Fluorescently Tagged Repressor Protein-Mediated Replication Blockage in Escherichia coli

Abstract

Source: Mettrick, K. A., Lawrence, et al., Inducing a Site-Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System. J. Vis. Exp. (2016)

This video demonstrates site-specific replication fork blockage in Escherichia coli using arabinose-induced expression of yellow fluorescent protein-tagged TetR that binds chromosomal tetO arrays.

Protocol

1. Blocking Replication with Fluorescence Repressor Operator System (FROS)

1. Inducing the Replication Blockage
   1. Dilute a fresh overnight culture of an E. coli strain carrying a tetO array and pKM118 to OD₆₀₀nm = 0.01 in a dilute complex medium (0.1% tryptone, 0.05% yeast extract, 0.1% NaCl, 0.17 M KH₂PO₄, 0.72 M K₂HPO₄) with antibiotics as required for selection.
      NOTE: Do not add tetracycline for selection as it is not compatible with this system. Make the volume of the culture equivalent to 10 ml per sample required. For example, the culture volume for the experimental design in Figure 1 is 60 ml.
   2. Grow at 30 °C with shaking until OD₆₀₀ nm = 0.05-0.1. Remove a 10 ml sample to serve as the uninduced control and add 0.1% arabinose to the remaining culture to induce the production of TetR-YFP from pKM1. Continue to grow both the uninduced and induced cultures. After 1 hr, check for the presence of a single focal point within each cell of the induced culture using fluorescence microscopy.
   3. If the induced cells are confirmed blocked (more than 70% of cells have a single focus), record the OD₆₀₀nm and take a 7.5 ml sample for analysis by 2-D gels. Take an equivalent sample from the uninduced control culture.

Results

Figure 1: Overview of the FROS Replication Block and Release Experimental Procedure. E. coli stra...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptone	Sigma-Aldrich	16922	Growth media component
Sodium Chloride	VWR	27810.364	Growth media component
Yeast extract	Sigma-Aldrich	92144	Growth media component
Potassium phosphate monobasic	Sigma-Aldrich	P9791	Growth media component; Potassium buffer component
Potassium phosphate dibasic	Sigma-Aldrich	P3786	Growth media component; Potassium buffer component
L-Arabinose	Sigma-Aldrich	A3256	For induction of TetR-YFP production
Axioskop 2 Fluorescence microscope	Zeiss	452310	Visualization of cells
eYFP filter set	Chroma Technology	41028	Visualization of YFP
CCD camera	Hamamatsu	Orca-AG	Visualization of cells
MetaMorph Software (Molecular Devices)	SDR Scientific	31282	Version 7.8.0.0 used in the preparation of this manuscript