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. Klargøring af udstyr <ol><li> Opnå en kemostat krukke (150-250 ml) eller en Erlenmeyerkolbe (250 ml) indeholdende en indløbsport og en udløbsport. Tilslut havnene med silicium slange giver mulighed for strømningshastigheder på 10-100 ml / time. Eventuelt bruge en udlufter, et luftudtag port, og temperaturregulerede vand indsugnings- og udstødningsporte. </li><li> Opnå en indretning egnet til chemostaten krukke, der sørger for omrøring og temperaturkontrol (eller brug en roterende rysteinkubator). <...

<ol>
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Mikroorganismer er i stand til at tilpasse sig næsten alle miljøer på grund af deres hurtige vækst og genetisk diversitet. Adaptive laboratorium evolution giver mikroorganismer til at udvikle sig under designede betingelser, som giver en måde at vælge individuelle organismer huser spontane mutationer, der er gavnlige under de givne betingelser. Chemostaten teknik er mere robust for at opnå kunstigt drevet evolution end overførselsteknikken af ​​følgende grunde: (a) en stabil mil...

Mikroorganismer kan overleve og tilpasse sig forskellige miljøer. Under alvorlig stress, kan tilpasningen ske via opkøb af gavnlige fænotyper ved tilfældige genomiske mutationer og efterfølgende positiv selektion 1-3. Derfor kan mikrobielle celler tilpasse ved at ændre metaboliske eller regulerende net for optimal vækst, som kaldes &quot;adaptiv evolution&quot;. Nylige vigtige mikrobielle tendenser, såsom udbrud af superbugs og forekomsten af ​​robuste mikrobielle stammer, er meget nært beslægtet med adaptive evolution under stressende forhold. Under definerede laboratorieforhold, er vi i stand til at studere mekanismerne i molekylær evolution og endda styre retningen af ​​mikrobiel evolution til forskellige applikationer. I modsætning til flercellede organismer, encellede organismer er velegnede til adaptiv laboratorium evolution (ALE) af følgende grunde: De regenerere hurtigt, de opretholder store befolkningsgrupper, og det er let at oprette og vedligeholde homogeneous miljøer. Kombineret med de seneste fremskridt inden for DNA-sekventering teknikker og avanceret teknologi, ALE giver mulighed for direkte observation af genomiske ændringer, der fører til systemiske lovgivningsmæssige ændringer. Mutations dynamik og en mangfoldighed af befolkningen er også observeres. Genetisk engineering strategier kan bestemmes ud fra en analyse af ALE stammer 4,5. Kemostatkultur er en metode, der anvendes til at opnå steady-state-celler og øge produktiviteten i fermenteringsprocesser 6. Frisk medium tilsættes, og dyrkningsbouillonen høstes under processen (sidstnævnte omfatter medium og biomasse). Langsigtet kemostatkultur imidlertid ændrer steady-state produktivitet af kulturen og bevirker en akkumulering af spontane mutationer og selektion under dyrkning (figur 1a). Under forskellige udvalg tryk (stressfaktorer), er ophobning af mutationer forbedret. En gradvis forøgelse af stress i en langsigtet<html> kemostat giver en kontinuerlig udvalg af mutationer, der arbejder imod de givne stressfaktorer, såsom temperatur, pH, osmotisk tryk, næringsstof sult, oxidation, giftige slutprodukter, osv Colony overførsel fra et fast medium og seriel overførsel fra et flydende medium (gentaget batch-kultur) tillader også forskere at opnå udviklet mikroorganismer (figur 1b og 1c). Selvom kemostatkultur kræver komplicerede metoder, puljen af ​​mangfoldighed (antal gentagelser og befolkningens størrelse) er højere end den, der opnås ved hjælp af koloni overførsel og serielle forflytningsteknikker. Den stabile stress udsættelse for individuelle celler og formindsket variation i den cellulære tilstand under kemostatkultur (steady state) er andre fordele ved ALE sammenlignet med batch kultur-baserede teknikker. Stress-induceret ALE af Escherichia coli udsat for høje succinat- betingelser er indført i denne artikel. Iles / ftp_upload / 54.446 / 54446fig1.jpg &quot;/&gt; Figur 1: Metoder til adaptiv laboratorium evolution (A) kemostat;. (B) seriel overførsel (C) koloni overførsel. De øverste figurer illustrerer begrebet metoderne til ALE, og de ​​nederste figurer illustrerer antallet af celler, der voksede i løbet ALE. <a href="https://www.jove.com/files/ftp_upload/54446/54446fig1large.jpg" target="_blank">Klik her for at se en større version af dette tal.</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 høj-succinat stress tilpasning, vildtype E. coli W3110 stamme blev dyrket i en chemostat ved D = 0,1 h-1 i 270 dage (figur 2). <img alt="Figur 2" src="/files/ftp_upload/54446/54446fig2.jpg" /> Figur 2: Høj-succinat stress tilpasning af E. coli W3110 hjælp kemostatkultur. Tynde pile angiver de tidspunkter, hvor koncentrationen af stressor blev forøget, og dristige pile angiver de tidsp...

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 præsenterer vi en protokol til at opnå adaptiv laboratorium udvikling af mikroorganismer under forhold ved hjælp kemostatkultur. Desuden er genomisk analyse af den udviklede stamme diskuteret.

Procedure for Adaptive Laboratory Evolution af mikroorganismer Ved hjælp af en kemostat

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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