Name: functional complementation analysis (fca): a laboratory exercise designed and implemented to supplement the teaching of biochemical pathways
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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 og MAS anerkender College of Science og Thomas H. Gosnell School of Life Sciences på Rochester Institute of Technology for støtte. Dette arbejde blev støttet delvist af USA National Science Foundation (NSF) pris til AOH MCB-1.120.541.

BEMÆRK: Forfatterne er villige til at give bakteriestammer og rekombinante plasmider for at lette indarbejdelsen af ​​funktionelle komplementering analyse til undervisningsbrug for personer, der er interesserede. Plasmiderne, der blev brugt til at lette funktionelle komplementerings- eksperimenter er anført i tabel 1. 1. Konstruktion af plasmider for Funktionel komplementering <ol><li> Kloning af diaminopimelat aminotransferase (dapL) for Funktionel komplementering. ...

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Mange af de kurser, der er en integreret del af bioteknologi og Molekylær Bioscience pensum på Rochester Institute of Technology har et laboratorium komponent ud over foredraget del af kurset. Studieordningen for det akademiske år 2014-2015 indeholder i alt 48 kurser, 29, der indeholder et laboratorium komponent, som udgør omkring 60%. En sådan kursus er Fundamentals of Plantebiokemi og Patologi (FPBP), en blandet foredrag / laboratorium kursus og Bioseparations: Principper og praksis (BPP), et laboratorium baseret...

Funktionel komplementering i forbindelse med afdækning funktion (er) / rolle (r) af et gen defineres som evnen af ​​en særlig homologe eller ortologe gen til at gendanne en bestemt mutant med en observerbar fænotype til vildtype-tilstand, når den homologe eller ortologe gen indføres i cis eller trans i mutanten baggrund. Denne teknik er blevet meget anvendt til at isolere og identificere den funktion (er) / rolle (r) af mange gener. Et særligt eksempel er isoleringen og identifikationen af orotidin-5-phosphat-decarboxylase fra Candida albicans under anvendelse af ura3 mutant af S. cerevisiae og pyrF mutant af E. coli. 1 Forfatterne har brugt denne teknik til at belyse funktionen af gener, der er involveret i metabolismen af aminosyrer, peptidoglycan og stringente reaktion i deres forskningsprogrammer og har indarbejdet denne teknik i deres undervisningsforløb i Biotechnology og Molekylær Bioscience (BMB) program på Rochester Institute of Technology (RIT). Forfatterne underviser Fundamentals of Plantebiokemi / Patologi (FPBP) (Hudson) og Bioseparations: principper og praksis (BPP) (Hudson / Savka), to øverste division elektiv laboratorium baserede kurser i BMB Program på RIT. Da nogle af de emner, der drøftes i kurserne er tilknyttet deres forskningsinteresser, har forfatterne indarbejdet mange af de teknikker og eksperimentelle værktøjer, der bruges i deres respektive forskningsprogrammer ind i disse to laboratorie-baserede kurser. Et sådant eksempel er funktionel komplementering som laboratorium øvelse at styrke undervisningsmateriale vedrørende aminosyre metabolisme fra planter, peptidoglycan og strenge svar stofskifte fra bakterier. Tre af aminosyre-veje fra planter, der diskuteres i FPBB kurset er, at lysin (Lys), tyrosin(Tyr) og phenylalanin (phe). Lys pathway er fremhævet i løbet på grund af betydningen af den aminosyre som en essentiel aminosyre for alle dyr navnlig mennesker siden dyr mangler den genetiske maskineri til at syntetisere Lys de novo. Derudover blev det for nylig opdaget, at planter anvender en vej for syntese af lys, der er væsentlig forskellig fra den for bakterier. Denne opdagelse blev delvist lettes ved funktionel komplementering af E. coli diaminopimelat (DAP) mutanter ved anvendelse af en gen, der koder for enzymet L, L-diaminopimelat aminotransferase (DapL) fra modellen planten Arabidopsis thaliana. 2 Varianten veje til syntese af lys gennem den mellemliggende diaminopimelat er vist i figur 1. Ud syntesen af lys letter gennem aspartat afledte familie af aminosyrer, som er stærkt reguleret. 3 Ud over deres betydning på protein Synthesis, de veje for tyr og phe er fremhævet givet deres betydning i at betjene som precursorforbindelser for anabolismen af phenylpropanoid forbindelser involverede syntesen af plante forsvar forbindelser såsom:. alkaloider, ligniner, flavonoider, isoflavonoider, hydroxykanelsyre blandt andre 4 tyr og phe veje fremhæves også at vise forskellen mellem anlægget og bakterielle anabolske veje. I bakterier er enzymet tyrosin-aminotransferase (TyrB) involveret i anabolisme af både aminosyrer, hvorimod i planter, enzymet primært involveret i katabolisme af tyr og phe og er ikke involveret i anabolisme af disse aminosyrer. (Figur 2). 4 Forskellene mellem Gram positive og Gram-negative bakterier vedrørende strukturen af ​​peptidoglycan (PG) er fremhævet i FPBP kurset. PG af Gram-negative bakterier er af interesse med hensyn plantepatologi baseret på det faktum, at de flesteplantepatogener er Gram negative. En nylig gennemgang vedrørende de 10 bakterielle phyto-patogener afslørede, at alle er Gram negative. Bakterierne var fra slægterne:. Pseudomonas, Ralstonia, Agrobacterium, Xanthomonas, Erwinia, Xylella, Dickeya og Pectobacterium 5 En af de kemiske forskelle ved sammenligning PG stilken af gramnegative og grampositive bakterier er forskellen mellem den tværbindende amino syrer med begge typer. Det første skridt for at forskellige tværbinding af PG forekommer i den cytoplasmatiske trinnet PG anabolismen og lettes af enzymet UDP N -acetylmuramoyl-L-alanyl-D-glutamat- meso -2,6-diaminopimelat ligase (MURE) (figur 3A). MURE katalyserer tilsætningen af en særlig diaminforbindelse på tredje position af peptidet stilken. 6 I de fleste Gram-negative bakterier, næstsidste lys precursor, meso -diaminopimelate ( <em&gt; m -DAP) fungerer som den tværbindende aminosyre og lys tjener den samme rolle i PG fleste gram-positive bakterier (figur 3B). 7. Dette skyldes det faktum, at både m -DAP og Lys besidder to amin grupper og kan danne to peptidbindinger til peptid stamceller tværbinding. I Bioseparations: principper og praksis (BPP) Selvfølgelig er forskellene mellem åbne og lukkede systemer til dyrkning af bakterier og hvordan næringsstoffer niveauer vil ændre sig betydeligt i begge systemer på grund af miljøændringer diskuteres. Disse begivenheder er knyttet til lovændringer kaldes en &quot;geare ned&quot; eller &quot;skifte op&quot; udløst af sult eller en tilstrækkelig forsyning af aminosyrer eller energi. &quot;Shift ned&quot; reaktion kan opstå, når en bakteriekultur overføres fra en rig og kompleks medium til et kemisk defineret medium med en enkelt kulstof kilde. Denne ændring i miljøet fører til den hurtige Cessation af tRNA og rRNA syntese. Dette ophør resulterer i manglende ribosomer, proteiner og DNA-syntese, selv om biosyntesen af ​​aminosyrer opreguleres. Efter &quot;geare ned&quot; svar, er de eksisterende ribosomer bruges til at producere nye enzymer til at syntetisere de aminosyrer, der ikke længere findes i mediet eller miljø. Efter en periode, er rRNA syntese og nye ribosomer samlet og populationen af ​​bakterieceller begynder at vokse, selv på en reduceret sats. Forløbet betegnes &quot;stringente reaktion&quot; eller &quot;streng kontrol&quot; og er et eksempel på global cellulær regulering og kan opfattes som en mekanisme til justering af cellens biosyntetiske maskineri for at kompensere for tilgængelighed af de nødvendige substrater og energibehov . 8 den strenge respons således gør det muligt for bakterier til at reagere hurtigt på fluxe af næringsstoffer i miljøet og bidrager og enhrelser evne bakterier til at konkurrere i miljøer, der kan ændre sig hurtigt med hensyn til næringsstoffer og eller substrat tilgængelighed. 8-9 Den stringente reaktion har en integrerende rolle i genekspression, når tilgængeligheden af aminosyrer, carbon, nitrogen, phosphat og fedtsyrer er begrænset. 8,10-14 Denne strenge reaktion koordineres af to nukleotider, guanosin tetraphosphat (ppGpp) og guanosin pentaphosphat (pppGpp) almindeligvis betegnet sammen som alarmone (p) ppGpp. For eksempel, når aminosyrer begrænset hvilket kan føre til en flaskehals i proteinsyntese-the alarmone, guanosin 3,5- (bis) pyrophosphat (ppGpp), afledt fra anabolisme af guanosin-3-diphosphat 5-triphosphat (pppGpp) akkumulerer i cellen. Ændringen i (p) ppGpp niveau er involveret i ekspression af gener, der regulerer reaktion afhjælpe manglen på substrater i miljøet, der er direkte involveret i cellevækst og udviklit. To af de gener, der er involveret i denne proces kaldes relA og Spot. RelA er et ribosom-forbundet (p) ppGpp-syntetaseaktivitet, der er involveret i responset til akkumulering af ikke-ladede tRNA&#39;er, som er resultatet af aminosyren begrænsning. Spot fungerer som et bifunktionelt (p) ppGpp-syntetaseaktivitet og hydrolase. Den syntetaseaktivitet SPOT er involveret i reaktion på den manglende kulstof og fedtsyre sult. 8 RelA / Spot homologer er udbredt i planter og bakterier og betegnes som Rsh for R ELA / S POT h omologs. 8,10 -12,16 en nylig manuskript viste, at der er en specifik Rsh protein involveret i syntesen af disse alarmones fra bakterien Novosphingobium sp Rr 2-17. 17 Her præsenterer vi fire biokemiske veje bundet til de funktionelle komplementeringsgrupper assays. Komplementeringen analyser skitseret i dette manuskript giver en avenue at EXPLmalm anvendelse af denne in vivo-assay som et middel til at identificere og karakterisere eller enzymer, der forudsiges at have ukendt / formodede funktion (er) eller som pædagogiske værktøjer til at supplere undervisningen i biokemiske veje.

<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

Funktionel komplementering assay (FCA) er en in vivo assay, der er almindeligt anvendt til at belyse funktionen / rolle af gener / enzymer. Denne teknik er meget almindelig i biokemi, genetik og mange andre discipliner. En samlet oversigt over den teknik til at supplere undervisningen i biokemiske veje vedrørende aminosyrer, peptidoglycan og den bakterielle strenge respons rapporteret i dette manuskript. To cDNA&#39;er fra modellen planteorganisme Arabidopsis thaliana, der er involveret i metabolismen af lysin (L, L-diaminopimelat aminotransferase (dapL) og tyrosin-aminotransferase (tyrB) involveret i metabolismen af tyrosin og phenylalanin er fremhævet. Derudover bakterielle peptidoglycan anabolske pathway er fremhævet gennem analyse af UDP- N -acetylmuramoyl-L-alanyl-D-glutamat- meso -2,6-diaminopimelat ligase (Mure) genet fra bakterien Verrucomicrobium spinosum involveret i indlægget-linking af peptidoglycan. Den bakterielle strenge reaktion er også rapporteret gennem analyse af rsh (r ela / s Pot h omolog) bifunktionel gen ansvarlig for en hyper-mucoid fænotype i bakterien Novosphingobium sp. Fire eksempler på FCA præsenteres. Videoen vil fokusere på tre af dem, nemlig lysin, peptidoglycan og stringent svar.

De bakteriestammer, der anvendes i de forskellige funktionelle komplementerings- analyser er anført i tabel 2. Funktionel komplementering analyse: L, L-diaminopimelat aminotransferase (dapL) E. coli dobbeltmutanten AOH1 (Δ dapD :: KAN2, dapE6) indeholder en mutation i dapE-genet og en komplet deletion af dapD-genet <stro...

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

Valideringen af ​​enzymatiske aktiviteter i forbindelse med biokemiske veje kan belyses ved hjælp af funktionel komplementering analyse (FCA). Beskrevet i dette manuskript er FCA-analysen viser den enzymatiske aktivitet af enzymer involveret i metabolismen af ​​aminosyrer, bakteriel strenge respons og bakteriel peptidoglycan biosyntese.

Funktionel Komplementeringsanalyse (FCA): A Laboratory Motion designet og implementeret til supplement af Undervisning i biokemiske veje

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 ...

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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

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

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

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

Bestemmelse af bidrag af Energy Systems under træning

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

Kortlægning Bakterielle Funktionelle Networks og spredningsveje i<em&gt; Escherichia Coli</em&gt; Hjælp Syntetiske Genetiske 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 ...

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

Flow cytometri af bimolekylær Fluorescens Complementation: En High Throughput Kvantitativ metode til at studere protein-protein interaktion

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

Isolering og funktionel analyse af mitokondrier fra dyrkede celler og mus Tissue

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

Måling af laktase enzymatisk aktivitet i undervisningen Lab

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

Påvisning af østrogene ligander i produkter til personlig pleje ved hjælp af en gær østrogen skærm optimeret for Undergraduate undervisning laboratorium

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

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