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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timescales+of+genetic+and+epigenetic+inheritance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+differential+adaptation+to+anaerobic+stress+via+genomic+mutations+by+Escherichia+coli+strains+K-12+and+B+lacking+alcohol+dehydrogenase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+and+evolution.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+evolution+of+Corynebacterium+glutamicum+resistant+to+oxidative+stress+and+its+global+gene+expression+profiling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+oxidative+stress-tolerant+Corynebacterium+glutamicum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+steady+states+of+microbial+growth+on+mixtures+of+substitutable+substrates+in+a+chemostat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+continuous+adaptation+of+Escherichia+coli+to+high+succinate+stress+and+transcriptome+analysis+of+the+tolerant+strain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+dynamics+during+experimental+evolution.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sequence+Alignment/Map+format+and+SAMtools.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genome+Analysis+Toolkit:+a+MapReduce+framework+for+analyzing+next-generation+DNA+sequencing+data.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mutations+in+laboratory-evolved+microbes+from+next-generation+sequencing+data+using+breseq.">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 &#34;adaptive evolution&#34;. 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="Figure 1" src="/files/ftp_upload/54446/54446fig1.jpg" /> 
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

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="Figure 2" src="/files/ftp_upload/54446/54446fig2.jpg" /> 
Figure 2: High-succinate stress adaptation of E. coli W3110 using chemostat culture. Thin arrows indicate the times at which the concentration of stressor was increased, and bold arrows i...

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Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

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) ...

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14.2K Views.  Korea Research Institute of Bioscience and Biotechnology (KRIBB). 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.

Video: Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions.

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments.

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Bioengineering

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain&#160;at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

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

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Experimental Evolution Protocol for Thermophile Adaptation Using Low-Cost Thermomixers

Adaptation at the Extremes of Life: Experimental Evolution with the Extremophile Archaeon Sulfolobus acidocaldarius

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures is a key requirement, not only for understanding fundamental evolutionary processes but also for developing S. acidocaldarius as a chassis for bioengineering. One major obstacle to conducting experimental evolution with thermophiles is the expense of equipment maintenance and energy usage of traditional incubators for high-temperature growth. To address this challenge, a comprehensive experimental protocol for conducting experimental evolution in S. acidocaldarius is presented, utilizing low-cost and energy-efficient bench-top thermomixers. The protocol involves a batch culture technique with relatively small volumes (1.5 mL), enabling tracking of adaptation in multiple independent lineages. This method is easily scalable through the use of additional thermomixers. Such an approach increases the accessibility of S. acidocaldarius as a model system by reducing both initial investment and ongoing costs associated with experimental investigations. Moreover, the technique is transferable to other microbial systems for exploring adaptation to diverse environmental conditions.

Author Spotlight: Understanding Microbe Adaptation Using Innovative Techniques for Exploring Thermophilic Evolution

Here, we present an experimental evolution protocol for adaptation in thermophiles utilizing low-cost, energy-efficient bench-top thermomixers as incubators. The technique is demonstrated through the characterization of temperature adaptation in Sulfolobus acidocaldarius, an archaeon with an optimal growth temperature of 75 &#176;C.

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures ...

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Summary

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