Name: procedure for adaptive laboratory evolution of microorganisms using a chemostat
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat at JoVE.com

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. Preparación del equipo <ol><li> Obtener un frasco quimiostato (150-250 ml) o un matraz Erlenmeyer (250 ml) que contiene un orificio de entrada y un puerto de salida. Conectar los puertos con tubo de silicona que permite velocidades de flujo de 10 a 100 ml / hr. Opcionalmente, utilizar una salida de aire, un puerto de salida de aire, y puertos de entrada y salida de agua de temperatura controlada. </li><li> Obtener un dispositivo adecuado para el frasco chemostat que proporciona para el control de la agitaci...

<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>

Los microorganismos son capaces de adaptarse a casi todos los entornos debido a su rápida tasa de crecimiento y la diversidad genética. la evolución adaptativa de laboratorio permite a los microorganismos a evolucionar en condiciones diseñadas, lo que proporciona una manera de seleccionar los organismos individuales que albergan mutaciones espontáneas que son beneficiosos en las condiciones dadas. La técnica quimiostato es más robusta para lograr la evolución impulsada artificialment...

Los microorganismos pueden sobrevivir y adaptarse a diversos ambientes. En situaciones de estrés severo, la adaptación puede ocurrir a través de la adquisición de fenotipos beneficiosos por mutaciones genómicas al azar y la selección positiva posterior 1-3. Por lo tanto, las células microbianas se pueden adaptar cambiando metabólica o las redes de regulación para un crecimiento óptimo, que se denomina &quot;evolución adaptativa&quot;. microbianas tendencias recientes importantes, tales como brotes de superbacterias y la aparición de cepas microbianas resistentes, están muy estrechamente relacionados con la evolución adaptativa en condiciones de estrés. En condiciones de laboratorio definidos, somos capaces de estudiar los mecanismos de la evolución molecular e incluso controlar la dirección de la evolución microbiana para diversas aplicaciones. A diferencia de los organismos multicelulares, los organismos unicelulares son muy adecuadas para la evolución adaptativa de laboratorio (ALE) por las siguientes razones: se regeneran rápidamente, mantienen grandes poblaciones, y es fácil de crear y mantener homogeneous entornos. En combinación con los recientes avances en las técnicas de secuenciación de ADN y las tecnologías de alto rendimiento, ALE permite la observación directa de los cambios genómicos que conducen a cambios en la regulación sistémicos. dinámica mutacional y una diversidad de la población también son observables. Estrategias de ingeniería genética se pueden determinar a partir del análisis de las cepas ALE 4,5. Cultura quimiostato es un método utilizado para obtener células en estado de equilibrio y aumentar la productividad en los procesos de fermentación de 6. se añade medio fresco y el caldo de cultivo se recoge durante el proceso (el último incluye medio y biomasa). Cultura quimiostato a largo plazo, sin embargo, cambia la productividad de estado estacionario de la cultura y provoca la acumulación de mutaciones espontáneas y la selección durante el cultivo (Figura 1a). Bajo diferentes presiones de selección (estrés), la acumulación de mutaciones es mayor. Un aumento gradual de la tensión en un largo plazo<html> chemostat prevé una selección continua de las mutaciones que trabajan contra los factores de estrés dadas, tales como temperatura, pH, presión osmótica, el hambre de nutrientes, la oxidación, los productos finales tóxicos, etc. transferencia de colonias a partir de un medio sólido y la transferencia en serie de un medio líquido (repetido cultivo por lotes) también permiten a los investigadores obtener microorganismos evolucionados (Figura 1B y 1C). Aunque la cultura chemostat requiere métodos complicados, la piscina de la diversidad (número de repeticiones y tamaño de la población) es mayor que la obtenida por transferencia de colonias y las técnicas de transferencia en serie. La exposición al estrés estable a las células individuales y la disminución de la variación en el estado celular durante el cultivo en quimiostato (estado estacionario) son otros beneficios de ALE comparación con las técnicas basadas en el cultivo por lotes. ALE inducida por estrés de Escherichia coli sometidos a altas condiciones de succinato se introduce en este artículo. iles / ftp_upload / 54446 / 54446fig1.jpg &quot;/&gt; Figura 1: Métodos de laboratorio de la evolución adaptativa (A) Chemostat;. (B) la transferencia en serie; (C) la transferencia de la colonia. Las máximas figuras ilustran el concepto de los métodos para ALE, y las cifras de abajo ilustran el número de células que crecieron durante la ALE. <a href="https://www.jove.com/files/ftp_upload/54446/54446fig1large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta 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 la adaptación de alta tensión succinato, la de tipo salvaje E. coli cepa W3110 se cultivó en un quimiostato a D = 0,1 h-1 por 270 días (Figura 2). <img alt="Figura 2" src="/files/ftp_upload/54446/54446fig2.jpg" /> Figura 2: High-succinato de adaptación al estrés de E. coli W3110 uso de la cultura quimiostato. flechas delgadas indican los momentos en que se aumentó la concentració...

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

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

A continuación, se presenta un protocolo para obtener la evolución adaptativa de laboratorio de microorganismos bajo condiciones de cultivo utilizando quimiostato. Además, se discute el análisis genómico de la cepa evolucionado.

Procedimiento para Adaptive Laboratory Evolución de microorganismos utilizando un quimiostato

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

The overall goal of this procedure is to enable a microorganism to evolve in laboratory conditions. This method can help answer key questions in the microbiology field, such as adrenal function relationships, stress responses, metabolic engineering, and of course, evolution. The main advantage of this technique is continuous selection of the most suaded descendant under specific laboratory conditions.Begin this procedure with the preparation of equipment, as well as initial medium, stress medium and high stress medium, as described in the text protocol. Inoculate a single colony or wild type E.coli in a 15-milliliter test tube containing four milliliters of initial medium. Incubate the test tube in a shaking incubator for 12 hours at 37 degrees celsius and 220 rpm.Incubate the chemostat jar providing for aeration and agitation at 37 degrees celsius for six hours. Following the incubation, aseptically connect the end of the silicon tubing from the pumps to the chemostat jar. Aseptically transfer one milliliter of pre-culture to the chemostat jar.Start the outlet pump and collect the culture in the exponential phase. Check the optical density at 600 nanometers of the culture from the outlet tubing. Then, start the inlet pump.Check the optical density of the culture at 600 nanometers from the outlet tubing every 24 hours. After operating the chemostat for 96 hours, which is a 9.6 full turnover, exchange the reservoir to the lowest concentration of high stress medium. When you exchange the reservoir to a higher stress medium, the cells may be shocked.If the cell density is too low, stop feeding the higher stress medium for some time to restore the cell density. If the optical density is lower than 0.2, stop the feeding inlet pump for six hours. Restart the inlet pump and check that the optical density is over 0.2.Gradually increase the concentration of the stressor by incrementally changing to a reservoir containing a higher stressor concentration. Take samples of the adapted culture whenever it reaches a stressor adaptation milestone and store for further genomic analysis. For sample storage, mix the 0.5 milliliter culture sample with 0.5 milliliters of a sterilized 80%glycerol solution and store it at 80 degrees celsius.Prepare 1.6%ager plate medium containing the same stressor at the same concentration as in the medium. Plate 0.1 milliliters of the outlet culture from the chemostat and incubate at 37 degrees celsius for 16 hours. Following incubation, pick single colonies from the plate using a sterile toothpick and inoculate them in 15 milliliter test tubes containing the same stressor and at the same medium concentration as in the chemostat.Incubate for six hours. Transfer one milliliter of the culture broth into a 250 milliliter erlenmeyer flask containing 50 milliliters of medium. Harvest 0.5 milliliters of the culture broth every hour and measure the optical density at 600 nanometers.Compare the growth rate of the adapted strain to that of the wild type strain given the stressor. The wild type E.coli strain was evolved in a high succinate stressed condition for 270 days, which corresponds to nearly 930 generations. Whenever higher succinate stress was added, the biomass concentration was immediately lowered and then restored.Shown here is a schematic diagram of the chemostat with a mutant that is tolerant against the high succinate stress. Most wild type cells are washed out under the stress conditions and the mutant grows more. Eventually, the population of the mutant descendant is increased.The second and third mutations are continuously selected for and eventually dominate the chemostat. This figure shows that the adapted strain grew without delay under high succinate stressed conditions, while the wild type strain did not. A similar strategy can be applied to adaptive laboratory evolution using a chemostat with a variation of stressors.After watching this video, you should have a good understanding of how to acquire and evolve the microorganism under a specified condition. Once mastered, this technique can be done in a couple of month. While attempting this procedure, it's important to remember not to wash out chemostat cells by adding too much stress at once and remember to check the optical density every day.Following this procedure are the methods like a genome analysis and transcriptome analysis can be performed in order to answer additional questions like what mutation contributes to the stress tolerant evolution?

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