Name: procedure for adaptive laboratory evolution of microorganisms using a chemostat
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat at JoVE.com

This study was financially supported by the Korean Ministry of Science, ICT and Future Planning (Intelligent Synthetic Biology Center program 2012M3A6A8054887). P. Kim was supported by a fellowship from the Catholic University of Korea (2015).

1. Utstyr Forberedelser <ol><li> Oppnå en kjemostat krukke (150-250 ml) eller en Erlenmeyer-kolbe (250 ml) inneholdende en innløpsport og en utløpsport. Koble portene med silikonrøret slik at for strømningshastigheter på 10 til 100 ml / time. Eventuelt bruke en luftventilen, en luftutløp port, og temperaturkontrollert vann innløps- og utløpsportene. </li><li> Skaff en enhet som passer for kjemostaten krukke som sørger for agitasjon og temperaturkontroll (eller bruk en roterende risteinkubator). </li><...

<ol>
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Mikroorganismer er i stand til å tilpasse seg nesten alle miljøer på grunn av sin raske vekst og genetisk mangfold. Adaptive laboratorium evolusjon gjør at mikroorganismer å utvikle seg i henhold til designet forhold, som gir mulighet til å velge individuelle organismer som bærer spontane mutasjoner som er gunstig under de gitte forhold. Kjemostaten teknikken er mer robust for å oppnå kunstig drevet evolusjon enn overføringsteknikker av følgende grunner: (a) en jevn miljø - fordi...

Mikroorganismer kan overleve og tilpasse seg ulike miljøer. Under alvorlig stress, kan tilpasning skje via oppkjøpet av gunstige fenotyper av tilfeldige genomiske mutasjoner og påfølgende positiv utvelgelse 1-3. Derfor kan mikrobielle celler tilpasse ved å endre metabolske eller regulatoriske nettverk for optimal vekst, som kalles &quot;adaptiv evolusjon&quot;. Nylige viktige mikrobielle tendenser, slik som utbrudd av super og forekomsten av robuste mikrobestammer, er meget nært beslektet med adaptive evolution under stressbetingelser. Under definerte laboratorieforhold, er vi i stand til å studere mekanismene for molekylær evolusjon og selv styre retningen av mikrobiell evolusjon for ulike bruksområder. I motsetning til flercellede organismer, encellede organismer er godt egnet til adaptive laboratorium evolusjon (ALE) av følgende grunner: de regenerere raskt, de opprettholde store bestander, og det er lett å lage og vedlikeholde homogeneous miljøer. Kombinert med nylige fremskritt i DNA-sekvensering teknikker og high-throughput teknologier, gjør ALE for direkte observasjon av genomisk endringer som fører til systemiske regulatoriske endringer. Mutasjons dynamikk og et mangfold av befolkningen er også observerbar. Genetic Engineering strategier kan bli bestemt fra analysen av ALE-stammer 4,5. Kjemostatkultur er en metode som brukes for å oppnå steady-state celler og øke produktiviteten i gjæringsprosessen 6. Friskt medium blir tilsatt og kulturmediet høstes i løpet av prosessen (den sistnevnte omfatter medium og biomasse). Long-term kjemostatkultur imidlertid endrer steady-state produktivitet av kulturen og forårsaker akkumulering av spontane mutasjoner og valg i kultur (figur 1a). Under forskjellige seleksjonstrykk (stressorer), er akkumulering av mutasjoner forbedret. En gradvis økning av stress i en langsiktig<html> kjemostat sørger for en kontinuerlig utvalg av mutasjoner som virker mot de gitte stressfaktorer, for eksempel temperatur, pH, osmotisk trykk, nærings sult, oksidasjon, giftige sluttprodukter, etc. Colony overføring fra et fast medium og serieoverføring fra et flytende medium (gjentatt batch kultur) også gi forskerne utviklet mikroorganismer (Figur 1b og 1c). Selv om kjemostatkultur krever kompliserte metoder, pool av mangfoldet (antall replikasjoner og populasjonsstørrelse) er høyere enn den som oppnås ved koloni-overføring og serieoverføringsteknikker. Den stabile stresset eksponering mot enkeltceller og redusert variasjon i celle tilstand under kjemostatkultur (steady state) er andre fordeler med ALE forhold til batch kulturbaserte teknikker. Stress-indusert ALE av Escherichia coli som utsettes for høye succinat betingelser er innført i denne artikkelen. iles / ftp_upload / 54446 / 54446fig1.jpg &quot;/&gt; Figur 1: Metoder for adaptive laboratorium evolusjon (A) kjemostat;. (B) seriell overføring; (C) koloni overføring. Den øverste tallene illustrerer konseptet av metodene for ALE, og de ​​nederste tallene illustrerer antall celler som vokste under ALE. <a href="https://www.jove.com/files/ftp_upload/54446/54446fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</a>

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mini-chemostat fermentor</td>
			<td style="width:71px;">Biotron Inc.</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">manufactured by special order</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">silicon tubing</td>
			<td style="width:71px;">Cole-Parmer</td>
			<td style="width:119px;">Masterflex L/S 13</td>
			<td>tubing size can be varied depending on the dilution rate and the size of fermentor jar.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">reservoir jar</td>
			<td style="width:71px;">Bellco</td>
			<td style="width:119px;">Media storage bottle&#160;</td>
			<td>20 L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">chemicals</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">-</td>
			<td>reagent grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glucose</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5767</td>
			<td>ACS reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NH4Cl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9434</td>
			<td>for molecular biology, suitable for cell culture, &#8805;99.5%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">746398</td>
			<td>ACS reagent, &#8805;99%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Na2HPO4&#183;2H2O</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">4272</td>
			<td>98.5-101%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">KH2PO4&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">795488</td>
			<td>ACS reagent, &#8805;99%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MgSO4&#183;7H2O</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">230391</td>
			<td>ACS reagent, &#8805;98%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CaCl2</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">793639</td>
			<td>ACS reagent, &#8805;96%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">thiamine&#183;HCl&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4625</td>
			<td>reagent grade, &#8805;99%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Na2&#183;succinate&#183;6H2O</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2378</td>
			<td>ReagentPlus, &#8805;99%</td>
		</tr>
	</tbody>
</table>

procedure for adaptive laboratory evolution of microorganisms using a chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

For høy succinate stresset tilpasning, villtype E. coli W3110-stamme ble dyrket i en kjemostat ved D = 0,1 hr -1 for 270 dager (figur 2). <img alt="Figur 2" src="/files/ftp_upload/54446/54446fig2.jpg" /> Figur 2: High-succinate stresset tilpasning av E. coli W3110 bruker kjemostatkultur. Tynne piler indikerer gangene hvor konsentrasjonen av stressor ble økt, og fet piler indikerer gangene h...

Watch this Scientific Journal Video about Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat at JoVE.com

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Her presenterer vi en protokoll for å skaffe adaptive laboratorium evolusjon av mikroorganismer under forhold som bruker kjemostatkultur. Dessuten er diskutert genomisk analyse av utviklet seg belastning.

Prosedyre for Adaptive Laboratory Utviklingen av mikroorganismer ved hjelp av en kjemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

The overall goal of this procedure is to enable a microorganism to evolve in laboratory conditions. This method can help answer key questions in the microbiology field, such as adrenal function relationships, stress responses, metabolic engineering, and of course, evolution. The main advantage of this technique is continuous selection of the most suaded descendant under specific laboratory conditions.Begin this procedure with the preparation of equipment, as well as initial medium, stress medium and high stress medium, as described in the text protocol. Inoculate a single colony or wild type E.coli in a 15-milliliter test tube containing four milliliters of initial medium. Incubate the test tube in a shaking incubator for 12 hours at 37 degrees celsius and 220 rpm.Incubate the chemostat jar providing for aeration and agitation at 37 degrees celsius for six hours. Following the incubation, aseptically connect the end of the silicon tubing from the pumps to the chemostat jar. Aseptically transfer one milliliter of pre-culture to the chemostat jar.Start the outlet pump and collect the culture in the exponential phase. Check the optical density at 600 nanometers of the culture from the outlet tubing. Then, start the inlet pump.Check the optical density of the culture at 600 nanometers from the outlet tubing every 24 hours. After operating the chemostat for 96 hours, which is a 9.6 full turnover, exchange the reservoir to the lowest concentration of high stress medium. When you exchange the reservoir to a higher stress medium, the cells may be shocked.If the cell density is too low, stop feeding the higher stress medium for some time to restore the cell density. If the optical density is lower than 0.2, stop the feeding inlet pump for six hours. Restart the inlet pump and check that the optical density is over 0.2.Gradually increase the concentration of the stressor by incrementally changing to a reservoir containing a higher stressor concentration. Take samples of the adapted culture whenever it reaches a stressor adaptation milestone and store for further genomic analysis. For sample storage, mix the 0.5 milliliter culture sample with 0.5 milliliters of a sterilized 80%glycerol solution and store it at 80 degrees celsius.Prepare 1.6%ager plate medium containing the same stressor at the same concentration as in the medium. Plate 0.1 milliliters of the outlet culture from the chemostat and incubate at 37 degrees celsius for 16 hours. Following incubation, pick single colonies from the plate using a sterile toothpick and inoculate them in 15 milliliter test tubes containing the same stressor and at the same medium concentration as in the chemostat.Incubate for six hours. Transfer one milliliter of the culture broth into a 250 milliliter erlenmeyer flask containing 50 milliliters of medium. Harvest 0.5 milliliters of the culture broth every hour and measure the optical density at 600 nanometers.Compare the growth rate of the adapted strain to that of the wild type strain given the stressor. The wild type E.coli strain was evolved in a high succinate stressed condition for 270 days, which corresponds to nearly 930 generations. Whenever higher succinate stress was added, the biomass concentration was immediately lowered and then restored.Shown here is a schematic diagram of the chemostat with a mutant that is tolerant against the high succinate stress. Most wild type cells are washed out under the stress conditions and the mutant grows more. Eventually, the population of the mutant descendant is increased.The second and third mutations are continuously selected for and eventually dominate the chemostat. This figure shows that the adapted strain grew without delay under high succinate stressed conditions, while the wild type strain did not. A similar strategy can be applied to adaptive laboratory evolution using a chemostat with a variation of stressors.After watching this video, you should have a good understanding of how to acquire and evolve the microorganism under a specified condition. Once mastered, this technique can be done in a couple of month. While attempting this procedure, it's important to remember not to wash out chemostat cells by adding too much stress at once and remember to check the optical density every day.Following this procedure are the methods like a genome analysis and transcriptome analysis can be performed in order to answer additional questions like what mutation contributes to the stress tolerant evolution?

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