Comp Biol. 54, 31-42 (2014).</li><li>Lee, J. Y., Seo, J., Kim, E. S., Lee, H. S., 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=Adaptive+evolution+of+Corynebacterium+glutamicum+resistant+to+oxidative+stress+and+its+global+gene+expression+profiling.">Adaptive evolution of Corynebacterium glutamicum resistant to oxidative stress and its global gene expression profiling.</a> Biotechnol Lett. 35, 709-717 (2013).</li><li>Lee, J. Y., 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=Artificial+oxidative+stress-tolerant+Corynebacterium+glutamicum.">Artificial oxidative stress-tolerant Corynebacterium glutamicum.</a> AMB Express. 4, 15 (2014).</li><li>Narang, 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+steady+states+of+microbial+growth+on+mixtures+of+substitutable+substrates+in+a+chemostat.">The steady states of microbial growth on mixtures of substitutable substrates in a chemostat.</a> J Theor Biol. 190, 241-261 (1998).</li><li>Kwon, Y. D., Kim, S., Lee, S. Y., 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=Long-term+continuous+adaptation+of+Escherichia+coli+to+high+succinate+stress+and+transcriptome+analysis+of+the+tolerant+strain.">Long-term continuous adaptation of Escherichia coli to high succinate stress and transcriptome analysis of the tolerant strain.</a> J Biosci Bioeng. 111, 26-30 (2011).</li><li>Barrick, J. E., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+dynamics+during+experimental+evolution.">Genome dynamics during experimental evolution.</a> Nat Rev Genet. 14, 827-839 (2013).</li><li>Li, 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=The+Sequence+Alignment/Map+format+and+SAMtools.">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li><li>McKenna, A., 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+Genome+Analysis+Toolkit:+a+MapReduce+framework+for+analyzing+next-generation+DNA+sequencing+data.">The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.</a> Genome Res. 20, 1297-1303 (2010).</li><li>Deatherage, D. E., Barrick, 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=Identification+of+mutations+in+laboratory-evolved+microbes+from+next-generation+sequencing+data+using+breseq.">Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq.</a> Methods Mol Biol. 1151, 165-188 (2014).</li></ol>

The authors have nothing to disclose.

Os microrganismos são capazes de se adaptar a quase todos os ambientes, devido à sua rápida taxa de crescimento e diversidade genética. evolução laboratório adaptável permite microorganismos a evoluir em condições destinadas, que fornece uma maneira de selecionar organismos individuais que abrigam mutações espontâneas que são benéficos nas condições dadas. A técnica quimiostato é mais robusto para a realização evolução de forma artificial do que as técnicas de transfe...

Os microrganismos podem sobreviver e se adaptar a diversos ambientes. Sob forte estresse, a adaptação pode ocorrer por meio de aquisição de fenótipos benéficos por mutações genómicas aleatórias e posterior selecção positiva 1-3. Portanto, as células microbianas podem se adaptar mudando metabólica ou redes de regulação para o crescimento ideal, que é chamado de &quot;evolução adaptativa&quot;. tendências microbianas importantes recentes, como surtos de superbactérias e a ocorrência de cepas microbianas robustos, estão intimamente relacionadas com adaptativa evolução sob condições estressantes. Em condições de laboratório definidos, somos capazes de estudar os mecanismos de evolução molecular e até mesmo controlar a direção da evolução microbiana para várias aplicações. Ao contrário de organismos multicelulares, organismos unicelulares estão bem adaptados à evolução laboratório adaptativo (ALE), pelas seguintes razões: eles regeneram rapidamente, eles manter grandes populações, e é fácil de criar e manter homambientes ogeneous. Combinado com os recentes avanços em técnicas de sequenciação de ADN e tecnologias de alto rendimento, ALE permite a observação direta de alterações genômicas que levam a mudanças regulatórias sistémicos. dinâmica de mutação e uma diversidade da população também são observáveis. Estratégias de engenharia genética pode ser determinado a partir da análise de estirpes de AEA 4,5. Cultura em quimiostato é um método usado para obter as células em estado estacionário e aumentar a produtividade em processos de fermentação 6. Adiciona- se meio fresco e caldo de cultura é colhida durante o processo (este último inclui médio e de biomassa). Quimiostato cultura de longo prazo, no entanto, muda a produtividade de estado estacionário da cultura e provoca a acumulação de mutações espontâneas e selecção durante a cultura (Figura 1a). Sob várias pressões de seleção (estressores), o acúmulo de mutações é reforçada. Um aumento gradual de estresse em um longo prazo<html> quimiostato prevê uma selecção contínua de mutações que trabalham contra os factores de stress dadas, tais como temperatura, pH, pressão osmótica, inanição nutriente, oxidação, produtos finais tóxicos, etc. Colony transferência a partir de um meio sólido e de transferência em série de um meio líquido (repetido cultura lote) também permitirá aos investigadores obter microorganismos evoluídos (Figura 1B e 1C). Embora a cultura em quimiostato requer métodos complicados, a piscina de diversidade (número de repetições, e o tamanho da população) é maior do que a obtida por técnicas de transferência em série de transferência de colónia e. A exposição ao estresse estável para células individuais e diminuição da variação no estado celular durante a cultura em quimiostato (estado estacionário) são outros benefícios do ALE em comparação com técnicas baseadas em culturas de diluição. ALE induzida pelo estresse de Escherichia coli submetida a altas condições succinato é introduzido neste artigo. iles / ftp_upload / 54446 / 54446fig1.jpg &quot;/&gt; Figura 1: Métodos de laboratório evolução adaptativa (A) quimiostato;. (B) a transferência de série; (C) a transferência de colónia. As figuras de topo ilustrar o conceito dos métodos para ALE, e as figuras de fundo ilustrar o número de células que cresceram durante a ALE. <a href="https://www.jove.com/files/ftp_upload/54446/54446fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mini-chemostat fermentor</td>
			<td style="width:71px;">Biotron Inc.</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">manufactured by special order</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">silicon tubing</td>
			<td style="width:71px;">Cole-Parmer</td>
			<td style="width:119px;">Masterflex L/S 13</td>
			<td>tubing size can be varied depending on the dilution rate and the size of fermentor jar.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">reservoir jar</td>
			<td style="width:71px;">Bellco</td>
			<td style="width:119px;">Media storage bottle&#160;</td>
			<td>20 L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">chemicals</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">-</td>
			<td>reagent grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glucose</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5767</td>
			<td>ACS reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NH4Cl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9434</td>
			<td>for molecular biology, suitable for cell culture, &#8805;99.5%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">746398</td>
			<td>ACS reagent, &#8805;99%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Na2HPO4&#183;2H2O</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">4272</td>
			<td>98.5-101%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">KH2PO4&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">795488</td>
			<td>ACS reagent, &#8805;99%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MgSO4&#183;7H2O</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">230391</td>
			<td>ACS reagent, &#8805;98%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CaCl2</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">793639</td>
			<td>ACS reagent, &#8805;96%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">thiamine&#183;HCl&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4625</td>
			<td>reagent grade, &#8805;99%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Na2&#183;succinate&#183;6H2O</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2378</td>
			<td>ReagentPlus, &#8805;99%</td>
		</tr>
	</tbody>
</table>

procedure for adaptive laboratory evolution of microorganisms using a chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Para adaptação ao estresse de alta succinato, o tipo selvagem E. coli W3110 estirpe foi cultivada num quimiostato em D = 0,1 h -1 durante 270 dias (Figura 2). <img alt="Figura 2" src="/files/ftp_upload/54446/54446fig2.jpg" /> Figura 2: adaptação ao estresse de alta succinato de E. coli W3110 usando cultura quemostato. setas finas indicam os tempos em que a concentração de estressor foi ...

Watch this Scientific Journal Video about Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat at JoVE.com

Procedimento para evolução laboratorial adaptativa de microrganismos usando um quimiostato | Genética | Vídeo

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Aqui, apresentamos um protocolo para obter a evolução adaptativa laboratório de microrganismos em condições usando cultura quemostato. Além disso, é discutida análise genômica da estirpe evoluiu.

Processo para o Adaptive Laboratório Evolução de microrganismos utilizando um quimiostato

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

procedure-for-adaptive-laboratory-evolution-microorganisms-using

Research

Education

JoVE Journal

JoVE Core

Biology

Genetics

Evolution

Evolutionary History

SCIENTISTS IN ACTION

14.1K visualizações.  Korea Research Institute of Bioscience and Biotechnology (KRIBB).  Aqui, apresentamos um protocolo para obter a evolução adaptativa laboratório de microrganismos em condições usando cultura quemostato. Além disso, é discutida análise genômica da estirpe evoluiu. 

Vídeo: Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

Concepção e utilização de um baixo custo, Automated Morbidostat para Adaptive Evolução das bactérias sob Antibiótico Seleção de Drogas

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

Genética

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

Integração de alvo CRISPR/Cas9-mediada na Vivo usando uma estratégia de baseada em juntar-se mediada por homologia final

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

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

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Testando o papel de plasmídeos multicópia na evolução da resistência aos antibióticos

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

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Vigilância de todo o genoma de erros de transcrição em organismos eucarióticos

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Comparação de alta resolução de frequências de conjugação bacteriana

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

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

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 for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

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

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

Produzindo a deleção do Gene na Escherichia coli por transdução P1 com fitas de resistência aos antibióticos sujeitos

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 ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

qKAT: quantitativa coeficiente digitação de assassino-célula Genes de receptores de imunoglobulina-como

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.

Here, we present a protocol to optimize CRISPR-Cas9 to achieve a higher specificity without the loss of on-target activity. We use a directed evolution approach called Sniper-screen to find a mutant Cas9 with the desired characteristics. Sniper-Cas9 is compatible with truncated single-guide RNAs and delivery in a ribonucleoprotein format, well-known strategies for achieving higher specificities.

Usando o Sniper-Cas9 para minimizar efeitos fora do alvo de CRISPR-Cas9 sem perda de atividade no alvo através de evolução dirigida

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion.

Um protocolo baseado em FACS para isolar o RNA das células secundárias de glândulas acessórias masculinas de Drosophila

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.

Here, we describe a genome-editing tool based on the temporal and conditional stabilization of clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) under the small molecule, Shield-1. The method can be used for cultured cells and animal models.

Um novo kit de ferramentas para avaliar funções genéticas usando estabilização condicional cas9

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory ...