Name: the use of chemostats in microbial systems biology
Uploaded: 2013-10-14T20:45:00
Description: Watch this Scientific Journal Video about The Use of Chemostats in Microbial Systems Biology at JoVE.com

Dette arbejde blev støttet af opstart midler fra New York University. Vi takker Maitreya Dunham og Matt Brauer, der oprindeligt blev udviklet brugen af ​​Sixfors bioreaktorer som kemostater.

Princippet om kontinuerlig dyrkning under anvendelse af en kemostat kan realiseres i en række forskellige implementeringer. I alle kemostater er det vigtigt at have 1) metoder til opretholdelse af sterilitet af alle komponenter, 2) et godt blandet kultur, 3) passende luftning af kultur fartøj og 4) en pålidelig hjælp af medier tilføjelse og fjernelse kultur. Her beskriver vi anvendelse af en Sixfors bioreaktor (Infors Inc) som en kemostat under anvendelse af fremgangsmåder, som let kan tilpasses til alternative op...

<ol>
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Kemostater muliggøre dyrkning af mikrober i vækst-kontrollerede steady-state betingelser. Cellerne vokser kontinuerligt ved en konstant hastighed resulterer i en invariant eksterne miljø. Dette er i modsætning til batch kultur metoder, hvor det eksterne miljø der konstant ændrer og hastigheden af ​​cellevækst bestemmes af det komplekse samspil mellem miljø og genotype. Således er en stor fordel ved dyrkning af mikrober i kemostater flere batchkulturer er evnen til eksperimentelt at kontrollere væksten af ?...

Væksten af celler er reguleret af komplekse netværk af interagerende genetiske og miljømæssige faktorer 1,2. Den multifaktoriel regulering af cellevækst nødvendiggør en strategi system-niveau til sin undersøgelse. Men den nøje undersøgelse af regulerede cellevækst udfordret af vanskeligheden ved eksperimentelt at styre den hastighed, hvormed cellerne vokser. Endvidere i selv de simpleste forsøg ekstracellulære betingelser er ofte dynamisk og kompleks som celler kontinuerligt ændre deres omgivelser, som de formere sig. En løsning på disse problemer er leveret af kemostaten: en metode til dyrkning af celler, der muliggør eksperimentel kontrol af celle vækstrater i definerede, invariante og kontrollerede miljøer. Metoden til kontinuerlig dyrkning ved hjælp af en kemostat blev selvstændigt beskrevet af Monod 3 og Novick &amp; Szilard 4 i 1950. Som oprindeligt udtænkt dyrkes celler i en fast mængde medier, der er conbestandigt fortyndet ved tilsætning af nye medier og samtidig fjernelse af gamle medier og celler (figur 1). Koblede ordinære differentialligninger (figur 2) beskriver ændringen i celle densitet (x), og koncentrationen af en vækst-begrænsende næringsstof (fer) i kemostat fartøjet. Vigtigere er det, dette system af ligninger forudsiger en enkelt (ikke nul) stabil steady-state (Figur 3) med den bemærkelsesværdige konsekvenser, at ved steady-state, den specifikke væksthastighed af cellerne (dvs. den eksponentielle vækst konstant) er lig med den sats hvor kulturen fortyndet (D). Ved at variere fortyndingsforholdet er det muligt at etablere steady state populationer af celler ved forskellige vækstrater og under forskellige betingelser for næringssaltbegrænsning. Den eksperimentelle styring af væksten ved hjælp kemostater var afgørende for udviklingen af ​​en forståelse af, hvordan celle fysiologi ændringermed vækstrater 5,6. Men denne tidligere grundpille i mikrobiologiske metoder blev stadig mere uklar under eksplosionen i molekylærbiologisk forskning i slutningen af ​​det tyvende århundrede. I dag har fornyet interesse i vækstkontrol i både mikrober og flercellede organismer og fremkomsten af ​​genom-skala for systemer niveau analyse fornyet motivation for anvendelse af kemostater. Her beskriver vi tre ansøgninger, der udnytter den præcise styring af celle vækstrater, og det eksterne miljø, som er unikt muligt ved hjælp kemostater. Først, vi beskrive brugen af ​​kemostater at undersøge, hvordan den overflod af tusindvis af biomolekyler - såsom udskrifter og metabolitter - er sideordnet reguleres med vækstrate. For det andet, vi beskriver, hvordan kemostater kan bruges til at indhente præcise estimater af vækst-rate forskelle mellem forskellige genotyper i næringsfattige begrænsede miljøer ved hjælp af konkurrence-eksperimenter. For det tredje, beskriver vi, hvordan kemostater kananvendes til at studere adaptive evolution af celler, der vokser i faste næringsfattige miljøer. Disse eksempler eksemplificerer de måder, hvorpå kemostater der muliggør undersøgelser af cellevækst regulering, gen af ​​miljø interaktioner og adaptive evolution systemer niveau.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Infors-HT Sixfors Chemostat</td>
			<td>Appropriate Technical Resources, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Bottle 9.5 L</td>
			<td>Fisher Scientific</td>
			<td>02-887-1</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Pinchcock</td>
			<td>Fisher Scientific</td>
			<td>05-867</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Stopper, Size 12, Green Neoprene</td>
			<td>Cole-Palmer</td>
			<td>EW-62991-42</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Straight Connector</td>
			<td>Cole-Palmer</td>
			<td>EW-30703-02</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>General purpose ties 4 in</td>
			<td>Fisher Scientific</td>
			<td>NC9557052</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Silicone Rubber</td>
			<td>Small Parts</td>
			<td>B000FMWTDE</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Silicone, 3/8 in OD</td>
			<td>Fisher Scientific</td>
			<td>02-587-1Q</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Silicone, 7/32 in OD</td>
			<td>Fisher Scientific</td>
			<td>02-587-1E</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Stainless Steel, 3/16 in OD</td>
			<td>McMaster-Carr</td>
			<td>6100K164</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Tubing, Stainless Steel, 3/8 in OD</td>
			<td>McMaster-Carr</td>
			<td>6100K161</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Hook Connectors</td>
			<td>Fisher Scientific</td>
			<td>14-66-18Q</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Ratchet Clamp</td>
			<td>Cole-Palmer</td>
			<td>EW-06403-11</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Luer, Female</td>
			<td>Cole-Palmer</td>
			<td>EW-45512-34</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Luer, Male</td>
			<td>Cole-Palmer</td>
			<td>EW-45513-04</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Millipore Aervent MTGR05010 62 mm Filter, 0.2 &mu;m</td>
			<td>Fisher Scientific</td>
			<td>MTGR05010</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>PTFE Acrodisc CR 13 mm filters, 0.2 &mu;m</td>
			<td>Fisher Scientific</td>
			<td>NC9131037</td>
			<td>For Media Vessel and Hosing</td>
		</tr>
		<tr>
			<td>Direct-Reading Flowtube for Air</td>
			<td>Cole-Palmer</td>
			<td>EW-32047-77</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Direct-Reading Flowtube for Nitrogen</td>
			<td>Cole-Palmer</td>
			<td>EW-32048-63</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Gas Proportioner Multitube Frames</td>
			<td>Cole-Palmer</td>
			<td>EW-03218-50</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Regulator, Two-Stage Analytical</td>
			<td>Airgas</td>
			<td>Y12-N145D580</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Hose Adaptor, Stainless Steel</td>
			<td>Airgas</td>
			<td>Y99-26450</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Hose Male Adaptor</td>
			<td>Airgas</td>
			<td>WES544</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Norprene Tubing</td>
			<td>US Plastics</td>
			<td>57280</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Tripod Base</td>
			<td>Cole-Palmer</td>
			<td>EW-03218-58</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Valve Cartridges</td>
			<td>Cole-Palmer</td>
			<td>EW-03217-92</td>
			<td>For Nitrogen Gas Setup</td>
		</tr>
		<tr>
			<td>Carboy 10 L</td>
			<td>Fisher Scientific</td>
			<td>02-963-2A</td>
			<td>For Media Preperation</td>
		</tr>
		<tr>
			<td>Steritop Sterile Vacuum Bottle-Top Filters, 1,000 ml, PES membrane; for 45 mm neck size</td>
			<td>Fisher Scientific</td>
			<td>SCGP-T10-RE</td>
			<td>For Media Preperation</td>
		</tr>
		<tr>
			<td>Media Bottle 100 ml, 45 mm neck size</td>
			<td>Fisher Scientific</td>
			<td>FB-800-100</td>
			<td>For Media Preperation</td>
		</tr>
		<tr>
			<td>calcium chloride&middot;2H2O</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>magnesium sulfate&middot;7H2O</td>
			<td>Sigma Aldrich</td>
			<td>230391</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>potassium phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>AC424205000</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>ammonium sulfate</td>
			<td>Fisher Scientific</td>
			<td>AC423400010</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>boric acid</td>
			<td>Sigma Aldrich</td>
			<td>B6768</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>copper sulfate&middot;5H2O</td>
			<td>Sigma Aldrich</td>
			<td>209198</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>potassium iodide</td>
			<td>Sigma Aldrich</td>
			<td>60400</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>ferric chloride&middot;6H2O</td>
			<td>Fisher Scientific</td>
			<td>I88-100</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>manganese sulfate&middot;H2O</td>
			<td>Sigma Aldrich</td>
			<td>230391</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>sodium molybdate&middot;2H2O</td>
			<td>Sigma Aldrich</td>
			<td>M7634</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>zinc sulfate&middot;7H2O</td>
			<td>Fisher Scientific</td>
			<td>Z68-500</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>biotin</td>
			<td>Fisher Scientific</td>
			<td>BP232-1</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>calcium pantothenate</td>
			<td>Fisher Scientific</td>
			<td>AC24330-1000</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>folic acid</td>
			<td>Sigma Aldrich</td>
			<td>F7876</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>inositol (aka myo-inositol)</td>
			<td>Fisher Scientific</td>
			<td>AC12226-1000</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>niacin (aka nicotinic acid)</td>
			<td>Sigma Aldrich</td>
			<td>N4126</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>p-aminobenzoic acid</td>
			<td>Fisher Scientific</td>
			<td>AC14621-2500</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>pyridoxine HCl</td>
			<td>Sigma Aldrich</td>
			<td>P9755</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>riboflavin</td>
			<td>Sigma Aldrich</td>
			<td>R4500-25G</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>thiamine HCl</td>
			<td>Fisher Scientific</td>
			<td>BP892-100</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>Leucine</td>
			<td>Sigma Aldrich</td>
			<td>L8000-100G</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma Aldrich</td>
			<td>U0750</td>
			<td>Media Reagents</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>DF0155-08-5</td>
			<td>Media Reagents</td>
		</tr>
	</tbody>
</table>

the use of chemostats in microbial systems biology

Celler regulere deres vækst som reaktion på signaler fra den ydre verden. Som cellen vokser, skal forskellige cellulære processer koordineres herunder makromolekylær syntese, metabolisme og i sidste ende engagement celledelingscyklussen. Kemostaten, en metode til eksperimentelt kontrollerende celle vækst, giver et stærkt middel til systematisk at studere, hvordan vækstrate påvirker cellulære processer - herunder genekspression og stofskifte - og de regulerende netværk, der styrer hastigheden af ​​cellevækst. Når opretholdes for hundreder af generationer kemostater kan bruges til at studere adaptive evolution af mikrober i miljøforhold, der begrænser cellevækst. Vi beskriver princippet om kemostat kulturer demonstrere deres drift og give eksempler på deres forskellige applikationer. Efter en stilstandsperiode efter deres indførelse i midten af ​​det tyvende århundrede, konvergensen af ​​genomet-skala metoder med en fornyet irente i reguleringen af ​​cellevækst og det molekylære grundlag for adaptiv evolution er stimulerende en renæssance i brugen af ​​kemostater i biologisk forskning.

En stor fordel ved kemostater er evnen til at kontrollere væksten af ​​celler eksperimentelt ved at variere fortyndingsforholdet. I spirende gær, Saccharomyces cerevisiae, morfologien af ​​en celle er informativ for fase celledelingscyklussen. Populationer med højere vækstrater indeholder en højere andel af aktivt delende celler som bestemt ved måling af fraktionen af knopskudte celler (figur 5A). Analyser af den globale mRNA-ekspression i kemostat kulturer har vist, at ekspressionen af ma...

Watch this Scientific Journal Video about The Use of Chemostats in Microbial Systems Biology at JoVE.com

The Use of Chemostats in Microbial Systems Biology

Cell vækstrate er en reguleret proces, og en primær faktor for celle fysiologi. Kontinuerlig dyrkning ved hjælp kemostater muliggør ydre kontrol af cellevækst sats ved næringssaltbegrænsning lette studiet af molekylære netværk, der styrer cellevækst og hvordan disse net udvikler sig til at optimere cellevækst.

Brug af kemostater i Mikrobiel Systembiologi

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

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