作者感谢罗伯托Kolter声称（哈佛医学院）的宝贵意见和共培养这种屏幕的发展过程中的协助。她还感谢马修权力来读取原稿的清晰度，以及嘉义程与获得图6的援助。

1。选择一个报告基因，并构建一个荧光转录记者 对于枯草芽孢杆菌： <ol><li>见参考图19描述的荧光转录记者在枯草芽孢杆菌建设协议的朱庇特的文章。 </li></ol> 对于其他细菌物种： <ol><li>确定是在感兴趣的生理反应上调的基因。这可以是基于现有的文献中或特定条件下，微生物的转录分析。 </li><li>构建荧光转录记者这个基因充当的表型变化的代理。该构建体应包括这个上调的基因的启动子驱动的生产合适的荧光蛋白（ 见图1）。 </li><li>整合该构建到染色体上的中性轨迹。这确保了原生章感兴趣的基因的ulation不被中断，并且避免了需要的质粒的选择机制（ 例如抗生素），可能与环境微生物的生长产生干扰。 </li></ol> 2。确定共培养条件 对于枯草芽孢杆菌P TAPA-YFP记者： <ol><li>使用0.1X LB，伦诺克斯（1克蛋白胨，0.5克酵母提取物，每升0.5克氯化钠）的媒介记者，因为B。枯草基质生产是最小的在Luria肉汤20。这种介质可以让B。枯草芽孢杆菌菌落长到亚毫米波但观察到的尺寸，同时允许从土壤中不同类群增长5。 </li><li>包括100毫米MOPS缓冲，以减少潜在的pH值的变化。 </li></ol> 对于其他细菌物种： <ol><li>利用转录公布的数据或实证检验各种培养条件，以确定一个地方的微生物生长，但推荐阅读。n个荧光记者是可以忽略不计（允许当记者株生长在共培养与诱导微生物被检测到其激活。） </li><li>筛选从贫营养环境（如土壤）环境微生物时，使用介质，养分含量低（相对于传统丰富的微生物培养基），因为具有高营养条件提出了21多的时候贫营养细菌不生长。低营养培养基也降低菌落大小，增加了屏幕的吞吐量。 </li><li>选择具有低背景荧光和良好的光学清晰度的媒介。 </li><li>优化生长温度，使双方的报告菌株和环境微生物同时增长。 </li><li>考虑加入一种缓冲剂。在板使用缓冲区将减少检测生理pH值为22-介导的变化的可能性，除非这种相互作用的兴趣。 </li></ol>3。记者准备等分 对于枯草芽孢杆菌P TAPA-YFP记者： <ol><li>从-80℃冷冻股票数报告菌株上用无菌牙签或涂药棒新鲜的LB平板。 </li><li>一夜之间成长在30°C </li><li>在液体培养基中进行连续稀释，尽量减少从生长在固体培养基上产生的背景荧光： <ol><li>接种LB的5毫升的液体培养和振荡，在37℃下生长</li><li>当培养物达到外径600〜0.6，稀释到5ml新鲜LB至0.02外径600。 </li><li>振荡培养直到达到外径600〜0.6再次增长在37℃。 </li><li>重复序列的增长稀释共有3倍的。 </li><li>让我们最后连续稀释培养成长为外径600〜0.4。 </li></ol></li><li>加入甘油至15-20％。 </li><li>分装50-200微升，用0.5 ml离心管中，并冻结在-80°C。 </li><li>妈（他们会二次筛选过程中需要）科相当于等份记者的非荧光母株。 </li></ol> 对于其他细菌物种： <ol><li>使用的细胞，具有低背景荧光（ 即正在条件下有自中，记者使用的启动子表达的小下生长）。 </li><li>令和冻结包含报告菌株已知CFU /毫升（菌落形成每毫升为单位）等分，使菌落适当数量可以在每个屏幕上共培养板中生长（参见上面的内容）。 </li><li>使相当于等分的非荧光亲株（他们将二次筛选过程中需要）。 </li></ol> 4。获取土壤样品<ol><li>收集到的土壤无菌锥形管或用刮刀无菌袋，丢弃前0.5厘米暴露表面的土壤。 </li><li>加无菌生理盐水（0.85％NaCl）中以每1克10ml的比例的土壤，使土壤泥浆。 </li><li>选择一个方法，从土壤颗粒赶走细菌：1）直接使用新鲜样品或b）样品冷冻后延迟使用。 <ol><li>可立即使用，涡流浆料1分钟。 </li><li>对于延迟使用，混合土壤泥浆搅拌机中三个1分钟周期，把搅拌杯冰上1分钟休止符混合周期之间。 </li></ol></li><li>让土壤泥浆沉降〜1分钟。 </li><li>移动上面的水层至新管。 </li><li>添加甘油至15％-20％的最终浓度。 </li><li>分装50-200微升，用0.5 ml离心管中，并冻结在-80°C。 </li></ol> 5。决定冻结记者土壤等分的CFU /毫升<ol><li>解冻冻结的土壤和记者等分。土壤中的微生物可以在冰上解冻。由于二枯草裂解，在4℃，这些等份应迅速解冻在RT，以尽量减少在低温下使用的时间。 </li><li>使两个重复赛瑞亚升稀释液（10 -8）的0.1倍LB或其他等渗缓冲。 </li><li>板5微升斑点各系列稀释到将被用于共培养筛选的相同培养基的琼脂平板上的。 </li><li>生长在RT（或将要使用用于筛选的温度）。 </li><li>第二天，计数菌落数的每个点内，并计算各冷冻等分试样的菌落形成单位/毫升。 </li></ol> 6。确认分装浓度为传播筛板<ol><li>解冻冷冻等分如步骤5.1。 </li><li>稀释至1×10 5，2.5×10 5，5×10 5，1×10 6和2.5×10 7 CFU /毫升。 </li><li>将每个稀释50μL点至个别板块的中心。这些应该产生板，每板25000菌落计算出的最佳值，以及2 - 和5 - 倍多和少，允许要确定的最好的实际稀释。 </li><li>加入约20无菌玻璃（3毫米），由珠轻轻拍打它们拖到板。珠提供了整个板块分布更均匀的菌落比热弯玻璃吊具一样。 </li><li>通过保持板在台式和他们摇晃来回，旋转它们，你的工作，直至液体被吸收分散细胞。不要继续摇晃珠一旦板是干燥的，否则它会开始杀死细菌。 </li><li>在翻转板和丢弃珠串成含乙醇的废物烧杯中。 </li><li>让板在正确的温度下生长的实验（ 如 24小时）的时间为您的检测（ 如 24℃/ RT）。 </li><li>用解剖体视镜，计数菌落在视图中的两个或多个字段的数量。 </li><li>计算每个区域的菌落数，并确定有多少菌落在每个板。 </li><li>调整将来的稀释是必要的。菌落的实际数量是不为对各可比屏板相同数量一样重要。 </li></ol><ol><li>记者解冻等分（如果冻土等分），如步骤5.1。 </li><li>在0.1倍LB或其他低营养，等渗溶液稀释记者（于浓度在第6优化）。 <ol><li> 冻土：稀释到浓度在0.1倍LB或其他低营养，等渗溶液第6条进行了优化。 </li><li> 对于新鲜的土壤：使新鲜的土壤泥浆稀释，基于预测的土壤泥浆的CFU / ml的范围可以从10 -4〜10 -9 CFU /毫升。 </li></ol></li><li>现货50微升的土壤和记者稀释到共培养屏板的中心。此外，独板的土壤和单独作为对照组的记者。 </li><li>传播与步骤6.4-6.6描述使用玻璃珠。 </li><li>如果有激活荧光报告，条纹或板块的记者在这些条件下，成长为在筛查阳性对照用已知的生长条件。 </l我&gt; <li>孵化在24℃，24 - 28小时（或适用于您的报告/分析） </li></ol> 8。屏幕共培养板的荧光<ol><li>使用明照明，集中你的体视镜，使殖民地是雪亮的。 </li><li>看土板只有以确定您的土壤样品是否已经自发荧光的菌落。如果是这样，这种土壤可能导致高误报率（在第二次筛选随之增加）。 <ol><li>使用高比率记者：在你的共培养板的土壤，以增加的可能性，诱导将由多个记者菌落包围，减少它们将被检测为假阳性的可能性（ 图2）。 </li><li>使用不同的荧光蛋白（一个发射在不同的频道）。 </li></ol></li><li>确定所述测定时间。对于大多数新记者，潜在诱导的定时是未知的，因此必须根据经验来确定。 <ol><li>立即开始殖民地成为清晰可见的解剖体视镜（放大倍率〜30X），并继续定期检查板，直到增长已经停止和/或背景荧光过高筛查荧光。 </li><li>一旦潜在的诱导的时间窗口被确定为一个特定的记者，它应该是相似的包含该记者所有共培养板，简化了屏板的监测。为B。枯草 P TAPA-YFP记者，适当的时候来检查板是24-28小时之间电镀细胞后，为B。枯草 P SSPB-YFP记者，这是26-32小时增长之间。 </li></ol></li><li>最大限度地发挥你的荧光灵敏度： <ol><li>确保你的荧光解剖范围是在一个黑暗的房间或遮光窗帘包围。感应可能会比一个组成生产的FP那么激烈，需要更高的敏感拉了一检测。 </li><li>等待一段时间以荧光灯为稳定和你的眼睛试图从你的共培养板（至少1-2分钟）荧光检测前适应黑暗。 </li></ol></li><li>采用阳性对照（如果有的话），请确保您所使用的放大倍率，可以检测荧光。放大倍率通常是最好的，如果你的视野大约是30-50X典型的菌落直径（200-400X）。 </li><li>关闭亮光，打开快门为您的荧光。 </li><li>之后，你的眼睛已经适应了黑暗，慢慢移动板来回穿过你的视野，寻找亮点。 <ol><li>开始从板的顶部，并使用之字形图案移动板一侧到另一侧，你向板的底部移动。 </li><li>实践移动板在明得到的感觉如何慢慢移动到板上，以确保您所覆盖整个河畔面对区域。 </li><li>移动板慢慢就好了殖民地不变得模糊。这是更好地过采样表面，而不是错过的地方。 </li><li>后一个完整的扫描，把板90°，重复。人类的眼睛是在通过这种方法检测，即使微弱的荧光非常好。 </li></ol></li><li>如果检测荧光，停止移动板回去找荧光区。 </li><li>转动明上慢慢地，确定与细菌菌落（而不是自身荧光土壤碎屑或媒体组件）的荧光是否相关联。如果是这样，非荧光菌落近端向荧光菌落是推定诱导有机体。 </li></ol> 9。隔离推定诱导有机体<ol><li>一旦诱发（荧光）的殖民地已经确定，隔离这些菌落分泌诱导化合物。 <ol><li>如果足够高的浓度记者菌落是长在板（&gt; 0.5:1记者：土壤菌落形成单位），则推定诱导的菌落会由多个荧光菌落包围（再次参见图2）。 </li><li>在情况下，共培养成长的复杂性使得它含糊其殖民地的诱导剂，隔离多个潜在诱导的菌落为随后的测试中次要屏幕。 </li></ol></li><li>本地化您想选择在视场中心的殖民地。 <ol><li>如果它们是靠近所述板的边缘，旋转板，使该板的唇距离的优势手（ 即 ，如果你是右利手，把该板的唇在左侧）。这可以让你接近殖民地以低角度，从而提高您采摘的准确性。 </li></ol></li><li>将新鲜的板，条痕假定诱导有机体到附近，以及废烧杯中所用的技巧丢弃到。 </li><li>离开荧光灯泡上，把明亮的灯光慢慢上，这样你就能够识别（通过形状和位置），荧光菌落和周围的假定诱导有机体。你可能需要去来回光几次能够确定你要挑的时候没有荧光，只有明亮的光线殖民地。 </li><li>用玻璃棒（200毫米直径的毫米（长）x 5）拿起无菌，圆200μL凝胶加载提示，并拿着它就像一支铅笔。这是采摘工具，你会用它来分离单个菌落。 </li><li>休息你的手掌外侧打击阶段，以稳定其工作表面上。将你的手的内侧，拇指侧另一只手来稳定你的采摘工具。 </li><li>如有必要，再次翻转荧光和明之间的意见，以确定你要挑菌落。 </li><li>保持板面以上枪头，把它移动到你的视野和居中的殖民地上面你想挑。针尖会失焦。 </li><li>用你的手的外缘（这是搁在显微镜载物台或工作表面），转动枪头慢慢下到殖民地被拾起。触摸它很轻，试图尽量减少多少其他殖民地的尖端接触。 </li><li>不旋转的拾取工具，条纹到新鲜平板的截面图。用柔软的触感蔓延到避免过切的琼脂。因为通过该方法转移细胞的数量是相当小的（菌落都远小于通常操作），一个单一的连续的条纹将导致隔离的菌落。 </li><li>与其他公认的诱导有机体重复此过程。 </li><li>孵育板在24°C（或您的检测的温度）。 </li></ol> 10。数推定诱导有机体获得分离的单菌落<ol><li>使用立体镜，确定 - 基于群体结构或形态 - 是否有包含在你的每一个假定的挑IND不同的群体类型ucing生物。 <ol><li>看看使用阶段，你可以照亮从两个以上，以及从下面的殖民地，以发现潜在的群体差异的殖民地。 </li></ol></li><li>重新划每个不同形态类型到一个新的板块和孵化，直到成长。 </li><li>重新划一次，让成长。如果不同的形态类型仍然存在，继续重新划到纯洁。 </li></ol> 11。复试推定诱导有机体中学屏<ol><li>重新测试所有的二级屏幕的假定诱导有机体，以确定哪些正在激活荧光转录记者。第二屏幕由一个草坪荧光转录记者株菌落，随着控制盘，其上公认的诱导有机体修补或发现的。 </li><li>设置三个相同的板：1含有荧光转录报告菌株的菌落草坪（在相同浓度下一个S为在共培养筛选使用）;含有无荧光报道与野生型亲代菌株的菌落草坪1，和一个不包含草坪。 <ol><li>标记每个板的背面的顶部，以确定板的方向。 </li><li>加为补丁/斑点的位置标记;高达10推定的诱导有机体可以在每个板组（ 图3）进行测试。 </li><li>解冻草坪等分（记者和非荧光亲株）如步骤5.1。 </li><li>用无菌小珠，如步骤6.4传播50μl的5×10 5 CFU / ml的稀释液到草坪板（或其它优化的稀释液）。 </li><li>让板干燥。 </li></ol></li><li>修补或现货推测诱导有机体： <ol><li>选择补丁如果一个更容易和更快的方法是需要的，并且它是可以接受的细胞沉积的不太精确的数目。修补： <ol><li>触摸无菌牙签将菌落测试 - 不要拿起所有的细胞。</li><li>补丁（做一个小的条纹）到空白盘。 </li><li>补丁重复用新鲜牙签到记者板上。 </li><li>补丁重复用新鲜牙签到控制板。 </li></ol></li><li>选择点状出血，如果一个定量的和可重复的方法是需要的。斑点允许进行归一化沉积的细胞数量（参见附图5为完整的细节），并允许不同的诱导有机体进行比较的相对效力。被发现： <ol><li>重悬推定诱导有机体的1毫升液体培养基的无菌塑料试管中。 </li><li>就拿外径600的再悬浮的。 </li><li>用公式X = 250÷（外径600 - 0.5），体积×各悬浮加入500微升液体介质得到一个解决方案，外径600 0.5。执行多个稀释时正常化外径的，因为你可以用相同体积（50此方法简化了所需的移液步骤0微升）为您的所有dilutants的。 </li><li>现货1微升每个外径标准化悬浮到每个三大板块组成。 </li></ol></li></ol></li><li>让成长在24℃，24〜28小时（或适用于您的报告/分析）。 </li><li>使用荧光解剖显微镜来识别假定的诱导有机体激活荧光报告菌株，但不是你父母的对照菌株。这些菌株是你的正面命中 - 分泌诱导你感兴趣的表型化合物对环境的微生物。 </li></ol>

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	<li>Berdy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactive+microbial+metabolites.">Bioactive microbial metabolites.</a> J. Antibiot. 58, 1-26 (2005).</li><li>Lopez, D., Vlamakis, H., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multiple+cell+types+in+Bacillus+subtilis.">Generation of multiple cell types in Bacillus subtilis.</a> FEMS Microbiol. Rev. 33, 152-163 (2009).</li><li>Vlamakis, H., Aguilar, C., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+fate+by+the+formation+of+an+architecturally+complex+bacterial+community.">Control of cell fate by the formation of an architecturally complex bacterial community.</a> Genes Dev. 22, 945-953 (2008).</li><li>Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yehuda, S., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruiting+body+formation+by+Bacillus+subtilis.">Fruiting body formation by Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 11621-11626 (2001).</li><li>Shank, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+interactions+that+result+in+Bacillus+subtilis+forming+biofilms+are+mediated+mainly+by+members+of+its+own+genus.">Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 1236-1243 (2011).</li><li>Piston, D. W., Patterson, G. H., Lippincott-Schwartz, J., Claxton, N. S., Davidson, 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=.">.</a> Introduction to Fluorescent Proteins. , (2013).</li><li>Chudakov, D. M., Matz, M. V., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+and+their+applications+in+imaging+living+cells+and+tissues.">Fluorescent proteins and their applications in imaging living cells and tissues.</a> Physiol. Rev. 90, 1103-1163 (2010).</li><li>Shaner, N. C., Patterson, G. H., Davidson, 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=Advances+in+fluorescent+protein+technology.">Advances in fluorescent protein technology.</a> J. Cell Sci. 120, 4247-4260 (2007).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Methods. 2, 905-909 (2005).</li><li>Nicolas, 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=Condition-dependent+transcriptome+reveals+high-level+regulatory+architecture+in+Bacillus+subtilis.">Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis.</a> Science. 335, 1103-1106 (2012).</li><li>Middleton, R., Hofmeister, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+shuttle+vectors+for+ectopic+insertion+of+genes+into+Bacillus+subtilis.">New shuttle vectors for ectopic insertion of genes into Bacillus subtilis.</a> Plasmid. 51, 238-245 (2004).</li><li>Shimotsu, H., Henner, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+single-copy+integration+vector+and+its+use+in+analysis+of+regulation+of+the+trp+operon+of+Bacillus+subtilis.">Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis.</a> Gene. 43, 85-94 (1986).</li><li>Guerout-Fleury, A. M., Frandsen, N., Stragier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmids+for+ectopic+integration+in+Bacillus+subtilis.">Plasmids for ectopic integration in Bacillus subtilis.</a> Gene. 180, 57-61 (1996).</li><li>Semsey, S., Blaha, B., Koles, K., Orosz, L., Papp, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+integrative+elements+of+rhizobiophage+16-3+can+integrate+into+proline+tRNA+(CGG)+genes+in+different+bacterial+genera.">Site-specific integrative elements of rhizobiophage 16-3 can integrate into proline tRNA (CGG) genes in different bacterial genera.</a> J. Bacteriol. 184, 177-182 (2002).</li><li>Charpentier, E., 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=Novel+cassette-based+shuttle+vector+system+for+gram-positive+bacteria.">Novel cassette-based shuttle vector system for gram-positive bacteria.</a> Appl. Environ. Microbiol. 70, 6076-6085 (2004).</li><li>Yang, H. Y., Kim, Y. W., Chang, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+an+integration-proficient+vector+based+on+the+site-specific+recombination+mechanism+of+enterococcal+temperate+phage+phiFC1.">Construction of an integration-proficient vector based on the site-specific recombination mechanism of enterococcal temperate phage phiFC1.</a> J. Bacteriol. 184, 1859-1864 (2002).</li><li>Choi, K. H., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mini-Tn7+insertion+in+bacteria+with+single+attTn7+sites:+example+Pseudomonas+aeruginosa.">mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa.</a> Nat. Protoc. 1, 153-161 (2006).</li><li>Craig, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn7:+a+target+site-specific+transposon.">Tn7: a target site-specific transposon.</a> Mol. Microbiol. 5, 2569-2573 (1991).</li><li>Garcia-Betancur, J. C., Yepes, A., Schneider, J., Lopez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+of+Bacillus+subtilis+biofilms+using+fluorescence+microscopy+and+flow+cytometry.">Single-cell analysis of Bacillus subtilis biofilms using fluorescence microscopy and flow cytometry.</a> J. Vis. Exp. (60), e3796 (2012).</li><li>Lopez, D., Fischbach, M. A., Chu, F., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structurally+diverse+natural+products+that+cause+potassium+leakage+trigger+multicellularity+in+Bacillus+subtilis.">Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 280-285 (2009).</li><li>Vartoukian, S. R., Palmer, R. M., Wade, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+culture+of+'unculturable'+bacteria.">Strategies for culture of 'unculturable' bacteria.</a> FEMS Microbiol. Lett. 309, 1-7 (2010).</li><li>Romano, J. D., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas-Saccharomyces+interactions:+influence+of+fungal+metabolism+on+bacterial+physiology+and+survival.">Pseudomonas-Saccharomyces interactions: influence of fungal metabolism on bacterial physiology and survival.</a> J. Bacteriol. 187, 940-948 (2005).</li><li>Branda, S. S., Chu, F., Kearns, D. B., Losick, R., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+protein+component+of+the+Bacillus+subtilis+biofilm+matrix.">A major protein component of the Bacillus subtilis biofilm matrix.</a> Mol. Microbiol. 59, 1229-1238 (2006).</li><li>Zengler, K., 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=Cultivating+the+uncultured.">Cultivating the uncultured.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 15681-15686 (2002).</li></ol>

作者宣称，他们有没有竞争的财务权益。

其中本协议的固有的局限性在于，它依赖于微生物有机体的培养力。由于已经有据可查24，大多数微生物的生命在这个星球上不能（还）进行探讨，以当日的培养条件下生长。因此，一个巨大的正在发生在自然环境中的微生物物种之间的相互作用的数量会使用这种方法去未被发现。然而，由于我们的愿望是，不仅确定这种相互作用的存在，但后来也学习参与调解他们的机制和分子，培养这...

我们感兴趣的是了解细菌分泌的代谢产物是如何影响相邻的微生物的生理和发育。许多代谢产物进行了表征他们对其他微生物的生物活性的影响。两个良好描述的实例包括抗生素，抑制其它微生物的生长，而群体感应分子，从而改变其它微生物的全基因表达。然而，细菌产生有没有已知的生物活性1许多其他的小分子天然产品。我们推测，细菌已经进化和保存产生一些代谢产物的能力，因为他们让他们在调节范围内大多数细菌存在于复杂的微生物群落的微生物邻居的细胞生理学。 枯草芽孢杆菌的细胞类型我们一直专注于研究，涉及Bacil化学介导的微生物的相互作用LUS芽孢杆菌。这不仅是因为其为革兰氏阳性菌的模型和用于它的操作所得到的遗传工具的状态，也由于其分化为特征的细胞类型的能力。例子包括细胞，分别是：游泳;生产所需要的强大的生物膜形成的细胞外基质，能吸收来自环境的DNA，并形成孢子，等等2。每种类型的细胞表达的特性的转录调节子，使它们的生理和/或物理上不同的从他们的遗传上相同的同胞。根据许多生长条件下，多种细胞类型中B的单个菌落中共存的各种亚群枯草芽孢杆菌细胞3。虽然许多种细菌可以表现出类似的细胞类型异质性，这种现象已被特别好地研究了B。枯草芽孢杆菌 。 尤其是，基因是不饱和聚酯树脂在每一个特定的B的egulated 枯草芽孢杆菌细胞类型已被确定。识别这样的上调基因是这里所描述的工作至关重要，因为许多这些利益微生物的表型是很难或不可能直接观察。例如，我们不能直观地检测出性状如固体（1.5％）琼脂平板上游泳，即使B的亚群枯草芽孢杆菌细胞产生这些条件3下鞭毛。另一个例子是生物膜基质生产。基质的产生可以通过菌落形态可视化（因为它导致宏观皱殖民地），但仅限于特定的生长培养基，只有经过增长4多天。然而，通过了解哪些基因分化过程中被上调，我们可以构建转录记者，作为标记物的细胞分化成这些类型的细胞。 记者构造这些荧光吨ranscriptional记者组成的启动子对细胞类型特异性基因驱动的生产报告基因，例如一个荧光蛋白。实例包括对HAG-YFP（游泳细胞），P TAPA-YFP（生物膜基质产生细胞）和P SSPB-YFP（对于形成孢子的细胞），其中，P x表示的启动子区域进行X基因。这些报道构建整合到染色体上的中立位点（ 图1和见下文），从而使表型的天然调节保持不变。但是，现在，当一个细胞表达这种表型，它也表现为一种荧光蛋白。这提供了一个可视化容易读出的特定表型行为的活化，使我们能够筛选的微生物，激活这个生理反应。虽然这样的记者，在微生物学通常被使用，它们没有被广泛地在屏幕上以便识别适用此方法之前微生物之间Ÿ代谢相互作用被描述5。 有许多的细胞类型特异性记者菌株的设计和施工的重要考虑因素。我们已经利用专门的转录荧光记者，虽然其他类型的结构是可能的。我们不鼓励使用翻译融合作为标记物在我们的屏幕细胞类型的分化，但是，有两个原因：1）离开原生细胞类型特异性蛋白沉着的欲望，和2）认识到弥漫，细胞广泛的荧光会更容易发现比细胞内的局部泪点（常见的翻译融合）。 报告基因的选择 在决定使用转录为读出后，将报告基因，必须选择（ 如 LacZ基因，荧光，或荧光素酶）。 LacZ的有需要的至少是特殊的优势美化版设备来检测，但有假阳性微生物的环境中高得多的可能性。在我们的手中，紫胶+生物土壤中的微生物本底水平是高的惊人（&gt;&gt;土壤微生物的10％是蓝色的（紫胶+）对X-gal的平板;数据未显示）。这可能是通过滴定培养基中的X-gal的浓度，这可被优化以允许使用的X-gal的记者，尽管我们并没有试图这样。荧光素酶提供检测灵敏度高，是最正交记者：几乎没有任何机会的环境是微生物本身发光。然而，我们发现它很难识别仪器在我们的机构，允许发光检测在整个培养皿，因为大多数被设计为仅扫描局部区域的多孔板。也可能存在于可视化发光殖民地的方式，也让同步物理是并发症olation诱导有机体。当使用受信人可能有这样的可能，我们反而选择使用荧光转录记者，这是在证明B.工作枯草芽孢杆菌 ，提供了检测和土壤生物体中低假阳性率的足够的灵敏度，并允许使用容易获得的仪器为可视化和分离步骤。 荧光基团的选择 所选的特定荧光团将取决于你的细菌种类，您所使用的琼脂培养基，和特定的荧光滤镜设置您有可用。随着我国仪器仪表中，我们发现，无论是B。枯草芽孢杆菌菌落自己和他们生长在展出较少的背景荧光，当被用于YFP（黄色荧光蛋白）过滤器，使得在我们手中了记者优于GFP（绿色荧光蛋白）的琼脂。荧光蛋白的密码子使用的是经常用于真核生物的优化，因此有必要选择荧光团从文献中已知的任一中的细菌物种的工作，或者使用组成型启动子明确地对其进行测试。是大量的不断变化的荧光蛋白变种现有6，已在多个源7,8，其中一些对实验9选择合适的荧光蛋白质明确地提供指导审阅。 启动子的选择 一个启动子的选择将在很大程度上取决于你的细胞类型或感兴趣的表型。对于生物体，如B。枯草芽孢杆菌 ，某些细胞类型特异性的报告基因已经建立在文献中。对于其他细菌菌株，研究微阵列或转录的数据将需要提供哪些基因的条件下是高度上调的信息，其中的利益，我的细胞类型s的体现。最近的一项研究编目B的转录根据使用平铺芯片10 104不同的生长条件芽孢杆菌 。本文提供全面的信息基因在不同条件下高度上调哪些，这是非常宝贵的不太充分表征的表型。 而不是绘制精确的启动子区域的每个感兴趣的基因，我们通常只需使用序列200-500 bp的基因的启动子上游。确切的序列长度依赖于基因组的上下文：较短的区域被使用时，必须避免包括上游编码区从邻近的开放阅读框。 中性位点和整合 如何保持你的细菌菌株记者构造成为设计的荧光转录报告菌株的最后一个问题。在细菌中，目的基因经常保持在使用抗生素的选择质粒。然而，它可能无法共存期间使用抗生素而不杀死的微生物环境。如果质粒稳定地保持在你的细菌种类，它可能会成长含有质粒携带的记者在抗生素存在的细菌以制备的记者进行筛选，然后在共存本身中消除抗生素，希望该质粒将被充分地维持，以允许荧光。然而，如果质粒容易丢失更多的细菌，或胁迫条件下会丢失，这不会是一个可行的选择。在许多情况下，最好的解决方法是将本报道构建整合到细菌染色体上，允许稳定地维持记者的即使在没有选择的。为了使整合不会破坏你感兴趣的基因的正常表达调控，我们建议在染色体上的整合到一个网站异位该CAÑ​​充当“中性位点。” B.中枯草这些整合位点是基因-突变时-表达的表型在某些基本培养基（允许不用抗生素的选择应确定整合体），但不改变在富媒体的增长或产孢率，并且包括这些基因作为AMYE，LACA，的thrC，pyrD，的gltA，和萨卡 （输送到利用淀粉的能力，β-半乳糖苷，苏氨酸，尿嘧啶，谷氨酸盐，蔗糖，分别）11-13。 而整合这些基因得到了可靠的使用多年的B。枯草芽孢杆菌 （尤其是在的AmyE和LACA），类似知识可能不提供对基因在许多其他细菌物种。利用噬菌体附着位点是中性染色体整合位点伟大的替代品：许多物种特异性14-16，以及一般的整合位点，如Tn7转附着部位（ATT Tn7转）有已经确定，并用于基因插入在许多种细菌17,18。 环境微生物我们利用土壤作为环境微生物为我们的共同培养​​屏幕的直接来源。土壤中含有微生物的高度多样性，许多这些生物是天然产物来源丰富。通过使用土壤的液体悬浮液直接置于板与我们的荧光转录报告菌株（不含从土壤细菌事先隔离），我们大大简化了实验方法。土可以被用来收割后立即，或者在-80℃下以供将来使用被冻结。立即使用具有微生物的更大的多样性可能会被种植的优势，包括那些将无法生存冻结很好。它的缺点是可耕土壤生物体从这些样品中的浓度是未知的，增加了必须使用屏板的数目。德尔AYED使用具有的优点是对菌落形成单位/毫升的各土壤源可以预先确定，从而允许在每个屏板将要生长的菌落的一个优化的数目。然而，它需要的土壤生物能力经受冻结。 需要注意的是多元化宗正（ 即土壤源）诱导池似乎是更有效地识别较深入的筛查在同一土壤中的新物种间的相互作用：更大的系统发育多样性，观察在我们的基质诱导屏确定为命中额外的土壤源进行了检查，而不是筛选同一土壤源更彻底（EA柄和R. Kolter声称，哈佛医学院，未公布结果）。 概观我们在这里描述的方法是直接在其技术要求方面。它包括：1）构建荧光转录记者在B。枯草芽孢杆菌或其他感兴趣的细菌菌种来说，2）识别条件下与记者没有被激活，3）制备本报告菌株和生物体被筛选的等份（在我们的情况下的土壤，但是其它来源可以被用来代替），4）将这些两组在固体培养基上，5）鉴定和分离推定的诱导有机体，和6），确认​​这些生物体确实激活这种表型在二级屏幕的微生物。一旦确定，这些微生物及其代谢产物为我们提供化学工具来调节细菌行为，研究细菌的生理和微生物的相互作用，并有可能作为新型支架的未来治疗性化合物。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>Any spectrophotomer capable of measuing OD600 absorbance values.</td>
		</tr>
		<tr>
			<td>Luria broth, Lennox</td>
			<td>VWR</td>
			<td>80017-484</td>
			<td>Alternative media sources may be necessary.</td>
		</tr>
		<tr>
			<td>Glass beads, 3 mm</td>
			<td>VWR</td>
			<td>26396-508</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel loading tips, round</td>
			<td>VWR</td>
			<td>29442-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass rods</td>
			<td>VWR</td>
			<td>59060-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence dissecting stereoscope</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>The author used a Zeiss Stemi SV6 dissection stereoscope with an EXFO X-cite 120 fluorescent light source, a long-pass YFP filter cube, an achromat 0.63X objective, 10X eyepieces, and an Axio HRC HR digital camera. Most screening was done with the focusing mount at 2.0-3.2X. Any dissecting stereoscope with fluorescence capabilities is fine, provided you have the correct filters for the FP you are using. It is best if there is a shutter that allows you to easily switch between brightfield and fluorescense, as well as a stage that allows illumination from above and below. If you want to capture images, an attached camera is also necessary.</td>
		</tr>
	</tbody>
</table>

using coculture to detect chemically mediated interspecies interactions

在自然界中，细菌很少孤立存在的，它们不是包围，从而改变当地环境通过分泌代谢物等微生物的多样化。这些代谢产物具有调节他们的邻居微生物的生理和分化的潜能，并在建立和维护复杂的微生物群落可能的重要因素。我们已经开发了一种基于荧光共培养筛选，以确定这些化学介导的微生物的相互作用。画面涉及结合荧光转录记者株在固体培养基上的微生物环境，并允许殖民地共培养成长。荧光转录记者被设计成使得当它被表达感兴趣的特定表型选择的细菌菌株发出荧光（ 即生物膜的形成，孢子形成，毒力因子的生产， 等等 ）被筛选生长的条件下进行的磨片再此表型是不表达的（并且因此报告菌株通常是无荧光）。当环境微生物分泌的代谢物激活此表型，它扩散通过琼脂并激活荧光报道构建体。这允许待检测的诱导代谢物生产的微生物：它们是无荧光的菌落最邻近的荧光菌落。因此，该屏幕允许产生的代谢物扩散激活在记者应变特定的生理响应环境微生物的鉴定。该出版物讨论如何：1）选择合适的共培养筛选条件，二）准备的记者和环境微生物的筛选，C）进行共培养屏，四）隔离推测诱导有机体，以及e）确认其在辅助屏幕活动。我们开发这个 ​​方法来筛选土壤生物激活生物膜基质生产的枯草芽孢杆菌 </em&gt;的，但是，我们还讨论了考虑应用这种方法对其他基因听话细菌。

这个屏幕被用来确定土壤生物体分泌，从而改变B的生理学化合物枯草芽孢杆菌 。此处描述的结果集中在B的基质产生细胞类型枯草芽孢杆菌 ，其产生所必需的强大的生物膜形成这种细菌的蛋白和胞外多糖。我们选择了TAPA-sipW-TASA操纵子的启动子为我们的荧光报告基因构建体（P TAPA-YFP）。此操纵子编码矩阵的蛋白质结构部件和生物膜基质生产23</s...

Watch this Scientific Journal Video about Using Coculture to Detect Chemically Mediated Interspecies Interactions at JoVE.com

Using Coculture to Detect Chemically Mediated Interspecies Interactions

细菌产生有可能影响到他们的邻居微生物的生理潜力分泌的化合物。在这里，我们描述了一个共同培养屏幕，可以检测这些化学介导的种间相互作用的混合土壤中的微生物与固体培养基上枯草芽孢杆菌的荧光转录记者株。

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

using-coculture-to-detect-chemically-mediated-interspecies

Research

JoVE Journal

Biology

13.5K 次观看.  University of North Carolina at Chapel Hill. 细菌产生有可能影响到他们的邻居微生物的生理潜力分泌的化合物。在这里，我们描述了一个共同培养屏幕，可以检测这些化学介导的种间相互作用的混合土壤中的微生物与固体培养基上枯草芽孢杆菌的荧光转录记者株。 

视频: Using Coculture to Detect Chemically Mediated Interspecies Interactions

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

科研

生物学

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

快速合成及其与绿色荧光蛋白为基础的记者化学激活的转录因子筛选

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

用于液相电子显微镜的化学固定哺乳动物细胞的石墨烯外壳

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

使用肠道器官模型研究猿特异性相互作用的文化方法

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

Bioinformatics Resources for the Study of Glycan-Mediated Protein Interactions

The Glyco@Expasy initiative was launched as a collection of interdependent databases and tools spanning several aspects of knowledge in glycobiology. In particular, it aims at highlighting interactions between glycoproteins (such as cell surface receptors) and carbohydrate-binding proteins mediated by glycans. Here, major resources of the collection are introduced through two illustrative examples centered on the N-glycome of the human Prostate Specific Antigen (PSA) and the O-glycome of human serum proteins. Through different database queries and with the help of visualization tools, this article shows how to explore and compare content in a continuum to gather and correlate otherwise scattered pieces of information. Collected data are destined to feed more elaborate scenarios of glycan function. Glycoinformatics introduced here is, therefore, proposed as a means to either strengthen, shape, or refute assumptions on the specificity of a protein glycome in a given context.

This protocol illustrates how to explore, compare, and interpret human protein glycomes with online resources.

聚糖介导的蛋白质相互作用研究的生物信息学资源

The Glyco@Expasy initiative was launched as a collection of interdependent databases and tools spanning several aspects of knowledge in glycobiology. In ...