Evaluating Mutation Frequency in Bacteria Using the Beta-Glucosidase Assay

Abstract

Source: Stefan, A., et al. The Multifaceted Benefits of Protein Co-expression in Escherichia coli. J. Vis. Exp. (96), (2015).

This video demonstrates the evaluation of mutation frequency in bacteria using a beta-glucosidase assay. Bacterial cultures expressing a proofreading-deficient DNA polymerase are collected over successive generations. Replication errors caused by reduced proofreading may activate a suppressed beta-glucosidase gene. After centrifugation and permeabilization, a substrate is added, and enzyme activity is measured to quantify mutation frequency across samples.

Protocol

1. Isolation of Escherichia coli or E. coli Co-transformants

1. Prepare electro-competent cells of the appropriate E. coli strain to be transformed. Transfer to 1 ml of Luria-Bertani (LB) medium (Tryptone, Yeast Extract, sodium chloride or NaCl at 10, 5, and 10 g/L, respectively) a single colony of the strain of choice, and incubate at 37 °C under shaking conditions of 180 rpm. Dilute the pre-culture 1:500 in 25 ml of fresh LB medium and incubate the culture at 37 °C.
2. At log-phase (0.6 O.D.), centrifuge the cell suspension at 5,000 x g for 20 min, and resuspe....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Sigma-Aldrich	A1296
Ampicillin	Sigma-Aldrich	A9518
Chloroform	Sigma-Aldrich	288306
EDTA	Sigma-Aldrich	EDS
Glycerol	Sigma-Aldrich	G5516
IPTG	Sigma-Aldrich	I5502
KCl	Sigma-Aldrich	P9541
L-Arabinose	Sigma-Aldrich	A3256
MgCl₂	Sigma-Aldrich	M2670
NaCl	Sigma-Aldrich	31434
PMSF	Sigma-Aldrich	P7626
PNP-gluc	Sigma-Aldrich	N7006
Tetracyclin	Sigma-Aldrich	87128
Trizma base	Sigma-Aldrich	T1503
Tryptone	Sigma-Aldrich	95039
Yeast extract	Fluka	70161
Cuvettes 0.1 cm	BioRad	1652089	For electroporation
Centrifuge 5415R	Eppendorf
Centrifuge Allegra 21R	Beckman
Chromatography apparatus GradiFrac	Pharmacia Biotech
Gene Pulser II electroporation	BioRad
Microplate Reader 550	Biorad
MiniProtean 3 cell	BioRad
Power Supply	BioRad