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Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Published: September 20th, 2016



1Super-Bacteria Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 2Microbiomics and Immunity Research Center, Korea Research Institute of Bioscience and Bioengineering (KRIBB), 3Department of Biotechnology, The Catholic University of Korea
* These authors contributed equally

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) 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.

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 ....

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1. Equipment Preparation

  1. 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.
  2. Obtain a device suitable for the chemostat jar that provides for agitation and temperature control (or use a rotary shaking incubator).
  3. Obtain two peristaltic pump.......

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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).

Figure 2
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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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.......

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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).


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Name Company Catalog Number Comments
Mini-chemostat fermentor Biotron Inc. - manufactured by special order
silicon tubing Cole-Parmer Masterflex L/S 13 tubing size can be varied depending on the dilution rate and the size of fermentor jar.
reservoir jar Bellco Media storage bottle  20 L
chemicals Sigma-Aldrich - reagent grade
glucose Sigma-Aldrich G5767 ACS reagent
NH4Cl Sigma-Aldrich A9434 for molecular biology, suitable for cell culture, ≥99.5%
NaCl Sigma-Aldrich 746398 ACS reagent, ≥99%
Na2HPO4·2H2O Sigma-Aldrich 4272 98.5-101%
KH2PO4  Sigma-Aldrich 795488 ACS reagent, ≥99%
MgSO4·7H2O Sigma-Aldrich 230391 ACS reagent, ≥98%
CaCl2 Sigma-Aldrich 793639 ACS reagent, ≥96%
thiamine·HCl  Sigma-Aldrich T4625 reagent grade, ≥99%
Na2·succinate·6H2O Sigma-Aldrich S2378 ReagentPlus, ≥99%

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