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.设备的准备<ol><li>得到恒化器罐子（150-250毫升）或含有一个入口的Erlenmeyer烧瓶（250ml）和一出口。用硅管允许10-100毫升/小时的流速连接的端口。任选地，使用空气通气口，出气口，和温度控制的水的入口和出口端口。 </li><li>获得适合的恒化器罐子提供用于搅拌和温度控制装置（或使用旋转式摇床）。 </li><li>以提供新鲜培养基，并收集培养获得两个蠕动泵。 </li><li>得到含有介质出口和一个吸入口的储存罐（10-20升）。 </li><li>获得适合于稀释率硅管（ 即 ，ID0.8毫米，流量范围0.06-36毫升/分钟; L / S 13管）。 </li></ol> 2.中型准备和灭菌<ol><li>初始介质<ol><li>溶解0.3克葡萄糖，0.08克NH 4氯，0.05克氯化钠，0.75克Na 2 HPO 4·2H 2 O，和0.3g KH 2 PO 4在90ml蒸馏水（DW）以恒化器罐子。 </li><li>密封罐子恒用夹子管子一起。不密封该通气孔。 </li><li>消毒在高压釜中的恒化器罐子在121℃下进行15分钟。灭菌后，在室温下储存的恒化器罐子。 </li><li>溶解0.02克用MgSO 4·7H 2 O 0.01克CaCl 2，并在10ml DW（溶液A）0.1毫克硫胺素。 </li><li>过滤溶液A使用注射器和一个预先灭菌的注射器过滤器（0.45微米孔径过滤器）。 </li><li>加入溶液A滤液于恒罐子。 </li></ol></li><li>中等压力<ol><li>溶解30克葡萄糖，将8g 氯化铵 ，5克氯化钠，75克Na 2 HPO 4·2H 2 O，将30g KH 2 PO 4，和300克琥珀酸二钠六水合物（钠2·琥珀酸盐·6H &lt;子&gt; 2 O;在这个实验中使用的应激）在9.9大号DW在贮罐。 </li><li>密封罐藏用夹子管子一起。不密封该通气孔。 </li><li>消毒在高压釜中贮存罐在121℃下15分钟。灭菌后，在室温下储存罐。 </li><li>溶解2克用MgSO 4·7H 2 O，1个克CaCl 2，并在100ml DW（溶液A）10毫克硫胺素。 </li><li>过滤溶液A用注射器和一个预先灭菌的注射器过滤器（0.45微米孔径过滤器）。 </li><li>加入溶液A滤液对储层罐子。 </li><li>无菌连接无菌硅管的水库罐子，装上蠕动泵。 </li></ol></li><li>高应力中<ol><li>制备培养基如2.2节，但与压力源的一个更大的浓度（ 即 ，3-5克/升的琥珀酸适应更高）。 注:该协议是适应应力日在可以经由介质递送。在物理应激如温度，搅拌，或照明的情况下，栽培应相应地设计。 </li></ol></li></ol> 3.初始培养<ol><li>接种野生型大肠杆菌的单个菌落大肠杆菌在含有4毫升起始培养基的15毫升试管中。 </li><li>孵育试管在振荡培养箱中于37℃12小时和220的转速。 </li><li>无菌转移1毫升预培养到恒罐子。 </li><li>孵育恒化器罐子，6小时提供用于充气（空气50ml /分钟）和搅拌（200rpm）下，在37℃下。 </li></ol> 4.适应压力<ol><li>无菌地连接来自泵至恒化器罐子的硅管的端部。 </li><li>启动出口泵（10毫升/小时或更高），并收集培养。 注意:培养应该在指数期，典型地初始培养后4-8小时。 </li><li>通道ECK从出口管培养物的光密度（600nm处）。 </li><li>启动入口泵（10毫升/小时，相当于0.1小时-1的稀释速率）。 </li><li>从出口管每24小时检查在600nm培养物的光密度。 </li><li>操作恒为96小时（9.6倍周转）以上。如果光密度是稳定的，交换包含高应力介质贮存器。如果光学密度低于0.2时，停止进料口泵6小时。重新启动进口泵，检查光密度超过0.2。 </li><li>逐渐改变到含有较高压力源浓度的储增加应激物的浓度。 </li><li>采取适于培养的样品时，它达到一个里程碑（ 例如 ，适于至100克/升琥珀酸应力应变），并存储用于进一步的基因组的分析。 </li><li>样品存储，用无菌80％甘油soluti混合培养样品（0.5毫升）中上（0.5毫升），并将其存储在-80℃。 注意:如果该微生物获取期间ALE过程降解应激的能力，在发酵罐的压力源的浓度是不一样的，在新鲜的贮存器。 </li></ol>应力 - 应变适应5.单菌落分离<ol><li>制备含有相同压力源，并在介质的相同浓度的琼脂平板培养基（1.6％琼脂）。 </li><li>板从恒化器的出口培养（0.1毫升）中，并在37°C孵育16小时。 </li><li>使用无菌牙签挑从板单菌落并且在含有相同应激15毫升试管，并在相同的培养基中的浓度如在恒化器接种他们，孵育6小时。 </li><li>传送1毫升培养液中成含有50毫升培养基的250毫升锥形瓶中。收获将0.5ml培养液的每1小时，并测量在600nm的OD值。比较适应strai的增长速度n至给定的压力源的野生型菌株的。 </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.

微生物能够适应，因为他们的速度快速增长和遗传多样性几乎所有的环境。自适应实验室进化使微生物设计条件，这提供了选择个体生物窝藏自发突变是在给定的条件下有利的方式下发展。 恒化器的技术是用于实现比转移技术人工驱动进化原因如下更健壮:（a）一种稳定的环境 - 因为传输技术基于分批培养物无论是在固体或在液体介质中，该细胞的环境批次期间变化文化而恒...

微生物可以生存和适应不同的环境。在严峻的压力，可以通过随机的基因组突变和随后的正选择1-3取得有益的表型发生调整。因此，微生物细胞可以通过改变代谢或调控网络对于最佳生长，这被称为"自适应进化"适应。最近的重要微生物的倾向，比如超级细菌爆发和强大的微生物菌种的发生，关系非常密切的压力条件下的适应性进化。下定义的实验室条件下，我们能够研究分子进化的机制和甚至控制各种应用微生物进化的方向。与多细胞生物，单细胞生物是非常适合自适应进化实验室（ALE），原因如下:他们很快再生，他们认为大量的人口，而且很容易创建和维护坎ogeneous环境。随着DNA测序技术和高通量技术最新进展相结合，ALE允许对基因组的变化，导致系统性监管政策的变化直接观察。突变动力学和人口的多样性也观察到。遗传工程策略可以从ALE菌株4,5的分析来确定。 恒化器培养是用于获得稳态细胞和发酵过程6提高生产率的方法。新鲜培养基，并将该过程（后者包括介质和生物量）中培养液收获。长期培养恒然而，改变了文化的稳态生产力和文化（ 图1a）期间带来的自发突变和选择的积累。在各种选择压力（压力），突变的积累被提高。应力的长期逐渐增加<html>恒化器提供了用于对给定的压力工作的突变，如温度，pH，渗透压，营养饥饿，氧化，有毒最终产品等菌落转移从液体介质中的固体培养基和串行传输（重复的连续选择分批培养），也让研究人员获得进化微生物（ 图1b和1c）。虽然恒化培养需要复杂的方法中，分集的池（复制和人口规模的数目）比由菌落转移和串行传输技术获得更高。稳定的压力接触到单个细胞和培养恒（稳态）进行比较，以一批以文化​​为基础的技术ALE其他福利过程中的细胞状态下降的变化。经受高琥珀酸条件大肠杆菌应激诱导ALE这篇文章中介绍。 尔斯/ ftp_upload / 54446 / 54446fig1.jpg"/&gt; 图1:自适应进化实验室方法 （A）恒化。 （B）串行传送; （C）菌落转移。顶部图说明为ALE方法的概念，底部数字说明ALE期间增长了细胞的数量。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54446/54446fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</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.

对于高琥珀酸应力适应，野生型E.大肠杆菌 W3110菌株是一个恒化器培养在D = 0.1小时-1 270天（ 图2）。 <img alt="图2" src="/files/ftp_upload/54446/54446fig2.jpg" /> 图2:E的高琥珀酸压力调适大肠杆菌 W3110采用恒化的文化。细箭头表示在该压力源浓度增加，与时俱进，大胆的箭头表示在该培养物保存的时间。用...

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

使用恒化器进行微生物适应性实验室进化的程序|遗传学 |视频

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

在这里，我们提出了一个协议，以获得使用恒化培养条件下微生物实验室适应性进化。此外，演进的株的基因组分析进行了讨论。

程序的微生物实验室自适应演化的恒化

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

Universidad de Cartagena UDEC

Research

Education

JoVE Journal

JoVE Core

Biology

Genetics

Evolution

Evolutionary History

SCIENTISTS IN ACTION

14.1K 次观看.  Korea Research Institute of Bioscience and Biotechnology (KRIBB). 在这里，我们提出了一个协议，以获得使用恒化培养条件下微生物实验室适应性进化。此外，演进的株的基因组分析进行了讨论。 

视频: 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.

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

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

科研

遗传学

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.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

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.

多拷贝产质粒在抗生素耐药性演变中的作用试验

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.

全基因组对真核生物转录误差的监测

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.

细菌结合频率的高分辨率比较

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.

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

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: 杀手细胞免疫球蛋白样受体基因的定量半自动分型

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.

利用 niper-cas9 在不损失目标活动的情况下通过定向进化, 最大限度地减少 crispr-cas9 的非目标效应

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.

从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

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.

使用有条件Cas9稳定评估基因功能的新工具包

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