AOH和MAS承认科学学院和生命科学在罗切斯特技术学院的支持托马斯H戈斯内尔学校。这项工作由美国国家科学基金会（NSF）奖AOH MCB-1120541一部分支持。

注:作者愿意提供菌株和重组质粒，以促进功能互补分析，为教学目的纳入为个人谁是感兴趣。了用来促进功能互补实验的质粒列于表1。 1质粒的构建功能互补<ol><li>对于功能互补二氨基庚二酸转氨酶（DAPL）的克隆。 <ol><li>从五扩增DAPL开放阅读框（ORF） 棘和cDNA的距离A.拟南芥和C.通过PCR 藻 。使用在94℃1个循环2分钟，随后为15秒的94℃30个循环，60℃30秒和72℃2分钟。包括引物各12皮摩尔，1毫用MgSO 4所示 ，每个4脱氧核苷三磷酸为0.5mM，和模板DNA为0.5ng和PFX的1个单位DNA聚合酶。 注:从工厂A.关于三个DAPL同源重组克隆的完整描述拟南芥 （ 在 -dapL），细菌Verrucomicrobium棘 （VS -dapL）和藻类莱茵衣藻（铬 -dapL）先前已公布。2,18-20 注意:是用于DAPL直向同源物的克隆的引物如下: VS DAPL F- 5&#39;-CCCCGAATTCATGGCCCTCATCAACGAAAACTTCCTCAAG-3&#39; VS DAPL - [R 5&#39;- CCCCGTCGACCTACTTCAGCGCGGCGATACGGCGGCAGAC-3&#39; 在 DAPL F- 5&#39;-GGGGCATTGGAAGGAGATATAACCATGGCAGTCAATACTTGCAAATGT-3&#39; 在 DAPL R-5&#39;GGGGGTCGACTCATTTGTAAAGCTGCTTGAATCTTCG-3&#39; 铬 DAPL F-5&#39;CCCCCGAATTCATGCAGCTCAACGTGCGGTCCACCGCCAGC- 3&#39; 铬 DAPL - [R -5&#39;- CCCCCAAGCTTCTAGTTACGCTTGCCGTAGGCCTCCTTAAA-3&#39; </li><li>克隆PCR片段入T他质粒pET30a中，以产生重组质粒，质粒pET30a :: VS DAPL，pET30a中:: 在 DAPL以及使用该引物，限制性内切酶和T4 DNA连接酶的pET30a :: 铬 DAPL。 <ol><li>简单地说，孵育插入件的50纳克并在1×连接酶缓冲液20纳克载体和1单位T4 DNA连接酶过夜17℃。变换连接入E.大肠杆菌 DH5α细胞和屏幕上的LB平板的菌落在37℃下培养24小时，补充有50微克/毫升卡那霉素-1 18-20 </li></ol></li><li>为了创建功能互补质粒，消化的pET30a :: VS DAPL和pET30a中:: 在 DAPL质粒用限制酶XbaI和SalI和XbaI和HindIII的pET30a中:: 铬 DAPL。结扎插入到使用T4DNA连接酶，以产生质粒pBAD33 :: VS DAPL质粒pBAD33; pBAD33 :: 在 DAPL和pBAD33 :: 铬 DAPL。 <ol><li>插入孵育50毫微克至20毫微克的在1×连接酶缓冲液矢量和1单位T4 DNA连接酶过夜17℃。变换连接入E.大肠杆菌 DH5α细胞和屏幕上的LB平板的菌落通过孵育37℃24小时补充有34微克/毫升-1卡那霉素。20 </li></ol></li></ol></li><li>从A酪氨酸转氨酶（tyrB）的克隆拟南芥的功能互补。 注:关于cDNA的从A克隆的完整细节拟南芥由At5g36160先前已描述的轨迹标记注解4 <ol><li>通过PCR扩增该cDNA。使用在94℃1个循环2分钟，随后为15秒的94℃30个循环，60℃30秒和72℃2分钟。包括引物各12皮摩尔，1毫硫酸镁 ，四个脱氧核苷酸三磷酸，和DNA模板为0.5ng和Pfx DNA聚合酶的1个单位的0.5毫米。 注意:用于CL的引物是At5g36160的oning如下: 在 tyrB F- 5&#39;-CCCCGAATTCATGGGAGAAAACGGAGCCAAGCGAT-3&#39; AttyrB R- 5&#39;CCCCGAATTCATGGGAGAAAACGGAGCCAAGCGAT-3&#39; </li><li>克隆该片段到质粒pET30a中消化用ECORI和SAL1 PCR片段以产生重组质粒pET30a中:: At5g36160;结扎在使用T4DNA连接酶如以下描述的片段到pET30a 4 </li><li>为了创建功能互补质粒消化的pET30a :: At5g36160用限制酶XbaI和HindIII，以及使用同样的限制性酶位点，以产生使用T4DNA连接酶将重组质粒pBAD33 :: At5g3160结扎插入到pBAD33。 <ol><li>孵育插入件的50纳克并在1×连接酶缓冲液20纳克载体和1单位T4 DNA连接酶过夜17℃。变换连接入E.大肠杆菌 DH5α细胞和屏幕上的LB平板菌落补充34微克/毫升-1氯仿溶剂通过孵育37℃24小时ramphenicol 4 </li></ol></li></ol></li><li>的克隆UDP- n在五 -acetylmuramoyl基-L-丙氨酰-D- glutamate- 消旋 -2,6-二氨基庚二酸连接酶（MURE）为棘功能互补。 注:牟礼ORF从五克隆棘已如前所述。18 <ol><li>通过PCR扩增开放阅读框。使用在94℃1个循环2分钟，随后为15秒的94℃30个循环，60℃30秒和72℃2分钟。包括引物各12皮摩尔，1毫硫酸镁 ，四个脱氧核苷酸三磷酸为0.5mM，0.5纳克模板DNA和Pfx DNA聚合酶的1个单位。然后克隆到质粒pET100D以产生质粒pET100D :: VS MURE。 注意:是用于VS MURE的克隆的引物如下: VS牟礼F- 5&#39;- CACCATGACCATTTTGCGCGATCTTATCGAGGGT -3&#39; VS牟礼R- 5&#39;- GTCGACTCACTGACGGTCATCCCTCCTTTGGCGTGC-3&#39; </li><li>为了生产用于功能互补的质粒，消化pET100D :: VS牟礼 用限制酶XbaⅠ和SAL1和结扎插入到pBAD33，以产生重组质粒pBAD33 :: VS MURE使用T4DNA连接酶。 <ol><li>孵育插入件的50纳克和20毫微克在1×连接酶缓冲液载体，用1单位T4 DNA连接酶，过夜17℃下沿。变换连接入E.大肠杆菌 DH5α细胞和屏幕用于通过孵育37℃24小时补充有34微克/毫升-1氯霉素在LB平板上的菌落。8 </li></ol></li></ol></li><li>从Novosphingobium SP的功能互补RELA / 秒壶（RSH）的克隆。 注意 :从Novosphingobium藻 RSH的克隆已如前所述17。 <ol><li>放大RSH ORF除了599个核苷酸upst在起始位点和46个核苷酸通过PCR终止点下游的一分子。使用在94℃1个循环2分钟，随后为15秒的94℃30个循环，60℃30秒和72℃2分钟。包括引物各12皮摩尔，1毫硫酸镁 ，四个脱氧核苷酸三磷酸为0.5mM，0.5纳克模板DNA和Taq DNA聚合酶的1个单位。然后克隆到质粒pCR2.1中，以产生的pCR2.1 :: NSP的rsh。 <ol><li>孵育1微升PCR扩增片段，1微升pCR2.1载体中的盐溶液1微升和1微升水。孵育在25℃下5分钟，2微升结扎到大肠杆菌的大肠杆菌细胞，并且屏幕上的LB平板的菌落通过孵育37补充有50微克/毫升卡那霉素-1 °下放置24小时。 注意:是用于RSH的克隆的引物如下: RSH F- 5&#39;- GTTGAAAAACGCCGAATAGC -3&#39; RSH R- 5&#39;- GAGACCTGTGCGTAGGTGGT -3&#39; </li><li>为功能互补，消化质粒pCR2.1中:: NSP的rsh用ECORI和结扎插入到广泛宿主范围pRK290，以产生重组质粒pRK290 :: NSP RSH使用T4DNA连接酶。 <ol><li>孵育插入件的50纳克并在1×连接酶缓冲液20纳克载体和1单位T4 DNA连接酶过夜17℃。变换连接入E.大肠杆菌 DH5α细胞和屏幕上的LB平板的菌落通过孵育37补充有10微克/毫升-1四环素 °下放置24小时。17 </li></ol></li></ol></li></ol></li></ol> 2.准备电能力的细菌细胞来促进转型注:电感受态细胞的制备是基于协议26为1.0L的培养物，可以按比例缩小到一个较小的体积（ 即，250毫升）。请注意，这个协议可以bË用来做主管，在这个手稿，以方便FCA中描述的所有菌株。22 <ol><li>接种入50ml液体培养基中具有单个菌落到烧瓶中并生长过夜，并确保在开始该步骤（ 表2）之前检查突变体的基因型。 </li><li>在第二天，接种1.0升的50过夜培养毫升合适的培养基（LB 大肠杆菌突变体与PD Novosphingobium SP），并生长至OD 600〜0.4-0.6（对数相），在30℃ C。 </li><li>收获细胞通过在4℃下以5000 xg离心离心15分钟，倒出上清液，并且重悬沉淀在500毫升无菌冰冷纯H 2 O的</li><li>离心将细胞以5,000 xg离心在4℃下20分钟，倒出上清液和重悬沉淀在250ml无菌冰冷的10％的甘油。 </li><li>离心细胞在5000 XG在4 20分钟 °C，倒出超游泳和重悬沉淀在2.0ml 10％甘油。 </li><li>通过转移50微升到微离心管分装细胞，并立即通过将管在干冰 - 乙醇浴冷冻。存储感受态细胞在-80℃下电。 </li></ol> 3.互补质粒的细菌细胞电穿孔<ol><li>对于电穿孔，添加重组质粒的1.0微升（10-50纳克）到感受态细胞的等分试样（50微升），并在冰上放置5分钟。 </li><li>通过移液转移混合物至电穿孔杯中和电穿孔装置设置为以下设置:25μF电容，2.5千伏和200欧姆电阻，并提供一个脉冲。 </li><li> ，增加1.0毫升回收介质（LB）的电穿孔杯中和转让的用移液管向15毫升锥形管中。在一个摇床轻柔旋转60分钟恢复。 </li><li>从RECO板100微升文化很跨上琼脂平板来选择吹打转化和使用无菌扩张蔓延。 注:确保在开始这一步，检查抗生素和基因型进行正确琼脂平板前检查表1和表2。 </li></ol> 4. AOH1的转型，以促进功能互补使用L，L-二氨基转氨酶（DAPL） <ol><li>用于互补分析，变换AOH1用空载体（pBAD33），并与使用在节3.0中概述的电协议独立的转化事件的DAPL的表达载体。 </li><li>通过电镀补充有50微克/毫升-1磷酸二铵和34微克/毫升-1氯霉素和50μg/ ml的卡那霉素-1 LB琼脂培养基上选择转化体。 </li><li>划线到LB我测试通过副本镀殖民地功能互补dium用0.2％具有和不具有50μg/ ml的-1磷酸二铵和34微克/毫升-1卡那霉素（重量/体积）阿拉伯糖。 </li><li>在30℃孵育板24小时，观察结果。 注意:结果应该表明，与载体仅控制时的突变是唯一能够在仅当DAPL基因在突变背景表示DAP可用媒体生长。 </li></ol> 5.转化TKL-11的要求促进功能互补使用UDP-Ñ-acetylmuramoylalanyl-D-谷氨酰基-2,6- 消旋 -diaminopimelate连接酶（MURE） <ol><li>变换用空载体（pBAD33）并使用在节3.0中概述的协议的MURE表达载体（pBAD33 :: VsmurE）的突变体。 </li><li> LB琼脂培养基上板，并选择转化体补充有50微克/毫升-1胸腺嘧啶和34微克/毫升-1氯霉素并在30℃下孵育板24小时。 </li><li>通过从对照和实验变换到两个LB培养基加上0.2％（重量/体积）阿拉伯糖和50微克/毫升-1胸腺嘧啶条纹或电镀菌落试验互补。 </li><li>孵育在30℃下一个板，另一个在42℃下放置24小时，以目视评估的生长表型。 注意:结果应该表明，该突变体是能够在42℃下生长，只有当MURE基因在突变背景表达相比，矢量仅控制。 </li></ol> 6. DL39的转型，以促进功能互补利用酪氨酸转氨酶（tyrB） <ol><li>在LB琼脂平板变换DL39与任一pBAD33或pBAD33 :: At5g36160，并选择转化体补充有50微克/毫升-1酪氨酸，50微克/毫升-1苯丙氨酸，和使用协议34微克/毫升-1氯霉素在部分所述3.0。 </li><li>复制品上最小（M9）媒体-plate菌落用50微克/毫升-1苯丙氨酸和50微克/毫升-1酪氨酸，50微克/毫升-1天冬氨酸盐，50微克/毫升-1亮氨酸，50微克/毫升-1缬氨酸， 50微克/毫升-1异亮氨酸，10微克/毫升-1尿嘧啶0.5％（重量/体积）甘油，0.2％（重量/体积）阿拉伯糖。上划线两个板的相同的菌落缺乏苯丙氨酸和酪氨酸板也复制品板菌落。 </li><li>在30℃下孵育板48小时，观察生长表型。 注意:结果应该表明，与载体仅控制时的突变是唯一能够在苯丙氨酸和酪氨酸的培养基生长，只有当tyrB基因表达的突变背景。 </li></ol> 7.转化Hx699，方便Novosphingobium的Hypomucoid表型SP的功能互补。应变Hx699 </p&gt; <ol><li>变换野生型菌株Novosphingobium 属 （Rr2-17）和突变Hx699与pRK290和pRK290 :: RSH NSP使用在节3.0中概述的变换协议2个独立的转化事件。 </li><li>板在马铃薯葡萄糖的转化（PD）琼脂补充有10微克/毫升-1四环素，孵育至少24小时或直到转化体出现。 </li><li>条纹两个变换中的新鲜的PD琼脂板上一个"X"图案，并且在30孵育 °℃至少4天。通过目视检查两个板的生长表型观察仅载体，和实验的表型。 注意:结果应该表明，当RSH在突变背景中表达时相比，矢量仅控制所述防粘液表型补充。 </li></ol>

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</ol>

作者宣称，他们没有竞争的经济利益。

许多是不可或缺的生物技术和分子生物学课程在罗切斯特技术学院的课程除了拥有课程的讲授实验室组件。学年2014-2015年的课程包含了共48场，其中29含有实验室组件，它代表了约60％。一个这样的课程是植物生物化学和病理学（FPBP），混合的讲座/实验课和生物分离的基本原理:原理与实践（BPP），以实验室为基础课程。 每门课程的实验室组件旨在加强讲课材料。例如，生化途径和代谢都严重强调FPBP和BPP。一些主题包括氨基酸代谢，合成肽，植物次生代谢，环境壁龛，除其他细菌的反应。作者已经集成功能COMPLEMentation在这两门课程实验练习巩固了在演讲和或实验室预演讲讨论的代谢途径的理解。讨论使用功能互补实验以分析参与赖氨酸，PG TYR，PHE和细菌的严格的响应的生化途径的基因的功能的四个例子。 有几个原因，作者已经集成了这个实验模块到他们的课程。首先，接触功能互补分析是加强或引入相关的遗传学，进化，基因组学，生物信息学和生物化学的主题一个很好的工具。其次，实验是轻便，并且可以在一个过程中的实验室环境来实现。所有被使用的试剂是安全和所使用的细菌被表示为生物安全水平1和不致病。因此，作者们愿意使试剂如质粒和巴提供给任何人谁是兴趣将功能互补作为自己的教学体验的一部分cterial株。应当注意的是，两个菌株（TKL-11和DL39）被直接从大肠杆菌遗传保藏中心（CGSC）（http://cgsc.biology.yale.edu/）中获得。 需要注意的是有对功能互补协议几个关键步骤是重要的。第一步是要确保的突变体（S）的能开始任何试验前，进行培养。其原因是，按表2中 ，有与每个突变体相关的多个基因型。为了生长的突变体的实验之前以制备感受态细胞，尽量具有或不具有所需的化学品和或温度要求关于在PG MURE突变体生长的突变体。这也是一个教学的时刻，因为这将证明该突变体是任何实验前效力。 它还应当指出的是，虽然这是测试基因的功能（多个）的优秀工具，它并不总是工作，由于多种因素，例如密码子的使用，其中感兴趣的基因（多个）不能正确地翻译由于缺乏在突变生物体的相应的tRNA。然而，这可以通过在克隆步骤中的开放阅读框或cDNA，它通常由通过改变核苷酸以匹配突变体（多个）的密码子使用合成的感兴趣的基因进行密码子优化来规避。另一个问题是，一些真核生物蛋白质需要功能翻译后修饰。翻译后修饰不是细菌的特征，因此人们可能不能够使用此技术来测试使用细菌的突变体的基因的功能（多个）。 该技术的，我们在课程强调的意义在于，FCA是检测到基因的功能在体内的绝佳方式。表征<html>酶主要基于体外研究，其中的酶是执行纯化和传统的酶测定。29也是传统酶试验是伟大的测量或检测酶活性，经常可以"逼食"与在市场上不能提供底物酶纯的或天然的酶。30 的体内系统如FCA提供关于基因的功能的更真实的证明给定的事实，即它是在生理条件如放在附近下操作，以在体外这通常是不生理条件下。2给定缺乏有关的许多基因的功能和事实的信息，大多数注释基于预测，31掌握该技术将促进实验以阐明该保持模糊或那些目前认为具有在各推定功能的许多基因的功能公共数据库。 jove_content"&gt;一的关于一个经常遇到在感受态细胞的制备的技术中的问题，其中一个应确保将细胞制备正确地保证有足够的细胞除了转化为确保细胞与水和甘油，以防止在电穿孔过程拱正常洗涤。人们也应该认识到的突变体的表型通过确保将细胞用适当的化学物质或在适当的温度下生长，以促进两者的初始生长突变，也为功能互补分析。 一旦这一技术被掌握，研究人员可以使用功能互补测定法，只要有可用以促进此分析生物体的适当突变，以评估基因的功能。请注意，在该手稿的协议描述了使用细菌。然而，其他生物如植物和哺乳动物细胞，等等，可用于评估基因的功能。32-33一会就必须确保适当的载体，除了用于优化细胞的转化和转染或以促进实验。

在基因的阐明功能（多个）/角色（多个）的上下文功能互补被定义为一个特定的同源或直系同源基因的当同源与观察到的表型来恢复一个特定的突变体与野生型状态的能力或直系同源基因在顺式或反式引入突变体背景。这种技术已被广泛地用于分离和鉴定的功能（多个）的许多基因的/角色（多个）。一个具体的例子是使用S的URA3突变的白色念珠菌乳清苷-5&#39;-磷酸脱羧酶的分离和鉴定酵母和大肠杆菌的的pyrF突变体大肠杆菌 1的作者已经使用此技术来阐明所涉及的氨基酸，肽聚糖和在他们的研究方案的严格响应代谢后，已把该技术进在B的教学计划的基因的功能艾讴和分子生物学（BMB）在罗切斯特技术学院（RIT）程序。 笔者教植物生物化学/病理学（FPBP）（哈德森）和生物分离的基本原理:原理与实践（BPP）（哈德森/ Savka），在BMB计划在RIT两个上师选修课实验室为主的课程。由于一些那是在课程中讨论的主题与他们的研究兴趣关联，作者纳入了许多了在各自的研究项目中使用到这两个基于实验室的课程，技术和实验工具。一个这样的例子是功能互补作为实验室锻炼来加强涉及氨基酸从植物，肽聚糖和从细菌中严格的响应代谢酸代谢的演讲材料。 从植物是在FPBB过程所讨论的氨基酸途径的三个是赖氨酸（Lys）的，酪氨酸（TYR）和苯丙氨酸（Phe）。的赖氨酸途径中强调过程，因为氨基酸的作为必须氨基酸对所有动物特别是人的重要性，因为动物缺乏遗传机械合成赖氨酸从头 。另外，最近发现，植物采用为赖氨酸的合成在与细菌显著不同的路径。这个发现是部分地由大肠杆菌的功能互补促进大肠杆菌二氨基庚二酸（DAP）使用从模型植物拟南芥编码酶L，L-二氨基庚二酸转氨酶（DAPL）的基因的突变体。2为通过中间二氨基庚二酸赖氨酸的合成的变体途径示于图1中 。另外，赖氨酸的合成通过氨基酸的天冬氨酸衍生的家族是高度调控便于3除了其重要性的蛋白质SYNThesis，对于TYR和苯丙氨酸的途径是高亮鉴于其重要性，作为前体化合物服务于苯丙化合物的合成代谢参与植物防御化合物，如合成:生物碱，木质素，类黄酮，异黄酮，等等羟基肉桂酸4 TYR和苯丙氨酸途径也突出显示的植物和细菌合成代谢途径之间的差异。在细菌中，酶酪氨酸转氨酶（TyrB）参与两个氨基酸的合成代谢，而在植物中，酶是主要参与酪氨酸和苯丙氨酸的代谢和不参与这些氨基酸的合成代谢。 （ 图2）。4 革兰氏阳性和革兰氏关于肽聚糖（PG）的结构阴性菌之间的差异突出了FPBP过程。革兰氏阴性细菌的PG是基于这样的事实，关于植物病理学的利益，大多数植物病原体是革兰氏阴性。对于排名前10位的细菌植物病原体最近的检查发现，一切都是革兰氏阴性。的细菌是从属: 假单胞菌属 ， 青枯 ， 土壤杆菌 ， 黄单胞菌属 ， 欧文氏菌属 ，Xylella，Dickeya和果胶杆菌属 5之一的化学差异比较革兰氏阴性和革兰氏阳性菌的PG干时是交联氨基之间的差这两种类型的氨基酸。为PG的不同交联的初始步骤中的PG合成代谢的细胞质步骤发生并且被酶UDP-Ñ-acetylmuramoyl基-L-丙氨酰-D- glutamate- 消旋 -2,6-二氨基庚二酸连接酶促进（MURE） （ 图3A）。 MURE催化加成6肽干的第三位置。一个特定的二胺化合物在大多数革兰氏阴性细菌的，倒数第二个赖氨酸前体， 内消旋 -diaminopimelate（ <eM&gt;米-Dap）作为交联氨基酸和赖氨酸服务于大多数革兰氏阳性菌的PG（ 图3B）相同的作用。7这是由于这样的事实，即两个M -Dap和赖氨酸具有两个胺基团和能够形成肽干交联两条肽键。 在生物分离:原理与实践（BPP）当然，讨论细菌的培养和水平如何营养将在这两个系统显著变化，由于环境变化开放和封闭系统之间的差异。这些事件被链接到被称为"降档"或"上移"监管的变化由饥饿或氨基酸或能源供应充足触发。当细菌培养从丰富和复合培养基转移到化学上确定的培养基用一个单一碳源可发生"下移"响应。这种变化在环境导致快速cessatRNA和rRNA的合成化。此停止导致缺乏核糖体，蛋白质和DNA合成的即使氨基酸的生物合成被上调。 继"下移"响应，现有的核糖体被用来产生新的酶合成氨基酸在培养基或环境不再可用。一段时间后，rRNA的合成和新核糖体组装和细菌细胞群开始以降低的速度虽然增长。事件的过程被称为" 严格的响应"或" 严格的控制"，并且是全球细胞调节的例子，并且可以被认为是作为一个机构，用于调节细胞的生物合成机器来补偿所需的底物和能量的需求的情况8的严格的响应从而使细菌迅速在环境营养素通量响应，并有助于与ENH细菌元代的环境中竞争，可以与关于营养和或基板供货迅速变化的能力。8-9 严格的响应具有时的氨基酸，碳，氮，磷，和脂肪酸的可用性被限制在基因表达的不可或缺的作用。8,10-14这种严格的响应是由两个核苷酸，鸟苷四磷酸（ppGpp的）和鸟苷的协调五磷酸盐（pppGpp）通常称为一起为alarmone（p）ppGpp的。例如，当氨基酸被限制 - 这可导致蛋白质合成的alarmone一个瓶颈，鸟苷3,5-（双）焦磷酸（ppGpp的），从鸟苷3-二磷酸5-磷酸的合成代谢衍生（pppGpp）累积在细胞中。在（P）ppGpp的电平的变化是参与调节响应克服缺乏直接参与细胞生长和发展在环境中基板的基因的表达吨。那些参与这一进程的基因的两个被称为RELA和现货。 RELA是核糖体相关（P）ppGpp的合成中所涉及的响应于不带电的tRNA的积累是氨基酸的限制的结果。点用作双功能（P）ppGpp的合成酶和水解酶。点的合成酶活性参与响应于缺少碳和脂肪酸饥饿8的RELA /点同系物是在植物和细菌广泛和被称为硫醇的R ELA / S的锅ħomologs。8,10 -12,16最近手稿表明有涉及从细菌Novosphingobium藻的Rr 2-17这些alarmones合成的特定硫醇的蛋白质。17 这里我们介绍拴在功能互补实验4生化途径。在这个手稿中概述的互补实验提供了一个渠道，以EXPL矿石采用这种体内测定为鉴定和或表征被预测为具有未知/推定功能（S）或作为教学工具来补充的生化途径的教学酶的手段。

<table><tbody><tr><td>&lt;em&gt;E. coli&lt;/em&gt; mutants</td><td>Hudson/Savka lab or CGSC (http://cgsc.biology.yale.edu/)</td><td></td><td></td></tr><tr><td>Electroporator</td><td>Biorad-USA</td><td>1652100</td><td></td></tr><tr><td>Electroporation Cuvettes</td><td>Biorad-USA</td><td>1652082</td><td></td></tr><tr><td>Temperature controlled incubator</td><td>Generic</td><td></td><td></td></tr><tr><td>Microcentrifuge</td><td>Generic</td><td></td><td></td></tr><tr><td>Luria Agar</td><td>Thermofisher Scientific</td><td>22700025</td><td></td></tr><tr><td>Luria Broth</td><td>Thermofisher Scientific</td><td>12795084</td><td></td></tr><tr><td>M9 Medium</td><td>Sigma-Aldrich</td><td>63011</td><td></td></tr><tr><td>Potato Dextrose Medium</td><td>Fisher Scientfic&amp;nbsp;</td><td>DF0013-15-8</td><td></td></tr><tr><td>Kanamycin</td><td>Sigma-Aldrich</td><td>K1377</td><td></td></tr><tr><td>Diaminopimelate</td><td>Sigma-Aldrich</td><td>92591</td><td></td></tr><tr><td>Thymine</td><td>Sigma-Aldrich</td><td>T0376</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C0378</td><td></td></tr><tr><td>Tyrosine</td><td>Sigma-Aldrich</td><td>T3754</td><td></td></tr><tr><td>Phenlylalanine</td><td>Sigma-Aldrich</td><td>P2126</td><td></td></tr><tr><td>Aspartate</td><td>Sigma-Aldrich</td><td>A9256</td><td></td></tr><tr><td>Valine</td><td>Sigma-Aldrich</td><td>V0500</td><td></td></tr><tr><td>Leucine</td><td>Sigma-Aldrich</td><td>L8000</td><td></td></tr><tr><td>Isoleucine</td><td>Sigma-Aldrich</td><td>I2752</td><td></td></tr><tr><td>Uracil</td><td>Sigma-Aldrich</td><td>U0750</td><td></td></tr><tr><td>Gylcerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Arabinose</td><td>Sigma-Aldrich</td><td>A3256</td><td></td></tr><tr><td>Tetracyline</td><td>Sigma-Aldrich</td><td>87128</td><td></td></tr><tr><td>Taq DNA polymerase</td><td>Thermofisher Scientific</td><td>10342-020</td><td></td></tr><tr><td>Platinum pfx DNA polymerase</td><td>Thermofisher Scientific</td><td>11708-013</td><td></td></tr><tr><td>T4 DNA ligase</td><td>Thermofisher Scientific</td><td>15224-041</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; Dh5-alpha</td><td>Thermofisher Scientific</td><td>18258012</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; Top10</td><td>Thermofisher Scientific</td><td>C4040-03</td><td></td></tr><tr><td>pET100D/topo vector</td><td>Thermofisher Scientific</td><td>K100-01</td><td></td></tr><tr><td>pCR2.1 Vector</td><td>Thermofisher Scientific</td><td>K2030-01</td><td></td></tr></tbody></table>

functional complementation analysis (fca): a laboratory exercise designed and implemented to supplement the teaching of biochemical pathways

功能互补测定（FCA）是一种体内测定，被广泛用来阐明基因/酶的功能/作用。这种技术是很常见的生物化学，遗传学等众多学科。该技术的全面介绍，以补充有关氨基酸，肽聚糖和细菌严格的响应在这个手稿报道的生化途径的教学。从所涉及的赖氨酸（L，L-二氨基庚二酸转氨酶（DAPL）以及参与酪氨酸和苯丙氨酸的代谢酪氨酸转氨酶（tyrB）的代谢模式植物生物体拟南芥两个cDNA被突出显示。另外，细菌肽聚糖合成代谢途径是通过UDP-ñ-acetylmuramoyl基-L-丙氨酰-D- glutamate- 消旋 -2,6-二氨基庚二酸连接酶（MURE）基因从参与跨细菌Verrucomicrobium棘的分析突出-linking肽聚糖。细菌严格的响应也通过RSH（R ELA / s的锅ħomolog）负责在细菌Novosphingobium藻一个超粘液型双功能基因的分析报告。FCA的四个实例。视频将专注于他们三个，分别是赖氨酸，肽聚糖和严格的响应。

了在各功能互补分析中使用的细菌菌株列于表2中 。 功能互补分析:L，L-二氨基庚二酸转氨酶（DAPL） 大肠大肠杆菌双突变AOH1（ΔDAPD :: Kan2，dapE6）窝藏在dapE的基因中的突变和DAPD基因的完全缺失（ 图1）。这样，突变体不能生长，除非提供DAP。由于这些突变，AOH1被视为营养缺陷型。AOH1适合升功能互补分析，L-二氨基庚二酸转氨酶（DAPL）直向同源物作为DAPL催化L的合成，L-二氨基庚二酸直接从THDP便于PG和赖氨酸合成（ 图1）。 AOH1突变体表达DapLs能够对L-生长，L- DAP-免费媒体表明该重组酶能够THDP转换为L时， L-DAP绕过在DAP /赖氨酸途径中DAPD和DAPE酶促步骤（ 图5）。 功能互补分析:UDP - N -acetylmuramoyl基-L-丙氨酰-D- glutamate- 消旋 -2,6-二氨基庚二酸连接酶（MURE） 消旋 -diaminopimelate（M -Dap）是革兰氏阴性细菌的PG中的交联的氨基酸。酶UDP-Ñ-acetylmuramoylalanyl-ð谷氨酰基-2,6- 消旋 -diaminopimelate连接酶（MURE）便于在这个过程中，除了米 -Dap（ 图3A - B）。 大肠大肠杆菌突变TKL-11藏着亩塔季翁在MURE基因，其导致所述突变体中的42中生长的能力 °Ç由于这样的事实，该变异型酶是不能够在此温度下正确折叠。 结果将证明，有在由控制和实验都在30℃的允许温度下生长。然而，只有表达MURE（实验）细胞将能够在42非允许温度下生长 ℃（ 图6）。 功能互补分析:酪氨酸转氨酶（tyrB） 拟南芥编码酪氨酸转氨酶，能够互变酪氨酸和4-羟基苯，以及苯丙氨酸和苯丙酮酸由At5g36160（ 图2）注释4的大肠杆菌关口1突变体（DL39）窝藏在tyrB基因，这使得营养缺陷为苯丙氨酸和酪氨酸的突变。24-26。应当注意的是，该突变体是营养缺陷型的氨基酸酪氨酸，苯丙氨酸，天冬氨酸，亮氨酸，异亮氨酸和缬氨酸，以及由于其他突变。应变是合适的，以评估由于来自大肠杆菌的TyrB酶同源物tyrB直向同源物的功能大肠杆菌是直接参与酪氨酸和苯丙氨酸的合成以促进蛋白质的合成。 结果应显示的是，虽然DL39突变体能够在M9生长提供酪氨酸和苯丙氨酸，只有当。然而，表达拟南芥 （At5g36160）的菌株能够在缺乏酪氨酸和苯丙氨酸表明所述酶是能够从4-羟基苯，和从苯丙酮酸吨苯丙氨酸合成酪氨酸的M9培养基中生长（Figure 7）4 功能互补分析:硫醇 （RELA和S锅同源） 从Novosphingobium SP的硫醇的蛋白质。 （Rr2-17）含有N-末端磷酸水解酶和第（p）ppGpp的合成酶功能域两者，并且因此具有类似大肠杆菌的SPOT拟议双功能作用大肠杆菌。这已使用大肠杆菌的互补分析证实大肠杆菌双突变体，CF1693利用Novosphingobium SP的RSH基因，窝藏突变RELA和点。在Rr2-17 RSH突变体的表型，命名为Hx699（RSH :: EZ-Tn5的，阚 R），结果在防粘液，这是由于非功能RSH。 Hx699的防粘液性状受评估 互补由本机RSH NSP基因促成了克隆到广宿主范围载体pRK290。与防粘液型相一致，Hx699窝藏空载体pRK290显示包含pRK290或pRK290 :: RSH NSP的由反式补充Hx699产生较少水溶性多糖（pRK290 :: RSH NSP）和Rr2-17（ 图8） 。 <img alt="图1" src="/files/ftp_upload/53850/53850fig1.jpg" /> 图1:赖氨酸合成代谢途径从天冬氨酸通过中间二氨基庚二酸（DAP）进行变体的途径是通过（A）的酰基的途径，（B）中的二氨基庚二酸脱氢酶（DDH）途径和（C）所述的L注解，L-二氨基庚二酸转氨酶（DAPL）途径。对于酶的缩写定义如下:天冬氨酸激酶（lysC的），天冬氨酸半醛脱氢酶（ASD），四氢吡啶二羧酸合酶（DAPA），四氢吡啶二羧酸还原酶（dapB的），四氢吡啶二羧酸酰基转移酶（DAPD），N -酰基-2-氨基-6- ketopimelate转氨酶（DAPC），N -酰基-L，L-2， 6-二氨基庚二酸脱酰酶（DAPE），二氨基庚二酸差向异构酶（dapF的）， 内消旋 -diaminopimelate脱氢酶（DDH）， 内消旋 -diaminopimelate脱羧酶（lysA的）中，L，L-二氨基庚二酸转氨酶（DAPL），UDP-ñ-acetylmuramoyl基-L-丙氨酰基D-谷氨酸: 内消旋 -2,6-二氨基连接酶（穆赫）和赖氨酰-tRNA合成酶（LysU） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53850/53850fig1large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53850/53850fig2.jpg" /> 图 2: 氨基酸初学者同化途径正弦和 ​​苯丙氨酸。（A）中通过中间分支酸和（B）通过中间arogenate植物途径中的细菌（ 大肠杆菌 ）途径。对于酶的缩写如下:分支酸变位酶/预苯酸脱水酶（PHEA），分支酸变位酶，预苯酸脱氢酶（TYRA），和酪氨酸转氨酶（TyrB）。该图通过并修改的形式帕布和Hudson，2010年4 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53850/53850fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53850/53850fig3.jpg" /> 图3:肽合成所述的细胞质步骤 （A）中的肽聚糖合成的细胞质步骤的示意图。缩写<html>酶如下:UDP-Ñ-acetylglucosamine enolpyruvyl转移酶（MURA），UDP-Ñ-acetylenolpyruvoylglucosaminereductase（MurB），UDP-Ñ-acetylmuramate -L-丙氨酸连接酶（MURC），UDP- -N-乙酰-muramoylalanine-D-谷氨酸连接酶（MurD），UDP-ñ-acetylmuramoyl-L-丙氨酰D-谷氨酸-2,6- 消旋 -diaminopimelate连接酶（MURE），UDP-ñ-acetylmuramoyl-三肽-D-丙氨酰-D-丙氨酸连接酶（MurF ），D-丙氨酸-D-丙氨酸连接酶（DDL）。 （B）的肽聚糖表示二糖Ñ-acetylglucosamine葡糖胺（GlcNAc）的单体单元的示意图和N -acetylmuramic（MurNAc）经由β-1,4糖苷键连接。在杆肽的3位上的氨基酸是参与交联大多数革兰氏阴性细菌和赖氨酸由内消旋 -diaminopimelate（M -Dap）表示在大多数革兰氏阳性菌。_blank"&gt;请点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53850/53850fig4.jpg" /> 图4:在细菌中严格的响应的示意图模型表示暴露到营养环境恶劣诱导硫醇（RELA或点）通过添加磷酸基团到的GTP（三磷酸鸟苷），以anabolize所述alarmone（P）ppGpp的（鸟苷五磷酸）。暴露于营养丰富的环境诱导硫醇的表达水解（p）的ppGpp的是一个生长抑制剂。该图获得通过，并从拉斯金等人修改，2007年27 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53850/53850fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" sRC ="/文件/ ftp_upload / 53850 / 53850fig5.jpg"/&gt; 图5:使用DAPL功能互补的AOH1 大肠杆菌的功能互补大肠杆菌突变体与L，L-二氨基庚二酸转氨酶（DAPL）基因从细菌中五棘 （VS DAPL），植物A.拟南芥 （ 在 DAPL）和藻类，C。莱茵衣藻 （CR DAPL）。所述突变体携带的质粒，pBAD33，pBAD33 :: VS DAPL，pBAD33 :: 在 DAPL和pBAD33 :: 铬 DAPL被复制铺板于补充有0.2％的LB琼脂平板（重量/体积）阿拉伯糖具有或不具有50μg/ ml的L，L -二氨基庚二酸，并且在30中生长 °下放置24小时。该图获得通过，并从Nachar 等修改，2012年18 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53850/53850fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图6:使用MURE功能互补的TKL-11 E的功能互补大肠杆菌突变株与来自五 ，UDP-Ñ-acetylmuramoyl基-L-丙氨酰-D- glutamate- 消旋 -2,6-二氨基庚二酸连接酶（MURE）基因棘。突变窝藏质粒BAD33或pBAD33 :: VsmurE在LB培养基中生长至OD 0.1 600和连续稀释至10 -1，10 -2和10 -3使用0.85％（重量/体积）盐水。五 各个稀释液的微升被复制铺板补充有0.2％（重量/体积）阿拉伯糖的LB培养基上并在30℃和42中生长评估 ℃24小时。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53850/53850fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </a&gt; <img alt="图7" src="/files/ftp_upload/53850/53850fig7.jpg" /> 图7:DL39 大肠杆菌 的功能互补 大肠杆菌 与从 A 轨迹标签At5g36160注明酪氨酸转氨酶基因 拟南芥 。被选定在补充有50微克/毫升-1酪氨酸，50微克/毫升-1苯丙氨酸的LB琼脂平板上携带质粒pBAD33或pBAD33 :: At5g36160的突变体，和34微克/毫升-1氯霉素。该菌株用0.85％（重量/体积）盐水在LB肉汤生长至OD外径1.0 600和连续稀释至10 -1，10 -2和10 -3。 5微升的各稀释液分别然后复制铺板到补充有50微克/毫升-1苯基的M9琼脂平板丙氨酸和50微克/毫升-1酪氨酸，0.5％（重量/体积）甘油，0.2％（重量/体积）阿拉伯糖，50微克/毫升-1天冬氨酸盐，50微克/毫升-1亮氨酸，50微克/毫升-1缬氨酸，50微克/毫升-1异亮氨酸，10微克/毫升-1尿嘧啶，并且还对缺乏苯丙氨酸和酪氨酸，并在30℃培养48小时平板。该图通过并修改的形式帕布和Hudson，2010年4 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53850/53850fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/53850/53850fig8.jpg" /> 图 8:Novosphingobium藻 的hypomucoid表型的功能互补 。突变株 Hx699。 ŧ他野生型菌株Novosphingobium 属 （Rr2-17）和突变菌株Hx699与pRK290或pRK290 :: RSH NSP进行转化。的菌株划线接种于一个"X"，并在30℃下的PD琼脂中培养至少4天，观察表型。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53850/53850fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>互补质粒</td><td>抗生素抗性标记</td><td>引文</td></tr><tr><td> pBAD33 </td><td>厘米ř </td><td> 21 </td></tr><tr><td> pBAD33 :: 在 DAPL </td><td>厘米ř </td><td> 2 </td></tr><tr><td> pBAD33 :: 铬 DAPL </td><td>厘米ř </td><td> 20 </td></tr><tr><td> pBAD33 :: VS DAPL </td><td>厘米- [R </td><td> 18 </td></tr><tr><td> pBAD33 :: At5g36160 </td><td>厘米ř </td><td> 4 </td></tr><tr><td> pBAD33 :: VS牟礼</td><td>厘米ř </td><td> 18 </td></tr><tr><td> pRK290 </td><td>春节ř </td><td> 28 </td></tr><tr><td> pRK290 :: RSH NSP </td><td>春节ř </td><td> 17 </td></tr></tbody></table> 表1: 本研究中使用的功能互补分析质粒的一览表中所用的功能互补质粒 。厘米R和四环素- [R分别表示氯霉素和四环素抗性。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>应变名</td><td>生物</td><td>基因型</td><td>表型</td><td>资源</td></tr><tr><td> AOH1 </td><td>大肠杆菌 </td><td> ΔDAPD :: Kan2，dapE6 </td><td>营养缺陷的二氨基</td><td>哈德森实验室</td></tr><tr><td> TKL-11 * </td><td> 大肠杆菌 </td><td> THR-1，leuB6（AM），murE1，fhuA21，codA1，lacY1的 ，TSX-95，glnV44（AS），λ - ，pyrF101， 他-108，thyA6，argG66，ilvA634，THI-1，deoC1 </td><td>生长敏感的表型是突变生长于30℃而不是42℃ </td><td> CGSC（＃5989） </td></tr><tr><td> DL39 * </td><td> 大肠杆菌 </td><td> LAM-，aspC13，FNR-25，RPH-1 ilvE12，tyrB507 </td><td>营养缺陷型的氨基酸;酪氨酸，苯丙氨酸，亮氨酸，异亮氨酸和缬氨酸</td><td> CGSC（＃6913） </td></tr><tr><td> Rr2-17 </td><td> Novosphingobium属 （野生型） </td&gt; <td> - </td><td>超粘液</td><td> Savka实验室</td></tr><tr><td> Hx699 </td><td> Novosphingobium藻。 </td><td> RSH :: EZ-Tn5的，阚ř </td><td>防粘液</td><td> Savka实验室</td></tr></tbody></table> 表2:用本研究中使用各自的基因型和表型的沿细菌菌株用于本研究的细菌菌株列表。请注意，由星号表示的菌株（TKL-11和DL39）可以直接从大肠杆菌遗传保藏中心（CGSC）（http://cgsc.biology.yale.edu/）获得。

Watch this Scientific Journal Video about Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways at JoVE.com

Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

所涉及的生化途径的酶活性的确认可以使用功能性互补分析（FCA）阐明。描述在这个手稿是FCA测定表明参与氨基酸，细菌严格的响应和细菌的肽聚糖的生物合成的代谢酶的酶活性。

功能互补分析（FCA）:实验室练习设计和实施，以补充生化途径的教学

Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very ...

functional-complementation-analysis-fca-laboratory-exercise-designed

Nanyang Technological University

Research

JoVE Journal

Biology

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

17.6K 次观看.  Rochester Institute of Technology. 所涉及的生化途径的酶活性的确认可以使用功能性互补分析（FCA）阐明。描述在这个手稿是FCA测定表明参与氨基酸，细菌严格的响应和细菌的肽聚糖的生物合成的代谢酶的酶活性。 

视频: Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

科研

生物化学

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

生物学

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

使用C2C12成肌细胞的生物能简介实验

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

双分子荧光互补

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

使用网关兼容双分子荧光互补（附设）系统在植物蛋白相互作用检测

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

功能互补分析（FCA）:实验室练习设计和实施，以补充生化途径的教学

摘要

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