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 studien ble støttet av en NSF Career Grant (MCB0954016) og en DOE bioenergiforskning Grant (DEFG0208ER64694).

1. Cellekultur (Figur 1) <ol><li> Grow celler i minimal medium med sporstoffer, salter, vitaminer og spesielt merket karbon underlag som er best for pathway etterforskning. Bruk enten risting flasker eller bioreaktorer for cellekultur. Organiske næringsstoffer, slik som gjærekstrakt, kan forstyrre målingen av aminosyren merking og kan derfor ikke være til stede i kulturen medium. </li><li> Monitor cellevekst ved den optiske tettheten av kulturen på en optimal bølgelengde (f.eks OD 730 for Cyanothe...

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Denne protokollen består av fôring cellen med en merket substrat og måle den resulterende isotopiske merking mønstre i aminosyrer via GC-MS. Siden MS data (m / z ratio) gir akkurat den totale mengden av merking av MS ioner, må vi vurdere isotopomer distribusjoner av aminosyrer ved å undersøke m / z forholdstall på begge ufragmentert (M-57) + og fragmentert aminosyrer (dvs. (M-159) + og (f302) +). Videre kan vi utføre flere cellekulturer med en kjemisk identisk medium, men underl...

<table border="1"><tbody><tr><th> Navn på reagens </th><th> Firma </th><th> Katalognummer </th><th> Kommentarer (valgfritt) </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> Merket karbon substrat </td><td> Cambridge isotoplaboratorier </td><td> Avhenge av eksperimentelle kravet </td><td> Nettside: <a href="http://www.isotope.com">http://www.isotope.com</a> </td></tr><tr><td> Gasskromatograf </td><td> Agilent Technologies </td><td> Hewlett-Packard, modell 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> For tørking aminosyre prøver </td></tr></tbody></table>

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

Mikrober har komplekse metabolske veier som kan undersøkes ved hjelp av biokjemi og funksjonell genomikk metoder. En viktig teknikk for å undersøke celle sentrale metabolisme og oppdage nye enzymer er 13 C-assistert metabolisme analyse 1. Denne teknikken er basert på isotop-merking, hvor mikrobene blir matet med en 13 C-merket underlag. Ved å spore atom overgangen stier mellom metabolitter i biokjemiske nettverket, kan vi fastslå funksjonell stier og oppdage nye enzymer. Som en komplementær metode for å transcriptomics og proteomikk, tilnærminger for isotopomer-assistert analyse av metabolske veier inneholder tre viktige trinn to. Først vokser vi celler med 13 C merket underlag. I dette trinnet, sammensetningen av mediet og utvalget av merket substrater er to viktige faktorer. For å unngå måling støy fra ikke-merket karbon i nærings kosttilskudd, Er en minimal medium med en eneste karbonkilde nødvendig. Videre er valget av en merket substrat basert på hvor effektivt det vil belyse veien blir analysert. Fordi romanen enzymer ofte innebære annen reaksjon stereokjemi eller mellomprodukter, generelt, enkeltvis merket karbon underlag er mer informative for påvisning av nye veier enn jevnt merket kodekene for påvisning av nye veier 3, 4. Second, analyserer vi aminosyre merking mønstre ved hjelp av GC -MS. Aminosyrer er rik på protein, og dermed kan fås fra biomasse hydrolyse. Aminosyrer kan derivatized av N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) før GC separasjon. TBDMS derivatized aminosyrer kan bli fragmentert av MS og resultere i ulike matriser av fragmenter. Basert på masse for å lade (m / z) forholdet fragmentert og ufragmentert aminosyrer, kan vi utlede den mulige merket mønstre av de sentrale metabolitter somforløperne til aminosyrer. tredje spor vi 13C karbon overganger i den foreslåtte stier og, basert på isotopomer data, bekrefte om disse banene er aktive 2. Måling av aminosyrer gir isotopiske merking informasjon rundt åtte avgjørende forløper metabolitter i sentrale metabolisme. Disse metabolske viktige noder kan reflektere funksjonene tilhørende sentrale veier. 13 C-assistert metabolisme analyse via proteinogenic aminosyrer kan bli mye brukt for funksjonell karakterisering av dårlig karakterisert mikrobiell metabolisme 1. I denne protokollen, vil vi bruke Cyanothece 51142 som modell påkjenning å demonstrere bruk av merket karbon underlag for å oppdage nye enzymatisk funksjoner.

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-isotopen merking er en nyttig teknikk for å bestemme cellen sentrale metabolisme for ulike typer av mikroorganismer. Etter at cellene har vært dyrket med en bestemt merket substrat, kan GC-MS måling avdekker funksjonelle metabolismeveier basert på unike merking mønstre i proteinogenic aminosyrer.

Metabolsk Pathway Bekreftelse og Discovery Through 13 C-merking av Proteinogenic Amino Acids

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

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

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