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 और मास विज्ञान के कॉलेज और थॉमस एच Gosnell समर्थन के लिए Rochester प्रौद्योगिकी संस्थान में लाइफ साइंसेज के स्कूल मानता है। इस काम में संयुक्त राज्य अमेरिका के राष्ट्रीय विज्ञान फाउंडेशन (NSF) AOH एमसीबी-1,120,541 को यह पुरस्कार द्वारा समर्थित किया गया था।

नोट: लेखकों व्यक्तियों, जो रुचि रखते हैं के लिए शिक्षण उद्देश्यों के लिए कार्यात्मक पूरक विश्लेषण के समावेश की सुविधा के लिए जीवाणु उपभेदों और पुनः संयोजक plasmids प्रदान करने के लिए तैयार हैं। Plasmids कि कार्य...

<ol>
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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. 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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. 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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. 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पाठ्यक्रम कि Rochester प्रौद्योगिकी संस्थान में जैव प्रौद्योगिकी और आणविक बायोसाइंस पाठ्यक्रम का अभिन्न अंग हैं में से कई पाठ्यक्रम के व्याख्यान हिस्से के अलावा एक प्रयोगशाला घटक है। शैक्षणिक वर्ष 2014-2015 क?...

<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

कार्यात्मक complementation परख (एफसीए) एक Vivo परख है कि व्यापक रूप से जीन / एंजाइमों के समारोह / भूमिका स्पष्ट करने के लिए प्रयोग किया जाता है। इस तकनीक जैव रसायन, आनुवंशिकी और कई अन्य विषयों में बहुत आम है। तकनीक की एक व्यापक सिंहावलोकन एसिड होता है, पेप्टीडॉग्लीकैन और बैक्टीरियल कड़े प्रतिक्रिया इस पांडुलिपि में सूचना दी है एमिनो से संबंधित जैव रासायनिक रास्ते के शिक्षण के पूरक के लिए। मॉडल संयंत्र जीव Arabidopsis thaliana कि लाइसिन (एल, एल diaminopimelate एमिनोट्रांस्फरेज (dapL) और tyrosine एमिनोट्रांस्फरेज (tyrB) tyrosine और फेनिलएलनिन के चयापचय में शामिल की चयापचय में शामिल कर रहे हैं से दो cDNAs डाला जाता है। इसके अलावा, बैक्टीरियल पेप्टीडॉग्लीकैन उपचय मार्ग पार करने में शामिल जीवाणु Verrucomicrobium spinosum से UDP- एन -acetylmuramoyl एल alanyl-D-glutamate- मेसो -2,6-diaminopimelate ligase (Mure) जीन के विश्लेषण के माध्यम से प्रकाश डाला हैपेप्टीडॉग्लीकैन की -linking। बैक्टीरियल कड़े प्रतिक्रिया भी RSH (आर इला / एस बर्तन ज omolog) bifunctional जीन जीवाणु Novosphingobium सपा में एक अति mucoid phenotype के लिए जिम्मेदार के विश्लेषण के माध्यम से सूचना दी है। एफसीए के चार उदाहरण प्रस्तुत कर रहे हैं। वीडियो उनमें से तीन पर ध्यान दिया जाएगा, अर्थात् लाइसिन, पेप्टीडॉग्लीकैन और कड़े प्रतिक्रिया।

जीवाणु उपभेदों कि विभिन्न कार्यात्मक पूरक विश्लेषण में कार्यरत हैं 2 टेबल में सूचीबद्ध हैं। कार्यात्मक पूरक विश्लेषण: एल, एल diaminopimelate एमिनोट्रांस्फरेज (dapL) <p class...

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

एंजाइमी जैव रासायनिक रास्ते में शामिल गतिविधियों की मान्यता कार्यात्मक पूरक विश्लेषण (एफसीए) का उपयोग कर elucidated जा सकता है। इस पांडुलिपि में वर्णित अमीनो एसिड, बैक्टीरियल कड़े प्रतिक्रिया और बैक्टीरियल पेप्टीडॉग्लीकैन जैवसंश्लेषण के चयापचय में शामिल एंजाइमों के enzymatic गतिविधि का प्रदर्शन एफसीए परख है।

कार्यात्मक complementation विश्लेषण (एफसीए): एक प्रयोगशाला व्यायाम डिजाइन और जैव रासायनिक रास्ते के शिक्षण के पूरक के लिए लागू

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

Zebrafish में लेंस प्रत्यारोपण और आँखों म्यूटेंट के विश्लेषण में इसके अनुप्रयोग

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 ...

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

Mutagenesis और आयन चैनल के कार्यात्मक विश्लेषण Heterologously स्तनधारी कोशिकाओं में व्यक्त

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

Bimolecular प्रतिदीप्ति complementation

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

व्यायाम के दौरान ऊर्जा प्रणालियों के योगदान निर्धारण

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

बैक्टीरियल कार्यात्मक नेटवर्क और रास्ते का मिलान<em&gt; Escherichia कोलाई</em&gt; सिंथेटिक जेनेटिक arrays का उपयोग

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

Bimolecular प्रतिदीप्ति complementation का प्रवाह cytometric विश्लेषण: प्रोटीन, प्रोटीन बातचीत का अध्ययन करने के लिए एक उच्च Throughput मात्रात्मक विधि

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

अलगाव और संवर्धित कोशिकाओं और माउस के ऊतकों से माइटोकॉन्ड्रिया के कार्यात्मक विश्लेषण

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

शिक्षण प्रयोगशाला में लैक्टेज एंजाइमी गतिविधि को मापने

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

एक खमीर एस्ट्रोजन स्क्रीन स्नातक शिक्षण प्रयोगशाला के लिए अनुकूलित का उपयोग कर व्यक्तिगत देखभाल उत्पादों में एस्ट्रोजन लाइगैंडों का पता लगाने

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

आनुवंशिक आंत सामग्री के लिए प्रयोगशाला प्रोटोकॉल समूह विशिष्ट rDNA प्राइमरों का उपयोग जलीय Macroinvertebrates का विश्लेषण

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