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06:03
Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat
Published on: September 20, 2016
Identification of mutations in evolved bacterial genomes
Liam Royce1, Erin Boggess, Tao Jin
1Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|February 19, 2013
Summary
Directed laboratory evolution aids in developing bacteria with enhanced traits, like toxin tolerance. This protocol details methods for identifying genetic mutations, including SNPs and indels, through DNA sequencing and verification.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Directed laboratory evolution is a powerful method for enhancing bacterial phenotypes.
- It complements rational engineering for complex traits, such as improved biocatalyst tolerance to toxic substances.
- Identifying the genetic basis of evolved traits requires robust reverse engineering strategies.
Purpose of the Study:
- To provide a comprehensive protocol for the identification of mutations in evolved bacterial strains.
- To outline methods for genomic DNA extraction, sequencing, and analysis.
- To detail techniques for verifying identified mutations.
Main Methods:
- Genomic DNA extraction and sequencing.
- Bioinformatic analysis of DNA sequence data to identify single nucleotide polymorphisms (SNPs), insertions/deletions (indels), duplications, and rearrangements.
- Mutation validation using Sanger resequencing.
Main Results:
- The protocol enables the systematic identification of various mutation types in evolved bacterial genomes.
- Successful application of the protocol allows for the characterization of genetic changes underlying desired phenotypes.
- Sanger resequencing confirms the accuracy of mutations detected through sequence data analysis.
Conclusions:
- This protocol offers a standardized approach for reverse engineering evolved bacterial strains.
- Understanding the genetic underpinnings of evolved traits is crucial for further strain improvement and application.
- The described methods facilitate the detailed characterization of mutations acquired during laboratory evolution.
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