Name: the use of chemostats in microbial systems biology
Uploaded: 2013-10-14T20:45:00.000+00:00
Duration: 13 min 19 s
Description: Watch this Scientific Journal Video about The Use of Chemostats in Microbial Systems Biology at JoVE.com

这项工作得到了启动资金形成纽约大学的支持。我们感谢弥勒邓纳姆和马特·布劳尔谁最初开发出了利用生物反应器SIXFORS作为恒化器。

可实现多种实现方式采用恒化连续培养的原则。在所有的恒化器就必须有1）方法用于保持所有组件的不育性，2）充分混合的培养，在培养容器的3）适当的通气和4）一个可靠的媒体除了和培养去除装置。在这里，我们描述了使用生物反应器SIXFORS（Infors公司）作为使用可以很容易适应替代设置方法恒化。 1。组装恒化船舶<ol><li>打开使用主开关的SIXFORS。 </li><li>彻底冲洗恒化器，搅拌器组件，并用去离子（DI）水连接管上。检查所有的管路和O形圈，并更换磨损件看。 </li><li>确保驱动轴支撑基座朝上的玻璃容器中，该驱动轴的顶端卡合到其上的搅拌器组件壳体。设置stirre[R装配到玻璃容器确保驱动轴的底部被固定在驱动轴支撑。使用该夹具的搅拌器组件固定到玻璃容器中。 </li><li>用约300毫升去离子水填满容器。 </li><li>从溶解氧取下镜头盖（DO 2）探针和拧下底部的外壳，并确保底部的外壳充满中途与电解质溶液检查电解液液位。重新拧紧底部外壳和插入的恒化器的端口。直到拧紧的手指。 </li><li>以一个pH探头，并从它的存储缓冲器（3米氯化钾）将其删除。移开电极帽和附加到发酵罐1。使用控制SIXFORS屏幕，导航到发酵罐参数菜单，然后选择“校准pH值”。将pH探头插入一个标准的pH值为7的缓冲并记录读数为高读。重复用pH4的缓冲并记录读数为低阅读。从发酵罐取下pH探头，洗净去离子水，将探头插入整型o在恒化器的端口。直到拧紧的手指。 </li><li>将恒化器在机架中。注意其中发酵罐（1-6）的pH探头连接至和校准的容器上。广场上的pH探头pH探头帽。使用铝箔紧紧地盖住做2探头的顶部。 </li><li>折叠箔过度附着在恒化器中的管的端部。 </li><li>重复步骤1.2-1.8的所有船只。 </li><li>通过高压灭菌他们在液体循环15分钟消毒的器皿。 </li></ol> 2。准备媒体<ol><li>建立浓度的营养通过种植分批培养在不同养分含量的限制范围内。养分被限制的范围内，其中的最终细胞密度为初始养分浓度（ 图4）的线性函数。以及在限制范围内选择营养浓度。与Saccharomyce研究标准介质组成物的例子酿酒酵母可在7-11。 </li><li>高压釜是后第一确保介质管路的一端覆盖在箔封端含一个空气入口和介质出口的橡胶塞的空瓶子。 </li><li>在一个单独的容器中制备10升介质。 </li><li>附加的500毫升过滤杯100毫升的媒体瓶子。用无菌镊子在乙醇中取出滤棉，并立即连接到端口的过滤介质上的瓶子。 </li><li>附加媒体大玻璃瓶到真空源和过滤器通过将其添加到过滤器杯杀菌媒体。 </li><li>当介质过滤完成后，关闭掉端口过滤用金属钳。 </li></ol> 3。校准做2个探头，并设立恒化<ol><li>之后的恒化器船只已灭菌并冷却下来代替容器在其相应的热护套。连接温度探头，pH探头，和做2个探头。让与电力船坐至少6小时，让做2个探头极化。 </li><li>将每个污水管的端部插入一个单独的收集容器。通过高压灭菌过滤器连接气源，并打开通风。在每个容器中的水应该流出的流出管，这表明所有密封件被适当地形成。 </li><li>根据所需的工作体积（ 例如 300ml）中调整的流出管的高度。我们使用的是标有校准到不同的工作卷管安置一把尺子。 </li><li>媒体的瓶子连接到恒化器。使用乙醇，以保持管端无菌越好。通过泵和开放钳螺纹泵管。手动按下泵，直到介质开始流入的恒化器中。从泵松开油管，媒体应该自由地流入恒化器。当媒体到达污水管，重新连接油管泵和钳。 </li><li>启动茹与叶轮设置为400转，并设置在30℃的温度nning的基本程序</li><li>为了校准做2个探头，关闭气源并切换到氮气。等待至少一个小时，记录测量值作为“低读”。切换回气源（ 即含环境氧浓度），等待至少一个小时并记录测量为“高读”。 </li><li>使用光圈软件启动数据记录。 </li></ol> 4。接种<ol><li>用70％乙醇消毒培养容器的顶部。 </li><li>在容器顶部和吸管1毫升过夜培养卸下螺钉拧入恒化器。重新拧紧螺丝。 </li><li>表明接种在虹膜的时间。 </li><li>等待大约24小时的培养，以达到早期固定相。作为培养生长，溶解氧和pH会下降。在葡萄糖限制媒体的情况下，溶解的氧气会返回到静止帕〜100％本身。对于其他营养物质限制溶解氧仍将&lt;100％的固定相。 </li></ol> 5。启动泵并实现稳态<ol><li>稀释率是通过将流量计算（多少介质流入每小时容器）由培养物体积。例如，使用300毫升的0.1的稀释率的体积意味着，加入30ml介质每隔一小时加入到培养。因为该系统是在正压力下的相同体积的容器确保培养物连续地稀释除去。稀释率（D）的范围可以使用不超过电池的最大生长速率（μ 最大 ），上面的哪些单元格将被淘汰的恒化的。 </li><li>选择泵的设置，其中指定的秒数泵是开启和关闭的数目，以建立所需的流速。该泵以〜0.11毫升/​​秒的速率传送媒体。 </li><li>定义使用整型SIXFORS程序erface指定该泵定时，温度和叶轮速度。启动该程序。 </li><li>倒空液体的收集容器，并记录时间。 </li><li>至少两小时后，用量筒测量多少媒体已经从容器中取出。这将等于已被添加到该恒化器容器的体积。计算稀释率（D = V 废水 / V 文化 /时间 ）。调整泵的设置，如果计算的稀释率不符合预期的稀释率。 </li><li>在稳定状态下，在恒化器的细胞密度随时间不发生变化。这可以在不通过采样流出扰动培养来测定。我们操作性定义达到稳态的是由至少一种群体倍增分离相同细胞密度的测量。文化稀释开始后达到稳定状态大约需要8文化几代人。在稳定状态下，细胞成倍增长（ 即在营养限制条件E。固定增长 ​​率在一段时间）。人口（生成时间）的倍增时间为LN（2）/ D。 </li><li>继续周期性地测量流出物体积为实验的持续时间，以确保恒定的稀释率得以维持。 </li></ol> 6。应用1：研究细胞生长以不同的速率在稳态条件<ol><li>在恒化器的稳态，细胞群体的生长速率等于稀释率。通过系统地改变稀释率，可以以不同的速率生长的细胞。这可让随生长速率，包括细胞体积，细胞周期阶段和抗逆性生理参数的系统研究。此外，全球的mRNA，蛋白质和代谢物水平的稳定状态的访问，可以测定细胞中以不同的速度生长。有两种方法获得的样本： </li><li>小样本可以是被动地观测通过将污水管的端部插入一个1.5毫升或15ml试管tained。可以在几分钟内获得的1-5毫升的样品（取决于流量）。许多生理参数，可以从这些样品进行测定。 </li><li>基因的表达，或代谢物的分析要求，必须尽可能快地获得较大的样本。将污水管的端入15ml锥形管中。松开螺丝固定的污水管的金属端推轻轻放下。一个约10毫升的样品会迅速填满你管。请记住，在恒化容器的体积发生了变化。如果许多连续抽取样本，可能有必要减少流量以维持恒定的稀释率。 </li></ol> 7。应用二：差异增长率基因型之间受控环境中使用流式细胞仪为基础的竞争性试验的精密测量<ol><li>恒化器可以被用来准确地量化养分浓度的影响在不同遗传背景的差异在增长率（ 即健身）。通过标记有不同的荧光蛋白共同培养的菌株的变化相对丰度在指数生长的速率是确定的。在进行这个实验在不同的稀释率（D）允许的养分浓度（S）对健身的差异影响的研究。 </li><li>建立两个菌株在不同的恒化容器具有相同的稀释率和300毫升的容积稳态文化。 </li><li>被动采样1毫升从每个容器。降速细胞，重新悬浮于磷酸盐缓冲盐水（PBS），并置于4℃这些样品含有同质的细胞样品，这对于随后的流式细胞分析的控制。 </li><li>将污水管从一个容器到高压灭菌量筒。松开螺丝固定的污水管的金属端，轻轻地向下推，从恒化器排出细胞。何时量达到150毫升，污水管的金属端返回到其原始位置（300毫升）中。重复与所述第二容器中，使用不同的量筒。 </li><li>用70％的乙醇以保持无菌，而在恒化器容器的顶部除去螺钉。将漏斗的开口，倒150毫升从其他文化融入恒化器。重新拧紧螺丝。重复与所述第二容器中，使用所述第二漏斗。每一恒化器现在包含两种菌株的混合物。 </li><li>使用被动采样每隔2-6小时，每艘船获得1毫升样品。降速细胞，重悬于PBS中，并储存于4°C。继续服用样品2-3天仔细记录每个样品所用的时间。 </li><li>在该实验中，声处理的结束和稀释样品至〜2×10 6个细胞/ ml。用流式细胞仪，测量两个菌株在每个样品中的比例。由于两个菌株呈几何级数增长，相对生长速率的区别是由LN的线性回归（strain1/strain2）与时间（以代测）来确定。回归的斜率为1株相对于其它的生长速率成比例的差值（ 即在健身优势）。 </li><li>由于混合培养可能需要一些时间来达到一个新的稳定状态，早期的时间点可能会很差适合的回归。这个问题最好通过允许2-3代，以开始采样或删除早期的点是明确的离群经过前解决。相反，一旦1菌株排挤其他的数据不再有用，这些点应该被排除。 </li></ol> 8。应用3：实验进化<ol><li>恒化器中进行实验进化选择的突变体在营养受限的环境中提高健身。一般进行过数百代的选择。 </li><li>建立使用应变稳态恒化培养的已知基因型（优选已知的基因组序列）和一个定义的营养限制。 </li><li>维持适配人口的“化石记录”被动采样恒化器每2-3天，并在样品中15％的甘油储存于-80℃。 </li><li>监测媒体水库和补充与必要的新鲜培养基中。 </li><li>保持成倍增长的文化数百代。 </li><li>选择板的细胞在固体琼脂平板上的一个样品完成后。经过细胞已成长为菌落，挑用牙签殖民地的一个无偏样本，克隆接种到96孔板中含有100微升的媒体的各个孔。允许克隆样生长过夜，在-80加入100μl30％甘油和存储°C。 </li><li>通过全基因组测序，并进行表型检测包括体能评估，采用通用荧光标记的参考菌株，如申请2所描述的特征的克隆。&lt;/ LI&gt; </ol>

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P., 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=When+transcriptome+meets+metabolome:+fast+cellular+responses+of+yeast+to+sudden+relief+of+glucose+limitation.">When transcriptome meets metabolome: fast cellular responses of yeast to sudden relief of glucose limitation.</a> Mol. Sys. Biol. 2, 49 (2006).</li><li>Tu, B. P., Kudlicki, A., Rowicka, M., McKnight, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Logic+of+the+yeast+metabolic+cycle:+temporal+compartmentalization+of+cellular+processes.">Logic of the yeast metabolic cycle: temporal compartmentalization of cellular processes.</a> Science. 310, 1152-1158 (2005).</li><li>Tzur, A., Kafri, R., LeBleu, V. S., Lahav, G., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Growth+and+Size+Homeostasis+in+Proliferating+Animal+Cells.">Cell Growth and Size Homeostasis in Proliferating Animal Cells.</a> Science. 325, 167-171 (2009).</li><li>Conlon, I., Raff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+control+in+animal+development.">Size control in animal development.</a> Cell. 96, 235-244 (1999).</li><li>Conlon, I. J., Dunn, G. A., Mudge, A. W., Raff, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+control+of+cell+size.">Extracellular control of cell size.</a> Nat. Cell Biol. 3, 918-921 (2001).</li><li>Fussmann, G. F., Ellner, S. P., Shertzer, K. W., Hairston, N. 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作者宣称，他们有没有竞争的财务权益。

恒化使微生物的生长控制的稳态条件下的培养。细胞在产生一个不变的外部环境的恒定速率连续地生长。这是相对于分批培养方法，其中外部环境是不断变化和细胞生长的速率由环境和基因型的复杂的相互作用决定。因此，在恒化器超过分批培养物中培养的微生物的一个主要优点是可以通过实验控制细胞的生长速率的能力。 在其中一个细胞生长的速率是包括养分传感，信号?...

细胞的生长是通过复杂的相互作用的遗传和环境因素1,2的网络监管。细胞生长的多因素调控需要一个系统级的方法来研究它。然而，调节细胞生长的严格的研究是通过实验控制在哪些细胞生长率的困难挑战。此外，即使是最简单的实验条件外经常是动态的，复杂的细胞不断地改变自己的环境，因为它们繁殖。培养细胞的方法，使实验对照的细胞生长率的定义，不变的和受控制的环境：如能解决这些问题，通过恒化器提供。 连续培养中使用恒化的方法是通过独立莫诺3和诺维克和西拉德4于1950年描述。按照最初的设想，细胞生长在媒体的一个固定体积是con通过添加新的媒体和同时去除旧的媒体和细胞（ 图1）tinually稀释。耦合的常微分方程（ 图2）描述了细胞密度（x）和限制生长的营养物质在恒化器（S）的浓度变化率。重要的是，方程系统预测单（非零）稳定稳态（ 图3）具有显着的含义，在稳定状态中，细胞（ 即指数增长速率常数）的特定生长率等于速率在该培养物稀释（D）。通过改变稀释率，可以在不同的生长速度和营养限制不同条件下建立的细胞稳态的人群。 实验对照采用恒化增长率为的细胞生理变化如何理解发展的关键随着增长5,6率。然而，微生物学方法这个前主体在二十世纪后期在分子生物学研究的爆炸过程中变得越来越模糊。今天，在生长控制在这两个微生物和多细胞生物和基因组规模的方法，系统级分析的出现新的兴趣一直延续的动力为使用恒化器中。在这里，我们描述了利用精确控制细胞生长速率和外部环境，是唯一可以使用恒化器三个应用程序。首先，我们描述了使用恒化器的研究如何以十万计的生物分子的丰度 - 如成绩单和代谢物 - 是协同调控与增长速度。第二，我们将描述如何恒化器可用于取得在使用竞争实验养分有限的环境不同基因型之间生长速率的差异的精确估计。第三，我们描述了如何能恒化器可用于研究细胞在不断的营养贫乏的环境中成长的适应性进化。这些例子体现在恒化正在使细胞生长调控，基因与环境的相互作用和适应性进化系统级的调查方式。

<table><tbody><tr><td>Infors-HT Sixfors Chemostat</td><td>Appropriate Technical Resources, Inc.</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Glass Bottle 9.5 L</td><td>Fisher Scientific</td><td>02-887-1</td><td>For Media Vessel and Hosing</td></tr><tr><td>Pinchcock</td><td>Fisher Scientific</td><td>05-867</td><td>For Media Vessel and Hosing</td></tr><tr><td>Stopper, Size 12, Green Neoprene</td><td>Cole-Palmer</td><td>EW-62991-42</td><td>For Media Vessel and Hosing</td></tr><tr><td>Straight Connector</td><td>Cole-Palmer</td><td>EW-30703-02</td><td>For Media Vessel and Hosing</td></tr><tr><td>General purpose ties 4 in</td><td>Fisher Scientific</td><td>NC9557052</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Silicone Rubber</td><td>Small Parts</td><td>B000FMWTDE</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Silicone, 3/8 in OD</td><td>Fisher Scientific</td><td>02-587-1Q</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Silicone, 7/32 in OD</td><td>Fisher Scientific</td><td>02-587-1E</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Stainless Steel, 3/16 in OD</td><td>McMaster-Carr</td><td>6100K164</td><td>For Media Vessel and Hosing</td></tr><tr><td>Tubing, Stainless Steel, 3/8 in OD</td><td>McMaster-Carr</td><td>6100K161</td><td>For Media Vessel and Hosing</td></tr><tr><td>Hook Connectors</td><td>Fisher Scientific</td><td>14-66-18Q</td><td>For Media Vessel and Hosing</td></tr><tr><td>Ratchet Clamp</td><td>Cole-Palmer</td><td>EW-06403-11</td><td>For Media Vessel and Hosing</td></tr><tr><td>Luer, Female</td><td>Cole-Palmer</td><td>EW-45512-34</td><td>For Media Vessel and Hosing</td></tr><tr><td>Luer, Male</td><td>Cole-Palmer</td><td>EW-45513-04</td><td>For Media Vessel and Hosing</td></tr><tr><td>Millipore Aervent MTGR05010 62 mm Filter, 0.2 &amp;mu;m</td><td>Fisher Scientific</td><td>MTGR05010</td><td>For Media Vessel and Hosing</td></tr><tr><td>PTFE Acrodisc CR 13 mm filters, 0.2 &amp;mu;m</td><td>Fisher Scientific</td><td>NC9131037</td><td>For Media Vessel and Hosing</td></tr><tr><td>Direct-Reading Flowtube for Air</td><td>Cole-Palmer</td><td>EW-32047-77</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Direct-Reading Flowtube for Nitrogen</td><td>Cole-Palmer</td><td>EW-32048-63</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Gas Proportioner Multitube Frames</td><td>Cole-Palmer</td><td>EW-03218-50</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Regulator, Two-Stage Analytical</td><td>Airgas</td><td>Y12-N145D580</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Hose Adaptor, Stainless Steel</td><td>Airgas</td><td>Y99-26450</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Hose Male Adaptor</td><td>Airgas</td><td>WES544</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Norprene Tubing</td><td>US Plastics</td><td>57280</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Tripod Base</td><td>Cole-Palmer</td><td>EW-03218-58</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Valve Cartridges</td><td>Cole-Palmer</td><td>EW-03217-92</td><td>For Nitrogen Gas Setup</td></tr><tr><td>Carboy 10 L</td><td>Fisher Scientific</td><td>02-963-2A</td><td>For Media Preperation</td></tr><tr><td>Steritop Sterile Vacuum Bottle-Top Filters, 1,000 ml, PES membrane; for 45 mm neck size</td><td>Fisher Scientific</td><td>SCGP-T10-RE</td><td>For Media Preperation</td></tr><tr><td>Media Bottle 100 ml, 45 mm neck size</td><td>Fisher Scientific</td><td>FB-800-100</td><td>For Media Preperation</td></tr><tr><td>calcium chloride&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>C79-500</td><td>Media Reagents</td></tr><tr><td>sodium chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td>Media Reagents</td></tr><tr><td>magnesium sulfate&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>230391</td><td>Media Reagents</td></tr><tr><td>potassium phosphate monobasic</td><td>Fisher Scientific</td><td>AC424205000</td><td>Media Reagents</td></tr><tr><td>ammonium sulfate</td><td>Fisher Scientific</td><td>AC423400010</td><td>Media Reagents</td></tr><tr><td>potassium chloride</td><td>Sigma Aldrich</td><td>P9541</td><td>Media Reagents</td></tr><tr><td>boric acid</td><td>Sigma Aldrich</td><td>B6768</td><td>Media Reagents</td></tr><tr><td>copper sulfate&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>209198</td><td>Media Reagents</td></tr><tr><td>potassium iodide</td><td>Sigma Aldrich</td><td>60400</td><td>Media Reagents</td></tr><tr><td>ferric chloride&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>I88-100</td><td>Media Reagents</td></tr><tr><td>manganese sulfate&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>230391</td><td>Media Reagents</td></tr><tr><td>sodium molybdate&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>M7634</td><td>Media Reagents</td></tr><tr><td>zinc sulfate&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>Z68-500</td><td>Media Reagents</td></tr><tr><td>biotin</td><td>Fisher Scientific</td><td>BP232-1</td><td>Media Reagents</td></tr><tr><td>calcium pantothenate</td><td>Fisher Scientific</td><td>AC24330-1000</td><td>Media Reagents</td></tr><tr><td>folic acid</td><td>Sigma Aldrich</td><td>F7876</td><td>Media Reagents</td></tr><tr><td>inositol (aka myo-inositol)</td><td>Fisher Scientific</td><td>AC12226-1000</td><td>Media Reagents</td></tr><tr><td>niacin (aka nicotinic acid)</td><td>Sigma Aldrich</td><td>N4126</td><td>Media Reagents</td></tr><tr><td>p-aminobenzoic acid</td><td>Fisher Scientific</td><td>AC14621-2500</td><td>Media Reagents</td></tr><tr><td>pyridoxine HCl</td><td>Sigma Aldrich</td><td>P9755</td><td>Media Reagents</td></tr><tr><td>riboflavin</td><td>Sigma Aldrich</td><td>R4500-25G</td><td>Media Reagents</td></tr><tr><td>thiamine HCl</td><td>Fisher Scientific</td><td>BP892-100</td><td>Media Reagents</td></tr><tr><td>Leucine</td><td>Sigma Aldrich</td><td>L8000-100G</td><td>Media Reagents</td></tr><tr><td>Uracil</td><td>Sigma Aldrich</td><td>U0750</td><td>Media Reagents</td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>DF0155-08-5</td><td>Media Reagents</td></tr></tbody></table>

the use of chemostats in microbial systems biology

调节细胞响应信号来自外部世界的增长速度。随着细胞生长，不同的细胞过程必须协调包括高分子合成，代谢，最终，承担细胞分裂周期。恒化器，实验控制细胞生长速率的方法，提供了强大的系统研究手段增长率影响细胞过程如何 - 包括基因表达和新陈代谢 - 和监管网络，控制细胞生长的速度。当维持数百代恒化器可​​用于研究环境条件的微生物，限制细胞生长的适应性进化。我们描述了恒化培养的原则，展示他们的操作，并提供他们的各种应用的例子。经过一段时间的废用他们在二十世纪中叶引进后，基因组规模方法的收敛重新票面​​细胞生长和适应性进化的分子基础的监管是刺激的复兴在生物学研究中使用恒化器中。

恒化器的主要优点是通过改变稀释率实验控制细胞的生长速度的能力。在芽殖酵母，酿酒酵母，细胞的形态是其信息在细胞分裂周期阶段。人群具有更高的生长速率包含的活跃分裂的细胞通过测量unbudded细胞的级分（ 图5A）所确定的比例较高。在恒化器培养物的全球mRNA表达的分析已经表明，许多基因的表达差异表达的生长速率（ 图5B和图5C）的函数。 <p cla...

Watch this Scientific Journal Video about The Use of Chemostats in Microbial Systems Biology at JoVE.com

The Use of Chemostats in Microbial Systems Biology

细胞生长速度是一个稳定的过程和细胞生理学的主要决定因素。利用恒化连续培养使细胞生长速度外在控制由营养限制有利于分子网络，控制细胞生长，以及这些网络的发展，优化细胞生长的研究。

恒化器中微生物系统生物学中的应用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

the-use-of-chemostats-in-microbial-systems-biology

Research

JoVE Journal

Biology

30.7K 次观看.  New York University. 细胞生长速度是一个稳定的过程和细胞生理学的主要决定因素。利用恒化连续培养使细胞生长速度外在控制由营养限制有利于分子网络，控制细胞生长，以及这些网络的发展，优化细胞生长的研究。 

视频: The Use of Chemostats in Microbial Systems Biology

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

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.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

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) ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

生物化学

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

生物工程

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

使用 Ever 设计自动化、高通量、连续细胞生长实验

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

在西图化学分析中，以检验水生生态系统中的微生物行为

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

环境科学

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

设计和自动照明，培养和取样系统实现微生物光遗传学的应用

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

生成受控动态化学景观，研究微生物行为

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

恒化器中微生物系统生物学中的应用

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