Name: fibs-enabled noninvasive metabolic profiling
Uploaded: 2014-02-03T15:15:00
Description: Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

Vi takker professor Wolf Frommer (Carnegie Institution for Science, Stanford University) for venligt giver os glukose FRET plasmid og Dr. Uwe Ludewig (Hohenheim Universitet) for venligt leverer glutamin FRET konstruere i pUTKan planteekspressionsvektor. AB er finansieret af BBSRC Målrettet program Prioriterede stipendier. Både CK og KP understøttes af RCUK stipendier i Biopharmaceuticals Processing. CK ønsker også at takke Lonza Biologics for deres økonomiske støtte. Center for Syntetisk Biologi og innovation er generøst støttet af EPSRC.

1.. Cellelinje Revival og vedligeholdelse <ol><li> Genoplive CHO celler i 9 ml CD-CHO medium suppleret med 8 mM L-glutamin og 10 ml / L 100x hypoxanthin / thymidin supplement (komplet vækstmedium). </li><li> Centrifuger ved 100 xg i 5 min. </li><li> Resuspender cellerne i 10 ml frisk komplet vækstmedium. </li><li> Fjern en 1 ml prøve og bestemme den levedygtige cellekoncentration ved lysmikroskopi under anvendelse af trypanblåt-farveeksklusion metode i et hæmocytometer. </li><li> Indled en kultur på en podningst...

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+cytosolic+glucose+and+ATP+levels+in+yeast+using+optical+sensors.">Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.</a> Biochem. J. 432, 399-406 (2010).</li><li>Yang, H. Y., Bogner, M., Stierhof, Y. D., Ludewig, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H(+)-independent+glutamine+transport+in+plant+root+tips.">H(+)-independent glutamine transport in plant root tips.</a> Plos One. 5, (2010).</li><li>Fehr, M., Takanaga, H., Ehrhardt, D. W., Frommer, W. 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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+hierarchical+relationship+of+Src+and+Rac+at+subcellular+locations+with+FRET+biosensors.">Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (38), 14353-14358 (2008).</li><li>Han, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+recombinant+Chinese+hamster+ovary+cells+grown+as+suspended+aggregates+in+stirred+vessels.">Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.</a> J. Biosci. Bioeng. 102 (5), 430-435 (2006).</li><li>Deuschle, K., Okumoto, S., Fehr, M., Looger, L. L., Kozhukh, L., Frommer, W. 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De FIBS muliggøre in vivo og in situ kvantificering af de vigtigste molekyler, i dette tilfælde vækstbegrænsende næringsstoffer, fjerne usikkerhedsmomenter, der skyldes afkølende og udvinding metoder. Resultaterne tyder på, at der er en god sammenhæng mellem FIBS signal og de intracellulære koncentrationer i området 1-5 mM for glukose og 0,3-2 mM for glutamin. I batch CHO cellekultur, er disse koncentrationer stødt på i de eksponentielle, stationære og tidlige tilbagegang faser. Den eksponentielle ...

Metabolite overvågning har forskellige formål i bioforarbejdning, herunder udvikling af processen, medier og design foder og metabolic engineering. Forskellige fremgangsmåder er tilgængelige for koncentrationsmålinger ved brug af enzymatiske 1, kemiske 2 eller bindingsassays 3. En interessant mulighed er brugen af fluorescerende protein-baserede in vivo biologiske sensorer som indberettere af den intracellulære koncentration af vigtige metabolitter. I ultralav volumen assays, fluorescens er et praktisk værktøj som miniaturisering faktisk forbedrer signal-til-støj-forhold 4,5 og protein-baserede sensorer kan være genetisk kodet således, at ingen exogene reagenser er nødvendige for metabolit-analyse. Forster Resonance Energy Transfer (FRET) biosensorer består af to fluorescerende proteiner forbundet med et ligandbindingsdomæne. FRET er en nonradiative overførsel af energi fra et foto-exciterede donor til en acceptor fluorescerende molekyle Statusmeddelelse<html> ed i umiddelbar nærhed (&lt;100). Ligand spaltning eller binding forårsager en konformationsændring i sensoren, hvilket igen inducerer en ændring i nærheden af ​​de fluoroforer, fører til en ændring i FRET effektivitet målt ved ændringen i emissionsspektret. FRET integrerede biologiske sensorer (FIBS) er konstitutivt produceres i cellen linje og deres spektrale egenskaber ændres baseret på koncentrationen af ​​metabolitten i cellen. FIBS har hurtige svartider gør dem ideelle til at tage målinger for dynamiske modeller 6. Tidligere ansøgninger omfatte overvågning single 7-9 og flere 10 metabolitter og leverer data om Spatiotemporal fordeling 11. Kan oprettes FIBS i to konfigurationer: Apo-Max, hvor ligandbinding forstyrrer nærhed fluorophorerne sænke energioverførsel og Apo-Min, hvor ligandbinding bringer de to fluoroforer i tættere kontakt (Figur 1). _content &quot;&gt; I dette arbejde, er en protokol fremlægges til konstruktion kinesisk hamster ovarie (CHO) cellelinier, der konstitutivt udtrykker en FIBS for en metabolit, glucose eller glutamin. En metode etableres til kalibrering af sensoren in vivo for at muliggøre fremtidig kvantitativ målinger af intracellulære metabolit koncentration, som beskrevet i figur 2.. Baseret på dette, den intracellulære koncentration af glukose eller glutamin kan bestemmes i fed-batch-CHO cellekulturer, som de to næringsstoffer blev tilføjet i høje koncentrationer i-proces. Resultaterne vise, at du bruger det fluorescerende signal fra FIBS og den tidligere konstrueret kalibreringskurven præcist forudsigelse af den intracellulære koncentration er mulig, som bekræftet af en uafhængig enzymatisk assay. Denne metode giver betydelige fordele i forhold til de nuværende analytiske teknologier, fordi det er non-invasiv, lave omkostninger og hurtig, hvilket giver en real-time signal for FIBS der kan være monitored hele kulturen.

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		<tr height="22">
			<td height="22" style="height:22px;width:239px;">CHO-S Cells</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;">11619-012</td>
			<td style="width:459px;">Cell line will vary depending on the goals of the study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">pCDNA4/TO vector&#160;</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">TransIT-PRO Transfection Reagent</td>
			<td style="width:120px;">Mirus Bio</td>
			<td style="width:120px;">MIR 5700</td>
			<td style="width:459px;">Transfections can be accomplished using any method suitable for the cell line under study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Zeocin</td>
			<td style="width:120px;">Invivogen</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">CD-CHO medium</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">10743-011</td>
			<td style="width:459px;">Cell growth medium is dependent upon the cells under study</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">100X HT Supplement</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">11067-030</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">L-Glutamine 200 mM (100X), Liquid</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">25030032</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">InfinitePRO 200 plate reader</td>
			<td style="width:120px;">Tecan</td>
			<td style="width:120px;">FLx800TBI</td>
			<td style="width:459px;">Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted</td>
		</tr>
		<tr height="21">
			<td height="190" rowspan="6" style="height:190px;width:239px;">Filters for plate reader</td>
			<td rowspan="6" style="width:120px;">Tecan</td>
			<td align="right" style="width:120px;">30000463</td>
			<td rowspan="6" style="width:459px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(520NM BW 10NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(430NM BW 35NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022787</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">(465NM BW 35NM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Maxiprep Plasmid Purification Kit</td>
			<td style="width:120px;">Qiagen</td>
			<td style="width:120px;">12163</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Amplex Red glucose/glucose oxidase assay kit</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:120px;">A22189</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">EnzyChrom&#160; Glutamine Assay Kit</td>
			<td style="width:120px;">BioAssay systems</td>
			<td style="width:120px;">EOAC-100</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Improved Neubauer haemocytometer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:120px;">MNK-420-010N</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Incubator&#160;</td>
			<td style="width:120px;">Nuaire</td>
			<td style="width:120px;">NU-5510E</td>
			<td style="width:459px;"></td>
		</tr>
	</tbody>
</table>

fibs-enabled noninvasive metabolic profiling

I en tid med bioinformatik, er nødvendigt med nye high throughput eksperimentelle systemer for at befolke og forfine modeller, så de kan valideres for prognoser. Ideelt set ville sådanne systemer være lav volumen, hvilket udelukker prøvetagning og destruktive analyser, når tiden kursus data skal opnås. Der er behov for en in situ overvågning værktøj, der kan indberette de nødvendige oplysninger i realtid og non-invasivt. En interessant mulighed er brugen af fluorescerende protein-baserede in vivo biologiske sensorer som indberettere af intracellulære koncentrationer. En særlig klasse af in vivo-biosensorer, der har fundet anvendelser metabolit kvantificering er baseret på Förster Resonance Energy Transfer (FRET) mellem to fluorescerende proteiner forbundet med et ligandbindingsdomæne. FRET integrerede biologiske sensorer (FIBS) er konstitutivt produceres i cellen linje, de har hurtige svartider og deres spektrale characteristics ændre baseret på koncentrationen af ​​metabolitten i cellen. I dette papir, er fremgangsmåden til konstruktion af kinesisk hamsterovarie (CHO)-cellelinier, som konstitutivt udtrykker en FIBS for glucose og glutamin og kalibrere FIBS in vivo i batch cellekultur for at muliggøre fremtidig kvantificering af intracellulært metabolitkoncentrationen beskrevet. Data fra fed-batch-CHO cellekulturer viser, at de FIBS var i stand til i hvert enkelt tilfælde registrerer den deraf følgende ændring i den intracellulære koncentration. Ved hjælp af fluorescerende signal fra FIBS og den tidligere konstrueret kalibreringskurve blev den intracellulære koncentration bestemmes nøjagtigt som bekræftet af en uafhængig enzymatisk assay.

En oversigt over den metode er vist i figur 2.. I arbejdet præsenteret heri, blev CHO-celler transficeret med FIBS vektor og stabile cellelinjer blev selekteret på et antibiotikum tryk på 400 ug / ml zeocin. To separate stabile cellelinier konstitutivt udtrykker glucose og glutamin sensorer blev derfor oprettet. Figur 1 viser forskellige konfigurationer af de to biosensorer anvendt i denne undersøgelse. Glukosesensoren er baseret på Apo-Max princip og det tilsvarende plasmid pRSET-...

Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

FIBS-enabled Noninvasive Metabolic Profiling

En beskrivelse af, hvordan du kalibrerer Förster Resonance Energy Transfer integrerede biologiske sensorer (FIBS) til in situ metabolisk profilering præsenteres. FIBS kan anvendes til at måle intracellulære niveauer af metabolitter noninvasively medvirken i udviklingen af ​​metaboliske modeller og high throughput screening af biologisk proces betingelser.

FIBS-aktiverede Noninvasiv stofskifteprofil

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

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