Name: metabolic pathway confirmation and discovery through 13c-labeling of proteinogenic amino acids
Uploaded: 2012-01-26T13:00:00
Description: Watch this Scientific Journal Video about Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids at JoVE.com

Denne undersøgelse blev støttet af en NSF Career Grant (MCB0954016) og en DOE Bioenergi Research Grant (DEFG0208ER64694).

1. Cellekultur (figur 1) <ol><li> Grow celler i minimal medie med sporstoffer, salte, vitaminer og specifikt mærket kulstof substrater, der er bedst for pathway undersøgelse. Brug enten ryster kolber eller bioreaktorer til cellekulturmedier. Organiske næringsstoffer, såsom gærekstrakt, kan interferere med måling af aminosyre mærkning og kan derfor ikke være til stede i dyrkningsmediet. </li><li> Overvåg cellevækst ved at den optiske tæthed af kulturen på en optimal bølgelængde (f.eks OD 730 ti...

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Denne protokol består af fodring cellen med en mærket substrat og måle den resulterende isotopisk mærkning mønstre i de aminosyrer via GC-MS. Siden MS data (m / z nøgletal) giver blot det samlede beløb for mærkning af MS ioner, er vi nødt til at vurdere isotopomer fordelinger af aminosyrer ved at undersøge m / z forhold for både fragmenteret (M-57) + og fragmenteret aminosyrer (dvs. (M-159) + og (f302) +). Desuden kan vi udføre flere cellekulturer med en kemisk identisk mediu...

<table border="1"><tbody><tr><th> Navn på reagenset </th><th> Firma </th><th> Katalog nummer </th><th> Kommentarer (valgfri) </th></tr><tr><td> TBDMS </td><td> Sigma-Aldrich </td><td> 19915 </td><td> - </td></tr><tr><td> THF </td><td> Sigma-Aldrich </td><td> 34865 </td><td> - </td></tr><tr><td> Mærket kulstof substrat </td><td> Cambridge isotoplaboratorier </td><td> Afhænger af den eksperimentelle krav </td><td> Hjemmeside: <a href="http://www.isotope.com">http://www.isotope.com</a> </td></tr><tr><td> Gaskromatograf </td><td> Agilent Technologies </td><td> Hewlett-Packard, model 7890A </td><td> - </td></tr><tr><td> GC kolonner </td><td> J &amp; W Scientific, Folsom, CA </td><td> DB5 (30m) </td><td> - </td></tr><tr><td> Massespektrometer </td><td> Agilent Technologies </td><td> 5975C </td><td> - </td></tr><tr><td> Reacti-VAP Evaporator </td><td> Thermo Scientific </td><td> TS-18825 </td><td> Til tørring aminosyre prøver </td></tr></tbody></table>

metabolic pathway confirmation and discovery through 13c-labeling of proteinogenic amino acids

Mikrober har komplekse metaboliske veje, som kan undersøges ved hjælp af biokemi og funktionel genomik metoder. En vigtig teknik til at undersøge cellens centrale stofskifte og opdage nye enzymer er 13 C-assisteret stofskifte analyse 1. Denne teknik er baseret på isotop-mærkning, hvor mikroorganismer er fodret med en 13 C-mærkede substrater. Ved at spore atomet overgangen stier mellem metabolitter i de biokemiske netværk, kan vi bestemme funktionelle veje og opdage nye enzymer. Som en supplerende metode til at transcriptomics og proteomics, strategier for isotopomer-assisteret analyse af stofskifteveje indeholder tre vigtige trin 2. Først, vi vokser cellerne med 13 C-mærkede substrater. I dette trin, er sammensætningen af ​​medium og udvælgelsen af ​​mærkede substrater to vigtige faktorer. For at undgå måling støj fra ikke-mærkede kulstof i næringsstof kosttilskud, Er en minimal medie med en eneste kulstofkilde påkrævet. Endvidere er valget af en mærket substrat er baseret på, hvor effektivt det vil belyse den vej at blive analyseret. Fordi romanen enzymer ofte indebærer forskellige reaktioner stereokemi og halvfabrikata, i almindelighed, enkeltvis mærket kulstof substrater er mere informativ til påvisning af nye veje end ensartet mærket dem til påvisning af nye veje 3, 4. For det andet analyserer vi aminosyre mærkning mønstre ved hjælp af GC -MS. Aminosyrer er rigelige på protein og dermed kan fås fra biomasse hydrolyse. Aminosyrer kan derivatized af N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) før GC adskillelse. TBDMS derivatized aminosyrer kan blive splittet på grund af MS og resultere i forskellige arrays af fragmenter. Baseret på massen til at oplade (m / z) forholdet mellem fragmenteret og fragmenteret aminosyrer, kan vi udlede den mulige mærket mønstre af de centrale metabolitter, der erforløbere for de aminosyrer. tredje, vi spore 13C kulstof overgange i det foreslåede veje, og baseret på isotopomer data, bekræfte, om disse veje er aktive 2. Måling af aminosyrer giver isotopiske mærkning oplysninger omkring otte afgørende forløber metabolitter i det centrale stofskifte. Disse metaboliske centrale knudepunkter kan afspejle de funktioner af associerede central-veje. 13 C-assisteret stofskifte analyse via proteinogene aminosyrer kan anvendes bredt til funktionel karakterisering af dårligt karakteriseret mikrobielle metabolisme 1. I denne protokol, vil vi bruge Cyanothece 51142 som den model stammen til at demonstrere brugen af mærket kulstof substrater for at opdage nye enzymatiske funktioner.

Watch this Scientific Journal Video about Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids at JoVE.com

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

13 C-isotop mærkning er en teknik til bestemmelse af cellens centrale stofskifte for forskellige typer af mikroorganismer. Efter at cellerne har været dyrket med en bestemt mærket substrat, kan GC-MS måling afslører funktionelle stofskifteveje baseret på unikke mærkning mønstre i proteinogene aminosyrer.

Metaboliseringsvej Bekræftelse og Discovery Gennem 13 C-mærkning af proteinogene aminosyrer

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

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