Name: functional complementation analysis (fca): a laboratory exercise designed and implemented to supplement the teaching of biochemical pathways
Uploaded: 2016-06-24T14:00:00
Description: 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

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...

<ol>
	<li>Gillum, A. M., Tsay, E. Y., Kirsch, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+the+Candida+albicans+gene+for+orotidine-5'-phosphate+decarboxylase+by+complementation+of+S.+cerevisiae+ura3+and+E.+coli+pyrF+mutations.">Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations.</a> Mol Gen Genet. 198, 179-182 (1984).</li><li>Hudson, A. O., Singh, B. K., Leustek, T., Gilvarg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+L,L-diaminopimelate+aminotransferase+defines+a+novel+variant+of+the+lysine+biosynthesis+pathway+in+plants.">An L,L-diaminopimelate aminotransferase defines a novel variant of the lysine biosynthesis pathway in plants.</a> Plant Physiol. 140, 292-301 (2006).</li><li>Jander, G., Joshi, V., Last, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspartate-derived+amino+acid+biosynthesis+in+Arabidopsis+thaliana.">Aspartate-derived amino acid biosynthesis in Arabidopsis thaliana.</a> The Arabidopsis Book. , (2009).</li><li>Prabhu, P., Hudson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+partial+characterization+of+an+L-Tyrosine+aminotransferase+from+Arabidopsis+thaliana.">Identification and partial characterization of an L-Tyrosine aminotransferase from Arabidopsis thaliana.</a> Biochemistry Research International. , 549572 (2010).</li><li>Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M., Verdier, V., Beer, S. V., Machado , M. A., Toth, I., Salmond, G., Foster, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top+10+pathogenic+bacteria+in+molecular+pathology.">Top 10 pathogenic bacteria in molecular pathology.</a> Molecular Plant Pathology. 13, 614-629 (2012).</li><li>McGroty, S. E., Pattaniyil, D. T., Patin, D., Blanot, D., Ravichandran, A. C., Suzuki, H., Dobson, R. C., Savka, M. A., Hudson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+Characterization+of+UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:+meso-2,6-diaminopimelate+ligase+(MurE)+from+Verrucomicrobium+spinosum+DSM+4136T.">Biochemical Characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE) from Verrucomicrobium spinosum DSM 4136T.</a> PLoS ONE. 8 (6), e66458 (2013).</li><li>Hutton, C. A., Perugini, M. A., Gerrard, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+lysine+biosynthesis:+an+evolving+antibiotic+strategy.">Inhibition of lysine biosynthesis: an evolving antibiotic strategy.</a> Mol Biosyst. 3, 458-465 (2007).</li><li>Potrykus, K., Cashel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(p)ppGpp:+still+magical.">(p)ppGpp: still magical.</a> Annu Rev Microbiol. 62, 35-51 (2008).</li><li>Dalebroux, Z. D., Svensson, S. L., Gaynor, E. C., Swanson, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp+Conjures+Bacterial+Virulence.">ppGpp Conjures Bacterial Virulence.</a> Microbiology and Molecular Biology Reviews. 74, 171-199 (2010).</li><li>Cashel, M., Gentry, D. J., Hernandez, V. J., Vinella, D., Neidhardt, F. C., Curtiss, R., Ingraham, J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Reznikoff, W. S., Riley, M., Schaechter, M., Umbarger, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stringent+response.">The stringent response.</a> Escherichia coli and Salmonella typhimurium: cellular and molecular biology. , 1458-1496 (1996).</li><li>Chatterji, D., Ojha, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+the+stringent+response,+ppGpp+and+starvation+signaling.">Revisiting the stringent response, ppGpp and starvation signaling.</a> Curr. Opin. Microbiol. 4, 160-165 (2001).</li><li>Jain, V., Kumar, M., Chatterji, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp:+stringent+response+and+survival.">ppGpp: stringent response and survival.</a> J. Microbiol. 44, 1-10 (2006).</li><li>Kramer, G. F., Baker, J. C., Ames, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-UV+stress+in+Salmonella+typhimurium:+4-thiouridine+in+tRNA,+ppGpp,+and+pppGpp+as+components+of+an+adaptive+response.">Near-UV stress in Salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and pppGpp as components of an adaptive response.</a> J. Bacteriol. 170, 2344-2351 (1988).</li><li>Braeken, K., Moris, M., Daniels, R., Vanderleyden, J., Michiels, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+horizons+for+(p)ppGpp+in+bacterial+and+plant+physiology.">New horizons for (p)ppGpp in bacterial and plant physiology.</a> Trends Microbiol. 14, 45-54 (2006).</li><li>Joseleau-Petit, D., Vinella, D., D'Ari, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+alarms+and+cell+division+in+Escherichia+coli.">Metabolic alarms and cell division in Escherichia coli.</a> J. Bacteriol. 181, 9-14 (1999).</li><li>Mittenhuber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+and+evolution+of+genes+encoding+bacterial+(p)+ppGpp+synthetases/hydrolases+(the+Rel,+RelA,+and+SpoT+proteins).">Comparative genomics and evolution of genes encoding bacterial (p) ppGpp synthetases/hydrolases (the Rel, RelA, and SpoT proteins).</a> J. Mol. Microbiol. Biotechnol. 3, 585-600 (2001).</li><li>Gan, H. M., Buckley, L., Szegedi, E., Hudson, A. O., Savka, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+rsh+Gene+from+a+Novosphingobium+sp.+Necessary+for+Quorum-Sensing+Signal+Accumulation.">Identification of an rsh Gene from a Novosphingobium sp. Necessary for Quorum-Sensing Signal Accumulation.</a> J. Bacteriol. 191, 2551-2560 (2009).</li><li>Nachar, V. R., Savka, F. C., McGroty, S. E., Donovan, K. A., North, R. A., Dobson, R. C., Buckley, L. J., Hudson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+biochemical+analysis+of+the+diaminopimelate+and+lysine+biosynthesis+pathway+in+Verrucomicrobium+spinosum:+Identification+and+partial+characterization+of+L,L-diaminopimelate+aminotransferase+and+UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-meso-diaminopimelate+ligase.">Genomic and biochemical analysis of the diaminopimelate and lysine biosynthesis pathway in Verrucomicrobium spinosum: Identification and partial characterization of L,L-diaminopimelate aminotransferase and UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-meso-diaminopimelate ligase.</a> Front. Microbiol. 3, 183 (2012).</li><li>Hudson, A. O., Gir&#242;n, I., Dobson, R. C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+and+preliminary+X-ray+diffraction+analysis+of+L,L-diaminopimelate+aminotransferase+(DapL)+from+Chlamydomonas+reinhardtii.">Crystallization and preliminary X-ray diffraction analysis of L,L-diaminopimelate aminotransferase (DapL) from Chlamydomonas reinhardtii.</a> Acta Cryst. 67, 140-143 (2011).</li><li>Dobson, R. C. J., Gir &#242;n, I., Hudson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L,L-Diaminopimelate+Aminotransferase+from+Chlamydomonas+reinhardtii:+A+Target+for+Algaecide+Development.">L,L-Diaminopimelate Aminotransferase from Chlamydomonas reinhardtii: A Target for Algaecide Development.</a> PLoS ONE. 6 (5), e20439 (2011).</li><li>Guzman, L. M., Belin, D., Carson, M. J., Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+regulation,+modulation,+and+high-level+expression+by+vectors+containing+the+arabinose+pBAD+promoter.">Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter.</a> J. Bacteriol. 177, 4121-4130 (1995).</li><li>Sambrook, J., Russell, D. 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> Molecular Cloning: A laboratory manual. , (2001).</li><li>Lugtenberg, E. J., van Schijndel-van Dam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+mutants+of+Escherichia+coli+K-12+with+low+activity+of+the+diaminopimelic+acid+adding+enzyme.">Temperature-sensitive mutants of Escherichia coli K-12 with low activity of the diaminopimelic acid adding enzyme.</a> J. Bacteriol. 110, 41-46 (1972).</li><li>Mavrides, C., Orr, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispecific+aspartate+and+aromatic+amino+acid+aminotransferases+in+Escherichia+coli.">Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli.</a> J. Biol Chem. 250, 4128-4133 (1975).</li><li>Gelfand, D. H., Steinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+mutants+deficient+in+the+aspartate+and+aromatic+amino+acid+aminotransferases.">Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases.</a> J. Bacteriol. 130, 429-440 (1977).</li><li>Whitaker, R. J., Gaines, C. G., Jensen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multispecific+quintet+of+aromatic+aminotransferases+that+overlap+different+biochemical+pathways+in+Pseudomonas+aeruginosa.">A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa.</a> J. Biol Chem. 257, 13550-13556 (1982).</li><li>Raskin, D. M., Judson, N., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+stringent+response+in+the+essential+function+if+the+conserved+bacterial+G+protein+CgtA+in+Vibrio+cholera.">Regulation of the stringent response in the essential function if the conserved bacterial G protein CgtA in Vibrio cholera.</a> Proc Natl Acad Sci, USA. 104, 4636-4641 (2007).</li><li>Ditta, G., Stanfield, S., Corbin, D., Helinski, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+host+range+DNA+cloning+system+for+Gram-negative+bacteria:+construction+of+a+gene+bank+of+Rhizobium+meliloti.">Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti.</a> Proc Natl Acad Sci, USA. 77, 7347-7351 (1980).</li><li>Watanabe, A., Suzuki, M., Ujiie, S., Gomi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+enzymatic+characterization+of+a+novel+&#946;-1,6-glucosidase+from+Aspergillus+oryzae.">Purification and enzymatic characterization of a novel &#946;-1,6-glucosidase from Aspergillus oryzae.</a> J Biosci Bioeng. , (2015).</li><li>Song, J. T., Lu, H., McDowell, J. M., Greenberg, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+key+role+for+ALD1+in+activation+of+local+and+systemic+defenses+in+Arabidopsis.">A key role for ALD1 in activation of local and systemic defenses in Arabidopsis.</a> Plant J. 40, 200-212 (2004).</li><li>Rost, B., Liu, J., Nair, R., Wrzeszczynski, K. P., Ofran, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+prediction+of+protein+function.">Automatic prediction of protein function.</a> Celuular and Molecular Life Sciences. 60, 2637-2650 (2003).</li><li>Norris, S. R., Shen, X., Penna, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementation+of+the+Arabidopsis+pds1+mutation+with+the+gene+encoding+p-Hyrdoxyphenylpyruvate+dioxygenase.">Complementation of the Arabidopsis pds1 mutation with the gene encoding p-Hyrdoxyphenylpyruvate dioxygenase.</a> Plant Physiol. 117, 1317-1323 (1998).</li><li>Mohler, W. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+and+cell+fusion+analyzed+by+lacZ+complementation+in+mammalian+cells.">Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells.</a> Proc Natl Acad Sci, USA. 93, 12423-12427 (1996).</li></ol>

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...

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 border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:223px;">E. coli mutants</td>
			<td style="width:372px;">Hudson/Savka lab or CGSC (http://cgsc.biology.yale.edu/)</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporator</td>
			<td>Biorad-USA</td>
			<td>1652100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporation Cuvettes</td>
			<td>Biorad-USA</td>
			<td>1652082</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temperature controlled incubator</td>
			<td>Generic</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Generic</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luria Agar</td>
			<td>Thermofisher Scientific</td>
			<td>22700025</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luria Broth</td>
			<td>Thermofisher Scientific</td>
			<td>12795084</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M9 Medium</td>
			<td>Sigma-Aldrich</td>
			<td>63011</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potato Dextrose Medium</td>
			<td>Fisher Scientfic&#160;</td>
			<td>DF0013-15-8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>K1377</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diaminopimelate</td>
			<td>Sigma-Aldrich</td>
			<td>92591</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thymine</td>
			<td>Sigma-Aldrich</td>
			<td>T0376</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyrosine</td>
			<td>Sigma-Aldrich</td>
			<td>T3754</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phenlylalanine</td>
			<td>Sigma-Aldrich</td>
			<td>P2126</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aspartate</td>
			<td>Sigma-Aldrich</td>
			<td>A9256</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Valine</td>
			<td>Sigma-Aldrich</td>
			<td>V0500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leucine</td>
			<td>Sigma-Aldrich</td>
			<td>L8000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoleucine</td>
			<td>Sigma-Aldrich</td>
			<td>I2752</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Uracil</td>
			<td>Sigma-Aldrich</td>
			<td>U0750</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gylcerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Arabinose</td>
			<td>Sigma-Aldrich</td>
			<td>A3256</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetracyline</td>
			<td>Sigma-Aldrich</td>
			<td>87128</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq DNA polymerase</td>
			<td>Thermofisher Scientific</td>
			<td>10342-020</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Platinum pfx DNA polymerase</td>
			<td>Thermofisher Scientific</td>
			<td>11708-013</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T4 DNA ligase</td>
			<td>Thermofisher Scientific</td>
			<td>15224-041</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E coli Dh5-alpha</td>
			<td>Thermofisher Scientific</td>
			<td align="right">18258012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E coli Top10</td>
			<td>Thermofisher Scientific</td>
			<td>C4040-03</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET100D/topo vector</td>
			<td>Thermofisher Scientific</td>
			<td>K100-01</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pCR2.1 Vector</td>
			<td>Thermofisher Scientific</td>
			<td>K2030-01</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?...

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 ...

Research

JoVE Journal

Biology

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

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

科研

生物学

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Bimolecular Fluorescence Complementation

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 ...

Determining the Contribution of the Energy Systems During Exercise

确定的能源系统，在运动的贡献

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

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

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

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 ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

双分子荧光互补流式细胞仪分析：高通量的定量方法研究蛋白质 - 蛋白质相互作用

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

实验教学中乳糖酶活性的测定

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

应用酵母雌激素筛检优选本科教学实验室的个人护理产品中的雌激素配体

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...