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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+the+hopf+bifurcation+in+a+live+predator-prey+system.">Crossing the hopf bifurcation in a live predator-prey system.</a> Science. 290, 1358-1360 (2000).</li><li>Cohen, E. P., Eagle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+chemostat+for+the+growth+of+mammalian+cells:+characteristics+of+cell+growth+in+continuous+culture.">A simplified chemostat for the growth of mammalian cells: characteristics of cell growth in continuous culture.</a> J. Exp. Med. 113, 467-474 (1961).</li></ol>

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 border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Infors-HT Sixfors Chemostat</td>
			<td>Appropriate Technical Resources, Inc.</td>
			<td>&nbsp;</td>
			<td>&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 &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 &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&middot;2H2O</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&middot;7H2O</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&middot;5H2O</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&middot;6H2O</td>
			<td>Fisher Scientific</td>
			<td>I88-100</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>manganese sulfate&middot;H2O</td>
			<td>Sigma Aldrich</td>
			<td>230391</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>sodium molybdate&middot;2H2O</td>
			<td>Sigma Aldrich</td>
			<td>M7634</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>zinc sulfate&middot;7H2O</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 ...

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30.5K 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

Investigating the Microbial Community in the Termite Hindgut - Interview

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels.

调查白蚁后肠中的微生物的社区 - 面试

Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes.     The ...

科研

生物学

Microbial Communities in Nature and Laboratory - Interview

微生物群落，在自然和实验室 - 面试

Biology of Microbial Communities  - Interview

微生物群落的生物 - 面试

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

使用微机电系统（MEMS）开发诊断工具

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

在临床的虚拟现实使用生物反馈：勇敢的项目

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

窗口上的一个微观世界：简单的微流体系统为研究多孔介质中的微生物运输

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Determining the Contribution of the Energy Systems During Exercise

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given physical activity. Although some sports are relatively easy to be reproduced in a laboratory (e.g., running and cycling), a number of sports are much more difficult to be reproduced and studied in controlled situations. This method presents how to assess the differential contribution of the energy systems in sports that are difficult to mimic in controlled laboratory conditions. The concepts shown here can be adapted to virtually any sport.
The following physiologic variables will be needed: rest oxygen consumption, exercise oxygen consumption, post-exercise oxygen consumption, rest plasma lactate concentration and post-exercise plasma peak lactate. To calculate the contribution of the aerobic metabolism, you will need the oxygen consumption at rest and during the exercise. By using the trapezoidal method, calculate the area under the curve of oxygen consumption during exercise, subtracting the area corresponding to the rest oxygen consumption. To calculate the contribution of the alactic anaerobic metabolism, the post-exercise oxygen consumption curve has to be adjusted to a mono or a bi-exponential model (chosen by the one that best fits). Then, use the terms of the fitted equation to calculate anaerobic alactic metabolism, as follows: ATP-CP metabolism = A1 (mL . s-1) x t1 (s). Finally, to calculate the contribution of the lactic anaerobic system, multiply peak plasma lactate by 3 and by the athlete&#x2019;s body mass (the result in mL is then converted to L and into kJ).
The method can be used for both continuous and intermittent exercise. This is a very interesting approach as it can be adapted to exercises and sports that are difficult to be mimicked in controlled environments. Also, this is the only available method capable of distinguishing the contribution of three different energy systems. Thus, the method allows the study of sports with great similarity to real situations, providing desirable ecological validity to the study.

This protocol allows researchers focused on exercise and sports sciences to determine the relative contribution of three different energy systems to the total energy expenditure during a large variety of exercises.

确定的能源系统，在运动的贡献

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

链接捕食风险，草食动物生理应激和植物凋落物的微生物分解

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...