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. 장비 준비 <ol><li> chemostat 항아리 (150-250 ml)로 또는 유입구를 함유하는 삼각 플라스크 (250 mL) 및 출구 포트를 얻었다. 실리콘 튜브는 10 ~ 100 ㎖ / hr의 유량을 허용하여 포트를 연결합니다. 선택적으로 통기, 공기 출구, 및 온도 조절 물 입구 및 출구 포트를 사용한다. </li><li> 교반 및 온도 제어를 제공하는 chemostat 항아리에 적합한 장치를 취득 (또는 회전 진탕 배양기를 사용). </li><li> 문화를 ?...

<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>

미생물 때문에 빠른 성장 속도와 유전 적 다양성의 거의 모든 환경에 적응시킬 수있다. 적응 진화 실험은 주어진 조건 하에서 유용 자연 돌연변이를 보유하는 개개의 유기체를 선택하는 방법을 제공하는 설계 조건 하에서 미생물 발전을 가능하게한다. chemostat 기술은 다음과 같은 이유로 이전 기법보다 인위적 구동 발전을 달성하기위한 더 강력 : (a) 정상 환경 - 전송 기술들?...

미생물이 생존하고 다양한 환​​경에 적응할 수 있습니다. 심한 스트레스, 적응은 임의의 게놈 변이 이후 긍정적 인 선택 1-3으로 도움이 표현형의 인수를 통해 발생할 수 있습니다. 따라서, 균체는 &quot;적응 진화&quot;이라한다 최적 성장을 위해 대사 또는 규제 네트워크를 변경하여 적용 할 수있다. 이러한 슈퍼 박테리아의 발생 및 강력한 미생물 균주의 발생으로 최근 중요한 미생물 경향은 매우 밀접하게 스트레스 조건에서 진화를 적응 적 관련이 있습니다. 정의 실험실 조건에서, 우리는 분자 진화의 메커니즘을 연구에도 다양한 용도 미생물 진화의 방향을 제어 할 수있다. 그들은 큰 개체군을 유지 빠르게 재생하고, 생성 및 혼을 유지하기 쉽다 : 다세포 유기​​체 달리 단세포 유기​​체 이유는 다음 실험실 적응 진화 (ALE)에 적합ogeneous 환경. DNA 시퀀싱 기술과 높은 처리량 기술의 최근 발전과 결합, ALE는 전신 규제 변화로 이어질 게놈의 변화를 직접 관찰 할 수 있습니다. 돌연변이 역학 및 인구의 다양성도 관찰 할 수 있습니다. 유전 공학 전략 ALE 균주 4,5의 분석으로부터 결정될 수있다. Chemostat 배양 정상 세포를 얻는 발효 공정 6의 생산성을 향상하기 위해 사용하는 방법이다. 신선한 배지를 첨가하고, 배양액의 처리 (후자는 중간 및 바이오 매스를 포함한다) 중에 수확된다. 장기 배양 chemostat 그러나 배양 정상 생산성을 변경하여 배양 (도 1a) 중 자발적인 돌연변이 및 선택의 축적을 초래한다. 다양한 선택 압력 (스트레스)에서 돌연변이의 축적이 향상된다. 장기 응력 점진적 증가<html> chemostat는 반복 액체 매질에서 고체 배지 및 시리얼 전송 (에서 온도, pH, 삼투압, 영양 결핍, 산화, 독성 최종 제품 등 식민지 전송으로 지정된 스트레스에 반대 돌연변이의 연속 선택을 제공합니다 배치 배양)도 연구 진화 미생물 (도 1B 및 1C)을 수득 할 수있다. chemostat 배양 복잡한 방법이 필요하지만, 다이버 시티 풀 (복제 인구 크기의 수) 콜로니 전송과 시리얼 전송 기술에 의해 얻어진 것보다 높다. 각각의 세포에 안정 스트레스 노출과는 chemostat 문화 (정상 상태) 일괄 문화 기반 기술에 비해 ALE의 다른 혜택입니다 동안 세포 상태의 변화를 감소. 높은 숙시 네이트 조건에 적용 대장균의 스트레스 유발 ALE는이 문서에서 소개된다. 세틸 / ftp_upload / 54446 / 54446fig1.jpg &quot;/&gt; 그림 1 : 적응 형 실험실 진화의 방법 (A) Chemostat;. (B) 시리얼 전송하는 단계; (C) 식민지 전송. 상단 그림은 ALE에 대한 방법의 개념을 설명하고, 아래 그림은 ALE 동안 성장 세포의 수를 보여줍니다. <a href="https://www.jove.com/files/ftp_upload/54446/54446fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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.

높은 숙시 응력 적응 야생형 E. 대장균 W3110 균주는 D에서 chemostat에서 배양 = 0.1 시간 -1 270일에 대한 (그림 2). <img alt="그림 2" src="/files/ftp_upload/54446/54446fig2.jpg" /> 그림 2 : E.의 높은 숙시 네이트 스트레스 적응 대장균을 이용하여 배양 chemostat W3110. 얇은 화살표는 스트레스의 농도가 증가하는 시간...

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

여기서는 chemostat 문화 사용 조건 하에서 미생물 적응 진화 실험을 얻을 프로토콜을 제시한다. 또한, 진화 된 균주의 게놈 분석은 설명한다.

Chemostat를 사용하여 미생물의 적응 연구소 발전을위한 절차

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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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

스나이퍼-Cas9를 사용 하 여 대상에서 감독된 진화를 통해 대상에 활동의 손실 없이 CRISPR Cas9의 영향을 최소화 하

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

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

체미애 남성 액세서리 땀샘의 이차 세포에서 RNA를 분리하는 FACS 기반 프로토콜

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

조건부 Cas9 안정화를 사용하여 유전자 기능을 평가하기 위한 새로운 툴킷

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