Name: Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat (Video) | JoVE Journal
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 6 min 3 s
Description: Learn adaptive laboratory evolution of microorganisms using chemostat culture with E. coli. This protocol enables continuous selection of evolved strains for studying stress responses and metabolic engineering applications.

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5 minutes

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. Equipment Preparation
<ol>
	<li>Obtain a chemostat jar (150-250 ml) or an Erlenmeyer flask (250 ml) containing an inlet port and an outlet port. Connect the ports with silicon tubing allowing for flow rates of 10-100 ml/hr. Optionally, use an air vent, an air outlet port, and temperature-controlled water inlet and outlet ports.</li>
	<li>Obtain a device suitable for the chemostat jar that provides for agitation and temperature control (or use a rotary shaking incubator).</li>
	<li>Obtain two peristaltic pumps in order to deliver fresh medium and collect the culture.</li>
	<li>Obtain a reservoir jar (10-20 L) containing a medium outlet port and an air inlet port.</li>
	<li>Obtain silicon tubing suitable for the dilution rate (i.e., ID 0.8 mm, flow range 0.06-36 ml/min; L/S 13 tubing).</li>
</ol>
2. Medium Preparation and Sterilization
<ol>
	<li>Initial Medium
	<ol>
		<li>Dissolve 0.3 g glucose, 0.08 g NH4Cl, 0.05 g NaCl, 0.75 g Na2HPO4&#183;2H2O, and 0.3 g KH2PO4 in 90 ml distilled water (D.W.) in a chemostat jar.</li>
		<li>Seal the chemostat jar along with the tubing using clamps. Do not seal the air vent.</li>
		<li>Sterilize the chemostat jar in an autoclave at 121 &#176;C for 15 min. After sterilization, store the chemostat jar at room temperature.</li>
		<li>Dissolve 0.02 g MgSO4&#183;7H2O, 0.01 g CaCl2, and 0.1 mg thiamine in 10 ml D.W. (solution A).</li>
		<li>Filter solution A using a syringe and a pre-sterilized syringe filter (a 0.45 &#181;m pore filter).</li>
		<li>Add the solution A filtrates to the chemostat jar.</li>
	</ol>
	</li>
	<li>Stress Medium
	<ol>
		<li>Dissolve 30 g glucose, 8 g NH4Cl, 5 g NaCl, 75 g Na2HPO4&#183;2H2O, 30 g KH2PO4, and 300 g disodium succinate hexahydrate (Na2&#183;succinate&#183;6H2O; the stressor used in this experiment) in 9.9 L D.W. in a reservoir jar.</li>
		<li>Seal the reservoir jar along with the tubing using clamps. Do not seal the air vent.</li>
		<li>Sterilize the reservoir jar in an autoclave at 121 &#176;C for 15 min. After sterilization, store the jar at room temperature.</li>
		<li>Dissolve 2 g MgSO4&#183;7H2O, 1 g CaCl2, and 10 mg thiamine in 100 ml D.W. (solution A).</li>
		<li>Filter solution A with a syringe and a pre-sterilized syringe filter (a 0.45 &#181;m pore filter).</li>
		<li>Add the solution A filtrates to the reservoir jar.</li>
		<li>Aseptically connect the sterilized silicon tubing to the reservoir jar and attach the peristaltic pumps.</li>
	</ol>
	</li>
	<li>High-stress Medium
	<ol>
		<li>Prepare the medium as in section 2.2, but with a greater concentration of stressor (i.e., 3-5 g/L higher in the succinate adaptation). 
		Note: This protocol is for adaptation to a stress that can be delivered via the medium. In the case of physical stressors such as temperature, agitation, or illumination, the cultivation should be designed accordingly.</li>
	</ol>
	</li>
</ol>
3. Initial Cultivation
<ol>
	<li>Inoculate a single colony of wild-type E. coli in a 15 ml test tube containing 4 ml of initial medium.</li>
	<li>Incubate the test tube in a shaking incubator for 12 hr at 37 &#176;C and 220 rpm.</li>
	<li>Aseptically transfer 1 ml of preculture to the chemostat jar.</li>
	<li>Incubate the chemostat jar, providing for aeration (air 50 ml/min) and agitation (200 rpm), at 37 &#176;C for 6 hr.</li>
</ol>
4. Stress Adaptation
<ol>
	<li>Aseptically connect the end of the silicon tubing from the pumps to the chemostat jar.</li>
	<li>Start the outlet pump (10 ml/hr or higher) and collect culture. 
	Note: The culture should be in the exponential phase, typically 4-8 hr after initial cultivation.</li>
	<li>Check the optical density (600 nm) of the culture from the outlet tubing.</li>
	<li>Start the inlet pump (10 ml/hr, corresponding to a rate of dilution of 0.1 hr-1).</li>
	<li>Check the optical density of the culture at 600 nm from the outlet tubing every 24 hr.</li>
	<li>Operate the chemostat for 96 hr (9.6-fold turnover) or more. If the optical density is stable, exchange the reservoir containing the high-stress medium. If the optical density is lower than 0.2, stop the feeding inlet pump for 6 hr. Restart the inlet pump and check that the optical density is over 0.2.</li>
	<li>Gradually increase the concentration of the stressor by changing to a reservoir containing a higher stressor concentration.</li>
	<li>Take samples of the adapted culture whenever it reaches a milestone (e.g., a strain adapted to 100 g/L succinate stress), and store for further genomic analysis.</li>
	<li>For sample storage, mix the culture sample (0.5 ml) with a sterilized 80% glycerol solution (0.5 ml) and store it at -80 &#176;C. 
	Note: If the microorganism acquires an ability to degrade the stressor during the ALE process, the stressor concentration in the fermentation jar is not the same as that in the fresh reservoir.</li>
</ol>
5. Single-colony Isolation of the Stress-adapted Strain
<ol>
	<li>Prepare agar plate medium (1.6% agar) containing the same stressor and at the same concentration of medium.</li>
	<li>Plate the outlet culture (0.1 ml) from the chemostat, and incubate at 37 &#176;C for 16 hr.</li>
	<li>Pick single colonies from the plate using a sterile toothpick and inoculate them in 15 ml test tubes containing the same stressor and at the same medium concentration as in the chemostat, and incubate for 6 hr.</li>
	<li>Transfer 1 ml of culture broth into a 250 ml Erlenmeyer flask containing 50 ml of medium. Harvest 0.5 ml of the culture broth every 1 hr, and measure the O.D. at 600 nm. Compare the growth rate of the adapted strain to that of the wild-type strain given the stressor.</li>
</ol>

<ol><li>Rando, O. J., Verstrepen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Timescales+of+genetic+and+epigenetic+inheritance%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Timescales of genetic and epigenetic inheritance.</a> Cell. 128, 655-668 (2007).</li>
<li>Kim, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short-term+differential+adaptation+to+anaerobic+stress+via+genomic+mutations+by+Escherichia+coli+strains+K-12+and+B+lacking+alcohol+dehydrogenase%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Short-term differential adaptation to anaerobic stress via genomic mutations by Escherichia coli strains K-12 and B lacking alcohol dehydrogenase.</a> Front Microbiol. 5, 476(2014).</li>
<li>Mendizabal, I., Keller, T. E., Zeng, J., Yi, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetics+and+evolution%5BTitle%5D)+AND+%22Integr+Comp+Biol%22%5BJournal%5D)">Epigenetics and evolution.</a> Integr Comp Biol. 54, 31-42 (2014).</li>
<li>Lee, J. Y., Seo, J., Kim, E. S., Lee, H. S., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adaptive+evolution+of+Corynebacterium+glutamicum+resistant+to+oxidative+stress+and+its+global+gene+expression+profiling%5BTitle%5D)+AND+%22Biotechnol+Lett%22%5BJournal%5D)">Adaptive evolution of Corynebacterium glutamicum resistant to oxidative stress and its global gene expression profiling.</a> Biotechnol Lett. 35, 709-717 (2013).</li>
<li>Lee, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artificial+oxidative+stress-tolerant+Corynebacterium+glutamicum%5BTitle%5D)+AND+%22AMB+Express%22%5BJournal%5D)">Artificial oxidative stress-tolerant Corynebacterium glutamicum.</a> AMB Express. 4, 15(2014).</li>
<li>Narang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+steady+states+of+microbial+growth+on+mixtures+of+substitutable+substrates+in+a+chemostat%5BTitle%5D)+AND+%22J+Theor+Biol%22%5BJournal%5D)">The steady states of microbial growth on mixtures of substitutable substrates in a chemostat.</a> J Theor Biol. 190, 241-261 (1998).</li>
<li>Kwon, Y. D., Kim, S., Lee, S. Y., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+continuous+adaptation+of+Escherichia+coli+to+high+succinate+stress+and+transcriptome+analysis+of+the+tolerant+strain%5BTitle%5D)+AND+%22J+Biosci+Bioeng%22%5BJournal%5D)">Long-term continuous adaptation of Escherichia coli to high succinate stress and transcriptome analysis of the tolerant strain.</a> J Biosci Bioeng. 111, 26-30 (2011).</li>
<li>Barrick, J. E., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome+dynamics+during+experimental+evolution%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Genome dynamics during experimental evolution.</a> Nat Rev Genet. 14, 827-839 (2013).</li>
<li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Sequence+Alignment%2FMap+format+and+SAMtools%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li>
<li>McKenna, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Genome+Analysis+Toolkit%3A+a+MapReduce+framework+for+analyzing+next-generation+DNA+sequencing+data%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.</a> Genome Res. 20, 1297-1303 (2010).</li>
<li>Deatherage, D. E., Barrick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+mutations+in+laboratory-evolved+microbes+from+next-generation+sequencing+data+using+breseq%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq.</a> Methods Mol Biol. 1151, 165-188 (2014).</li>
</ol>

The authors have nothing to disclose.

Microorganisms are capable of adapting to almost all environments because of their rapid growth rate and genetic diversity. Adaptive laboratory evolution enables microorganisms to evolve under designed conditions, which provides a way of selecting individual organisms harboring spontaneous mutations that are beneficial under the given conditions.
The chemostat technique is more robust for achieving artificially driven evolution than transfer techniques for the following reasons: (a) a steady e...

Microorganisms can survive and adapt to diverse environments. Under severe stress, adaptation can occur via acquisition of beneficial phenotypes by random genomic mutations and subsequent positive selection1-3. Therefore, microbial cells can adapt by changing metabolic or regulatory networks for optimal growth, which is termed "adaptive evolution". Recent important microbial tendencies, such as outbreaks of superbugs and the occurrence of robust microbial strains, are very closely related to adaptive evolution under stressful conditions. Under defined laboratory conditions, we are able to study the mechanisms of molecular evolution and even control the direction of microbial evolution for various applications. Unlike multicellular organisms, single-celled organisms are well suited to adaptive laboratory evolution (ALE) for the following reasons: they regenerate quickly, they maintain large populations, and it is easy to create and maintain homogeneous environments. Combined with recent advances in DNA sequencing techniques and high-throughput technologies, ALE allows for the direct observation of genomic changes that lead to systemic regulatory changes. Mutational dynamics and a diversity of the population are also observable. Genetic engineering strategies can be determined from the analysis of ALE strains4,5.
Chemostat culture is a method used to obtain steady-state cells and increase productivity in fermentation processes6. Fresh medium is added and culture broth is harvested during the process (the latter includes medium and biomass). Long-term chemostat culture, however, changes the steady-state productivity of the culture and brings about the accumulation of spontaneous mutations and selection during culture (Figure 1a). Under various selection pressures (stressors), the accumulation of mutations is enhanced. A gradual increase of stress in a long-term chemostat provides for a continuous selection of mutations that work against the given stressors, such as temperature, pH, osmotic pressure, nutrient starvation, oxidation, toxic end products, etc. Colony transfer from a solid medium and serial transfer from a liquid medium (repeated batch culture) also allow researchers to obtain evolved microorganisms (Figure 1b and 1c). Although chemostat culture requires complicated methods, the pool of diversity (number of replications and population size) is higher than that obtained by colony transfer and serial transfer techniques. The stable stress exposure to individual cells and decreased variation in the cellular state during chemostat culture (steady state) are other benefits of ALE compared to batch culture-based techniques. Stress-induced ALE of Escherichia coli subjected to high succinate conditions is introduced in this article.
<img alt="Chemostat culture diagram with cell growth graphs; serial and colony transfer methods." src="/files/ftp_upload/54446/54446fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1: Methods of adaptive laboratory evolution. (A) Chemostat; (B) serial transfer; (C) colony transfer. The top figures illustrate the concept of the methods for ALE, and the bottom figures illustrate the number of cells that grew during ALE. <a href="https://www.jove.com/files/ftp_upload/54446/54446fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Mini-chemostat fermentor</td><td>Biotron Inc.</td><td>-</td><td>manufactured by special order</td></tr><tr><td>silicon tubing</td><td>Cole-Parmer</td><td>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><td>reservoir jar</td><td>Bellco</td><td>Media storage bottle</td><td>20 L</td></tr><tr><td>chemicals</td><td>Sigma-Aldrich</td><td>-</td><td>reagent grade</td></tr><tr><td>glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>ACS reagent</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma-Aldrich</td><td>A9434</td><td>for molecular biology, suitable for cell culture, &amp;ge;99.5%</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>746398</td><td>ACS reagent, &amp;ge;99%</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>4272</td><td>98.5-101%</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>795488</td><td>ACS reagent, &amp;ge;99%</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>230391</td><td>ACS reagent, &amp;ge;98%</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>793639</td><td>ACS reagent, &amp;ge;96%</td></tr><tr><td>thiamine&amp;middot;HCl</td><td>Sigma-Aldrich</td><td>T4625</td><td>reagent grade, &amp;ge;99%</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;&amp;middot;succinate&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>S2378</td><td>ReagentPlus, &amp;ge;99%</td></tr></tbody></table>

procedure for adaptive laboratory evolution of microorganisms using a chemostat

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 high-succinate stress adaptation, the wild-type E. coli W3110 strain was cultured in a chemostat at D = 0.1 hr-1 for 270 days (Figure 2).
<img alt="Bacterial growth curve, OD600 measurement, time series data, chart with peaks and arrows, 0-250 days." src="/files/ftp_upload/54446/54446fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2: High-succinate stress adaptation of E. coli...

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

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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Learn adaptive laboratory evolution of microorganisms using chemostat culture with E. coli. This protocol enables continuous selection of evolved strains for studying stress responses and metabolic engineering applications.

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

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