Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Summary

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.

Abstract

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.

Introduction

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 selection^1-3. 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 dire....

Protocol

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 pumps in order to deliver fresh medium and collect the culture.
4. Obtain a reservoir jar (10-20 L) containing a medium outlet port and an air inle....

Results

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: High-succinate stress adaptation of E. coli W3110 usi.......

Discussion

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.......

Materials

List of materials used in this article

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%