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Related Experiment Video

Updated: Oct 11, 2025

Measuring Microbial Mutation Rates with the Fluctuation Assay

Measuring Microbial Mutation Rates with the Fluctuation Assay

Published on: November 28, 2019

Measuring Mutator Enzyme Activity Using an E. coli-Based Colony Formation Assay.

Mei-Chen Liu¹, Sebastian D Fugmann^2,3,4,5

¹Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.

Methods in Molecular Biology (Clifton, N.J.)

|December 6, 2021

Summary

This summary is machine-generated.

This study presents new E. coli-based assays to measure mutator enzyme activity, crucial for understanding DNA/RNA sequence alteration and its link to cancer. These versatile methods aid in defining enzyme functions outside their natural biological context.

Keywords:

AID APOBEC Cytidine deaminase Deaminase Mutator enzyme Somatic hypermutation

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Last Updated: Oct 11, 2025

Measuring Microbial Mutation Rates with the Fluctuation Assay

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Published on: November 28, 2019

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

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Published on: March 16, 2011

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Published on: February 24, 2014

Area of Science:

Molecular Biology
Enzymology
Genetics

Background:

Mutator enzymes modify DNA/RNA sequences, playing roles in immunity and host optimization.
Dysregulation of these enzymes is implicated in various cancers.
Accurate measurement of mutator enzyme activity requires context-independent assays due to low in vivo mutation rates.

Purpose of the Study:

To provide detailed protocols for two E. coli-based assays to detect cytidine deaminase activity.
To enable the study of mutator enzymes independent of their biological context.
To offer a versatile platform for analyzing enzyme-driven mutations.

Main Methods:

Utilized E. coli as a host system for ectopic expression of mutator enzymes.
Employed two distinct reporter genes: an extrachromosomal kanamycin-resistance gene and the endogenous chromosomal rpoB gene.
Quantified mutation generation using a colony formation assay.

Main Results:

Successfully detected and quantified the activity of ectopically expressed cytidine deaminases.
Demonstrated the utility of both reporter gene systems for measuring enzyme-induced mutations.
Established robust and versatile assays for mutator enzyme characterization.

Conclusions:

The developed E. coli-based assays provide a robust and versatile method for defining mutator enzyme activities.
These assays are valuable for studying enzymes like cytidine deaminases and can be adapted for other mutator enzymes.
This work facilitates research into the role of mutator enzymes in biological processes and disease.