The authors would like to thank Prof. Sze-Bi Hsu and Ms. Zhenzhen for useful discussions and help in the theoretical analysis and numerical simulation. Y. T. Y. would like to acknowledge funding support from the Ministry of Science and Technology under grant numbers MOST 103-2220-E-007-026 and MOST 104-2220-E-007-011, and from the National Tsing Hua University under grant numbers 103N2042E1, 104N2042E1, and 105N518CE1.

1.大会和预测试的Morbidostat设备的<ol><li>在Morbidostat的大会<ol><li>按机器上的文化小瓶用18G的注射器针头帽3孔。切三块聚乙烯管材在〜长度为7厘米插入这三件聚乙烯管上的盖。 </li><li>用带子把盖的边缘以作为铸造的聚二甲基硅氧烷（PDMS）混合物。通过用牙签手动搅拌混合将5g组分和0.5g在150毫升的塑料容器中的PDMS B成分的。加载混合物到10ml注射器中。 <ol><li>倒在与注射器的帽与PDMS混合物。烤整个帽在70℃固化的PDMS在烘箱8小时。 </li></ol></li><li>焊接LED的阳极与24号线和LED阴极用烙铁碳电阻。焊接一个680Ω碳质电阻用24克线。用绝缘电工胶带的电线连接。 </li><li>所以lder连续用24克丝和带100kΩ的碳电阻光电检测器的发射器的光检测器的收集器。用绝缘电工胶带的电线连接。 </li><li>放置在培养小瓶保持器的发光二极管。通过遵循补充 图2的电路原理图，连接发光二极管和电源它5伏的供电确保LED的工作原理是使用数码相机，可以检测IR（ 例如，一个手机摄像头）。 </li><li>放置在培养小瓶保持器的光检测器。通过遵循补充 图2的电路原理图，连接光电探测器和电源它与5 V的电源线连接到光电检测电阻器两端测量通过电控板的电压。 </li><li>胶上的冷却风扇，其作为磁力搅拌器单元的轴上的磁体。注意，alignm风扇到培养瓶中，培养瓶和磁铁之间的间​​距的轴的耳鼻喉科是磁力搅拌装置的正常功能非常关键的。 </li><li>每一块培养小瓶的聚乙烯管连接到用于微型泵，介质瓶和废液瓶的相应片硅管的。打开泵1小时，并测量总体积从培养基瓶到培养瓶中被泵送，以确定所述泵速率。注意典型的泵送速率应〜4个毫升/小时，相当于稀释率（D）= 0.33小时-1为12 mL工作的培养小瓶的体积。 </li><li>将整个组件放在中型（34厘米x 34英寸厘米×43.5厘米）摇床孵化器。 </li></ol></li><li>在预试Morbidostat <ol><li>凉风扇设置电源电压为5 V开始其电机测试磁力搅拌棒。注意，该搅拌作用可进行目视检查。该驱动器的电压S中的冷却风扇电机精确由脉冲宽度调制（PWM）控制。 </li><li>准备了一系列野生型大肠杆菌的大肠杆菌样品在600光密度通常nm的范围从0.07〜0.3进行校准。将测试E.在培养瓶的大肠杆菌样品并记录光检测器电压。 <ol><li>地方〜100微升在平板阅读器相同的样品，并记录光密度。使用光检测器电压与光密度数据绘制校正曲线。注意，典型地，在光密度一单位变化对应于〜7.6 v更改在光检测器与这里所用的偏置方案。 </li></ol></li></ol></li></ol> 2.运行Morbidostat <ol><li>准备开始每日实验前<ol><li>具有三个超耐化学腐蚀的聚乙烯管材与内径1/32英寸和外径3/32英寸准备培养小瓶入口形成器件与第1.1和补充图1。 </li><li>准备M9基本培养基（0.2％葡萄糖）和甲氧苄啶（TMP）含药培养基。消毒在121℃的高压釜中的介质，所述介质瓶，药物介质瓶，废液瓶和培养瓶中。 注:TMP是一个储液用于以后达到表1中的浓度和含介质在TMP药物未高压灭菌。 </li></ol></li><li>运行Morbidostat <ol><li>在实验的第一天，解冻1毫升冷冻野生型大肠杆菌大肠杆菌 MG1655细胞在室温和移交120微升细胞含有培养瓶中的5分钟〜12毫升M9生长培养基。第一天之后，解冻1毫升冰冻E的大肠杆菌前一天5分钟，并转移120微升细胞包含新培养瓶中的细胞〜12毫升M9生长培养基。 </li><li>打开摇动孵化并设置坦佩<html>叉涂抹到30℃，无晃动。 </li><li>通过接通微型泵，磁搅拌单元，和光密度测量开始morbidostat操作。 注:在操作过程中，一个反馈阈算法用于控制药物注入。当所测量的光密度超过预设阈值时，药物注射泵将被打开，并且该介质的泵将被关闭。否则，药物注射泵保持关闭，介质泵上。 </li><li>监测从时间的电压以时间，以确保有微生物生长。 注意:由于冻融循环，E的大肠杆菌细胞每一天的生长过程中表现出〜4小时的延迟。 </li><li> 〜23小时后，通过关闭其电源电压停止微型泵，并采取了文化小瓶。 15％甘油添加到培养瓶中，并在-80℃储存冻结的样品。 </li><li>重复步骤2.2.1至2.2.5。 </li></ol></li></ol>乐"&gt; 3。药敏测试在96孔板注:在平板阅读器96孔板进行的增长速度测量，以确定抗生素抗性水平。 <ol><li>解冻在室温下每日冷冻样品5分钟，并转移15微升细胞的含〜15毫升M9培养基培养物小瓶。让他们成长，直到600的光密度达到0.1〜。分15 ml接种入1 ml瓶装，在该步骤中添加的TMP药物以获得如表1中所示的所需的药物浓度。 </li><li>取来自步骤3.1和代替样品200微升每孔的板读取器，并允许他们生长12小时5。在波长600nm处的光密度被测量期间自动记录。 注:对于各数据点，一式三份测量被记录并平均，以确保数据的一致性。对时间上的光密度数据能够bË用来获取的增长速度。 </li><li>绘制增速数据相对于药物浓度，以获得50 IC。需要注意的是集成电路50被定义为药物浓度在该生长速率是最大生长速率5的百分之五十。 </li></ol> 4.单细胞形态及药敏检测的微流体装置<ol><li>如其他地方15所述，执行经由来自PDMS多层软光刻技术的微流体器件的制造。 <ol><li>使用光刻法，使为控制和流层中的光致抗蚀剂模。注意，对于控制层，可以使用负性光刻胶（〜15微米高）和正性光刻胶（〜8微米的高度），以在裸硅晶片上产生的模具。 </li><li>制备的PDMS的混合物与5交联率:1和20:1，用于分别控制层和流层。把混合物成干燥器在真空下，以除去任何气泡为1小时。 </li><li>露出硅晶片上的控制层光致抗蚀剂模具三甲基氯硅烷（TMCS）蒸汽。通过铸造与PDMS混合物作〜厚度为6mm的控制层（具有交联比5:1）上的控制层光致抗蚀剂模在硅晶片上。在80℃下烘烤40分钟，控制层在烘箱1小时。 </li><li>上的流动层光致抗蚀剂模:（1，旋转速度3000rpm下进行60秒，交联比20）通过旋涂将PDMS混合物使流动层。在80℃下烘烤30分钟的流动层在烘箱1小时。 </li><li>用剃刀刀片切割控制层成所需的芯片尺寸（通常为3厘米×2厘米），并手动剥离控制层。放置在流动层的顶部的控制层和在立体显微镜下将它们对齐。在烤箱过夜固化对齐组件在80℃。然后，浸泡在装配在250ml的去离子水在塑料容器5小时，以溶解残余TMCS。 </li><li>接着，用冲床20G针的进气孔和接合整个弹性体涂覆有空白的PDMS层的载玻片（PDMS交联比5:1，旋转速度2000rpm下进行30秒），在40秒由空气等离子体处理1.2托气压。 </li><li>广泛开展烘烤36小时，在80℃，以减少从PDMS的细胞毒作用。请注意，此烘烤步骤是关键的，以尽量减少毒性的细菌细胞。 </li></ol></li><li>片上生长实验<ol><li>加载到微流体装置之前，用去离子水冲洗它为1小时和M9培养基1小时。 </li><li>解冻在室温下每日冷冻样品5分钟，并接种新试管用新鲜的M9培养基。放置在摇床培养箱样品在37℃下过夜。 </li><li>稀释微生物样品10倍，M9培养基并在5磅施压微生物样本容器其加载到微流体装置。然后加载TMP药物介质到芯片通过以5psi的加压药物介质容器。通过致动两个腔室之间的微机械阀的微流体芯片上的15分钟执行微生物和药物之间的混合。 </li><li>放置在安装在倒置显微镜的显微镜培养箱微流体芯片。获得在生长室安装在倒置显微镜每小时为与CCD摄像机8小时的细胞图像。 </li><li>使用自定义程序脚本，通过对细胞的图像数据18阈值算法计算出细胞数量。注意，该细胞数数据的平均值通常在三个微流体的腔室。 </li></ol></li></ol>

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</ol>

The authors have nothing to disclose.

从低成本分量的低足迹morbidostat设备是证明。由设备注册的耐药水平的增加是与以前的报告5一致。设计为耐药性的进化研究，该装置是可能适用于许多其他的实验。首先，药物诱导的突变的综合数据库可为一大组的临床相关抗生素来建立。例如，多药耐药性的进化途径可以通过简单地增加的实验中所用的药物的数量进行研究。这里所报告的模块的主要优点是它的灵活配置性，可实现大型化，平行实验不同的实验条件下。 虽然概念在微流体测试表明，调查权力可以通过与微流体技术流体相结合的23提高。每个成本测量（经济规模）急剧的降低试剂消耗，降低了劳动减少。这里，单细胞形态的数据可从微流体平台被提取。近来，单个细胞的微流体形态的测试已成功在临床微生物实验室实施，并且可比执行到标准肉汤系列稀释的测试24。随着微流体设备的日益普及，技术相结合将使得大规模，高通量实验室进化实验。 几个步骤是运行该morbidostat至关重要。首先，由于磁力搅拌单元从磁体和冷却风扇形成的，关键的是要在风扇的轴对准到培养瓶中。另外，培养瓶和磁铁之间的距离是非常关键的，以确保稳定操作。其次，切换到在每一天的实验开始一个新的培养瓶中也是重要避免生物膜的形成。否则，生物膜的形成可在2〜3天出现。第三，由于大肠杆菌大肠杆菌样品每天的实验过程中从冷冻样品每日解冻，以确保一致性，来设置至少4小时的延迟时间，以避免共洗出的微生物是十分重要的。可替代地，人们可以选择不冻结E.大肠杆菌样本，直接用样品接种新的培养瓶，开始一种新的文化。 在实际的环境，目前的制度设计必须克服一些限制扩大其潜在用途。整个系统的体积目前由每天消耗的介质限制（chemostatic操作期间，一种文化小瓶在0.33小时-1的典型的稀释率消耗大约0.5升每天介质的）。为了减少工作容积，进一步优化可以通过仔细的模拟与plumbi药物抑制人口动态实现NG参数25。 最后，morbidostat是容易适应范围广泛的细菌培养实验。例如，通过附着的发光二极管，该系统可以被重新配置为一个光生物反应器，使蓝细菌或微藻26的进化监控。该模块可与吸附剂通过在电池组中无线地操作该装置可以扩展到厌氧和微需氧培养在气密容器中。作为另一实例，一种毒素的浓度可以逐渐增加来设计是与靶化学27抗性菌株。

自推出第一种抗生素药物青霉素，微生物的耐药性已经发展成为一个全球性的健康问题1。尽管抗生素抗性的获得可在体内回顾性研究，这些实验的条件通常不会在整个演化2控制。另外，自适应进化实验室可以从抗生素药物3显示环境下的应力或选择压力微生物物种的分子进化。最近，抗生素耐药性的许多良好控制的进化实验已经阐明抗生素耐药性的出现。例如，奥斯汀的小组展示了在一个适当设计的微流体隔间4环境迅速出现。最近开发的morbidostat诱导药物的选择压力下5,6系统性的突变。该morbidostat，微生物SELEC该连续调整抗生素浓度维持几乎恒定的人口和灰装置，是从在微生物学7,8中使用的波动测试的一大进步。在波动测试，抗生素药物以高浓度注入，幸存的突变体进行筛选并计数。相反，在morbidostat微生物都在不断的挑战，并获得多个突变。 该morbidostat功能类似恒，由诺维克和Szliard于1950年发明了一种培养装置，通过同时稀释微生物种群连续9提供营养保持一个恒定的人口。自推出以来，恒化已提前和完善。目前的微流控恒化已经达到了纳升和单细胞的能力。然而，这些装置不适合于适应进化实验，这需要许多突变事件10,11的大细胞群体。近日，迷你用约10 mL工作体积恒化器也已开发以补升规模的生物反应器和微流体恒化器12,13之间的间隙。 在这里，我们提出了一种抗生素耐药性的研究设计和使用成本低，自动化morbidostat。所提出的模块可以在摇床培养箱，在微生物实验室通过更少的硬件要求使用。开源固件也很容易适合于适应进化的特定的应用，如代谢工程3。最后，morbidostat被集成到抗生素药敏试验14复用微流体平台。

<table><tbody><tr><td>Environmental Shaker Incubator</td><td>BioSan</td><td>ES-20</td><td></td></tr><tr><td>Arduino Leonardo board</td><td>Arduino</td><td>Leonardo</td><td></td></tr><tr><td>680 Ohm Carbon Resistor</td><td>Digikey</td><td></td><td>Bias resistor for LED</td></tr><tr><td>100k Ohm Carbon resistor</td><td>Digikey</td><td></td><td>Bias resistor for phototransistor</td></tr><tr><td>940 nm light emitting diode</td><td>Bright LED Electronic</td><td>BIR-BM13E4G-2</td><td>Optical density measurement</td></tr><tr><td>940 nm phototransistor</td><td>Kodenshi&amp;nbsp;</td><td>ST-2L2B</td><td>Optical density measurement</td></tr><tr><td>Darlington pair IC Toshiba</td><td>Mouser</td><td>ULN2803APG</td><td>this IC drives micropumps and magnetic stirring unit</td></tr><tr><td>5 V DC brushless fan</td><td>ADDA</td><td>AD0405LX-G70</td><td>spec: 5 V supply voltage and 80&amp;nbsp;mA; available www.jameco.com</td></tr><tr><td>Piezoelectric micropump</td><td>CurieJet</td><td>PS15I-FT-5L</td><td>Pressure &gt; 3&amp;nbsp;kPa; Flow rate &gt;5 ml/min</td></tr><tr><td>Tygon 3350 Tuning</td><td>Saint Gobain</td><td>ABW00001</td><td>ID: 1/32&quot; OD: 3/32&quot; L:50&#039;&amp;nbsp;</td></tr><tr><td>Magnetic Stir bar</td><td>COWIE</td><td></td><td>tapered shape dim: 10&amp;nbsp;mm x 4 mm</td></tr><tr><td>Glass scintillation 20 ml vial</td><td>DGS</td><td></td><td>Pyrex glass 28 mm (diameter) x 61 mm (height)</td></tr><tr><td>Culture vial holder</td><td></td><td></td><td>Custom made from Polyformaldehyde&amp;nbsp;</td></tr><tr><td>Silicone</td><td>Dow Corning</td><td>Sylgald 184</td><td>used to seal the glass vial</td></tr><tr><td>Medium bottle</td><td>VWR</td><td>66022-065</td><td></td></tr><tr><td>Difco M9 minimal salt 5x</td><td>BD</td><td></td><td>Medium</td></tr><tr><td>Cadamino Acid</td><td>BD</td><td></td><td>Medium</td></tr><tr><td>glucose</td><td>Sigma</td><td></td><td></td></tr><tr><td>Agar Bateriological</td><td>Oxoid</td><td></td><td>for agar plate</td></tr><tr><td>Luria Bertani medium</td><td></td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Leica Microsystems</td><td>Leica DMI-LED</td><td>used for microfluidic measurement Use 40X objective NA = 0.55</td></tr><tr><td>Microscope Incubator</td><td>Live Cell Instrument</td><td>CU-109</td><td>used for microfluidic measurement</td></tr><tr><td>Solenoidal valves</td><td>Pneumadyne</td><td>S10MM-31-12-3</td><td>Normally open 1.3 Watt 12 Vdc</td></tr><tr><td>USB interface card</td><td>Hobby Engineering</td><td>USBIO24-R Digital I/O Module&amp;nbsp;</td><td>for microfluidics measurement</td></tr><tr><td>Air compressor</td><td>Rocker Scientific</td><td>ROCKER 440</td><td>Pressure source for microfluidcs; Max. Pressure 80 psi</td></tr><tr><td>Male luer-lock fittings to 1/8&quot; barb</td><td>ValuePlastics.com</td><td>MTLL230-1</td><td>used for microfluidic control</td></tr><tr><td>1/8&quot; barb to 10-32 threaded port</td><td>ValuePlastics.com</td><td>B-1</td><td>used for microfluidic control</td></tr><tr><td>Female luer-lock fittings to 10-32 threaded port</td><td>ValuePlastics.com</td><td>KFTL-1</td><td>used for microfluidic control</td></tr><tr><td>NPN darlington transistor 500 mA, 40&amp;nbsp;V (2N6427)</td><td>DigiKey.com</td><td>2N6427GOS-ND</td><td>used for microfluidic control</td></tr><tr><td>10 kOhm, carbon film resistor, 0.25 W</td><td>DigiKey.com</td><td>P10KBACT-ND</td><td>used for microfluidic control</td></tr><tr><td>Tantalum capacitor, 10&amp;nbsp;&amp;mu;F, 25 V, 10%</td><td>DigiKey.com</td><td>478-1841-ND</td><td>used for microfluidic control</td></tr><tr><td>Andor CCD camera</td><td>Andor</td><td>Zyla 4.2 Plus SCMOS</td><td>used for microfluidic on chip imaging</td></tr><tr><td>ELISA plate reader</td><td></td><td></td><td></td></tr><tr><td>two component Silicone&amp;nbsp;</td><td>Momentive</td><td>RTV 615</td><td>used for microfluidic chip fabrication</td></tr><tr><td>SU-8 photoresist</td><td>Micrchem</td><td>SU8 2015</td><td>used for microfluidic chip fabrication</td></tr><tr><td>AZ4620 photoresist</td><td>Clariant</td><td>AZ 4620</td><td>used for microfluidic chip fabrication</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC 32G</td><td>used for microfluidic chip fabrication</td></tr><tr><td>20 Gauge Syringe Needle</td><td>BD</td><td></td><td>used for microfluidic chip fabrication</td></tr><tr><td>Labcycler</td><td>Sensoquest</td><td>Labcycler</td><td>PCR</td></tr><tr><td>DNA polymerase</td><td>Toyobo</td><td>KDO Plus</td><td>PCR amplification</td></tr><tr><td>Trimethoprim</td><td>Sigma</td><td></td><td></td></tr><tr><td>Plate reader</td><td>Biotek</td><td>Synergy H1 hybrid&amp;nbsp;</td><td>antibiotic resistane measurement</td></tr></tbody></table>

design and use of a low cost, automated morbidostat for adaptive evolution of bacteria under antibiotic drug selection

我们描述用于表征抗生素耐药性的进化途径一种低成本，可配置morbidostat。的morbidostat是连续监测细菌的生长并动态调整的药物浓度，因为它们进化获得抗药性不断挑战细菌细菌培养装置。该器件具有的〜10毫升的工作容积，并且完全自动化，并配备了光学密度测量结果和微型泵为介质和药物递送。为了验证平台，我们测得的逐步收购大肠杆菌 MG 1655耐甲氧苄啶，以及集成设备与复用微流体平台来研究细胞形态及药敏。该方法可以按比例放大的抗生素耐药性的实验室研究，并可扩展至适应性进化为代谢工程和其它细菌培养试验菌株改进。

上述morbidostat在图1中概略显示。在共同morbidostat操作，包括实验进化，药敏试验和细胞形态的检查，在大肠杆菌进行了验证大肠杆菌 MG1655文化受到甲氧苄氨嘧啶（TMP），常用的抗生素药物5,6。 TMP诱导耐药性非常鲜明逐步增加，并且该突变围绕二氢叶酸还原酶（DHFR）基因簇。因此，TMP是用于验证morbidostat动作的高效标准药物。每一天，微生物预计增长高于预设光密度阈值，并触发药物注射， 如图2a中 。药物注射预期抑制生长，其结果，降低了光密度。可能需要几个试运行，以确定最佳的药物浓度。作为一个经验法则，我们的人口增加Ë发现该药物中未能抑制增长药后介质的浓度为5倍。在实验的整个过程后，每日冷冻样品解冻并用于确定IC 50值，将药物抗性水平的定量测量。在图2b的曲线图显示了在抗生素耐药性的时间增加。耐药性增加了约1500倍，1000〜微克/毫升以上〜12天，在实验室进化5以前的研究结果一致，临床分离19。在最后一天的突变体的DHFR点突变通过Sanger测序测量，并在表2中列出，其中一个突变发现位于启动子区，并表示在启动子区域的单点突变可以显著改变DHFR的表达水平酶20。在位置49910另一个突变位于DHFR基因区，靠近行为在DHFR的香港专业教育学院网站21酶。具有多个突变的耐药性的获取证明morbidostat的有用性，作为在含有琼脂平板药物的选择倾向赋予唯一的单一突变。 作为细胞形态的更灵敏的读出，与抗生素易感性单细胞形态检查在平行进行的，复用的微流体平台14。 图3示出了复用的微流体芯片的设计。细菌细胞的显微照片（ 图5）揭示了下TMP的亚抑制浓度的野生型形态的改变和突变菌株。野生型菌株表现出显著成丝，而突变株没有显示出显著成丝。在单细胞水平这种形态变化所用的快速诊断抗生素抗性的使用。 图4显示器上的智在不同浓度的TMP的对生长曲线。突变的样品显示了在抗生素药物抗性的显著增加。 图4中的芯片上生长数据也与从图2b中显示的板读取器的IC 50值一致。 <img alt="图1" src="/files/ftp_upload/54426/54426fig1.jpg" /> 图 1: 示意了morbidostat的 morbidostat是测量微生物群体的光密度，并相应地调整的药物浓度的连续培养装置。该器件采用药物注射方式和药物稀释模式下运行。 <a href="https://www.jove.com/files/ftp_upload/54426/54426fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54426/54426fig2.jpg"/&gt; 图2: 生长曲线和抗生素耐药水平的增加 （a）根据反馈代表生长曲线。微生物的生长是由抗生素药物，其注入由一个阈值算法触发的抑制。当正的增长率上升到超过预设的阈值的光密度（OD TH = 0.086），器件可以切换到药物注射方式。否则，该设备在药物稀释模式下运行。红色和紫色的箭头表示分别转换到药物注射和药物稀释模式。 （b）在暴露于甲氧12天的细胞的抗性水平。该IC 50显示抗生素耐药性的独特逐步增加。 <a href="https://www.jove.com/files/ftp_upload/54426/54426fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img al<html> T ="图3"SRC ="/文件/ ftp_upload / 54426 / 54426fig3.jpg"/&gt; 图3: 为调查细胞形态和片药敏微流体装置 （a）该芯片布局的显微。绿色和蓝色舱室的生长和药物介质腔室，分别与红色布局表示控制层。生长室的尺寸为450微米（ 长 ）×400微米（W）×8微米（ 小时 ）。生长室装有细菌和药物腔装有TMP。比例尺为1厘米。 （ 二 ）放大查看相同的芯片。比例尺= 500微米。 （ 三 ）放大图两院之前和混合后， <a href="https://www.jove.com/files/ftp_upload/54426/54426fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "SRC ="/文件/ ftp_upload / 54426 / 54426fig4.jpg"/&gt; 图4: 大肠杆菌 的片上生长曲线 大肠杆菌 暴露于各种浓度的TMP:（ 一 ）野生型祖应变和（b）突变株（在12天）。突变样本显示抗生素耐药性一个显著上升。 <a href="https://www.jove.com/files/ftp_upload/54426/54426fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54426/54426fig5.jpg" /> 图5: 单细胞形态 （ 一 - d）与TMP的亚抑菌水平在治疗后的细胞的明场图象。注意野生型细胞在显著成丝（b）中 ，这是在突变体缺失（ 四 </s仲&gt;）。面板（E - H）是数字化的图像（A - D）。所有细菌的图像是由CCD摄像头，支持40倍的放大倍率的目标。 <a href="https://www.jove.com/files/ftp_upload/54426/54426fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 补充图1:尺寸和文化小瓶架装配 <a href="https://www.jove.com/files/ftp_upload/54426/Supplemental_Figure_1.pdf" target="_blank">请点击这里下载此文件。</a> 补充图2:对于LED和光电探测器电路原理图 <a href="https://www.jove.com/files/ftp_upload/54426/Supplementary_Figure_2.PNG" target="_blank">请点击这里下载此文件。</a> 补充代码:自定义脚本来算从微流控芯片获得的细胞图像细胞数 <a href="https://www.jove.com/files/ftp_upload/54426/renamed_40eb7.pdf" target="_blank">请点击这里下载此文件。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2">样品</td><td colspan="11"> TMP药（微克/毫升） </td></tr><tr><td colspan="2"> 1-3日</td><td> 0 </td><td> 0.06 </td><td> 0.12 </td><td> 0.25 </td><td> 0.5 </td><td> 1 </td><td> 2 </td><td> 4 </td><td> 8 </td><td> 16 </td><td> 32 </td></tr><tr><td colspan="2">第4天</td><td> 0 </td><td> 1 </td><td> 2 </td><td> 3 </td><td>五</td><td> 10 </td><td> 20 </td><td> 40 </td><td> 80 </td><td> 160 </td><td> 320 </td></tr><tr><td colspan="2"> 5-7日</td><td> 0 </td><td> 2 </td><td> 4 </td><td> 8 </td> &lt;TD&gt; 15 </td><td>三十</td><td> 60 </td><td> 120 </td><td> 240 </td><td> 480 </td><td> 960 </td></tr><tr><td colspan="2"> 8-12日</td><td> 0 </td><td> 3 </td><td> 7 </td><td> 15 </td><td>三十</td><td> 60 </td><td> 125 </td><td> 250 </td><td> 500 </td><td> 1000 </td><td> 2000 </td></tr></tbody></table> 表1:用于确定IC 50 的板读取器测量药物浓度的微克/毫升单位的药物浓度显示为每日冷冻样品。随着细胞培养更高耐药性，所使用的药物浓度也相应增加。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>没有</td><td>位置</td><td> SNP </td><td>注意</td></tr><tr><td> 1 </td><td> 49894 </td><td> C-&gt; T </td><tD&gt; DHFR基因</td></tr><tr><td> 2 </td><td> 49910 </td><td> T-&gt;一个</td><td> DHFR基因</td></tr><tr><td> 3 </td><td> 49795 </td><td> C-&gt; T </td><td> DHFR启动</td></tr></tbody></table> 表2:突变的DHFR由Sanger测序鉴定的代表性序列数据显示在最后一天突变体（12天）三DHFR点突变。启动子和基因区域含有一个与2个SNPs，分别。在PCR中使用的正向和反向引物为3&#39;TCTAGAGAGATCATTACCGA GGACCGCGAACATCTGTCAT5&#39;和3&#39;ACTAGT TTACCGCCGCTC CAGAATCTCAA AGCAATAGCTG5&#39;。

Watch this Scientific Journal Video about Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection at JoVE.com

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 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 ...

design-use-low-cost-automated-morbidostat-for-adaptive-evolution

Nanyang Technological University

Research

JoVE Journal

Genetics

9.7K 次观看.  National Tsing Hua University. 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.

视频: Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

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) ...

科研

遗传学

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 ...

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 ...

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

实验进化方法中疟原虫介导抗生素耐药性的定量化

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

免疫与感染

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions.

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments.

大肠杆菌长期进化实验中的每日转移、存档种群和测量适应性

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

生物学

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

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 ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

复用的恒化器阵列的设计与应用

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

用于宿主靶向治疗结核病临床前检测的自动化培养系统

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...