Isolation of DNA from Bacillus subtilis Using the Wizard® Plus SV Miniprep DNA...

Introduction

 We used an E. coli-B. subtilis shuttle vector to express a gene from Streptomyces coelicolor  (Figure 1) in Bacillus subtilis, a similar bacterial species in that it is a Gram positive, soil-dwelling bacterium. This article highlights the method we used to isolate the plasmid DNA from the transformed B. subtilis cells.

Figure 1. Streptomyces coelicolor A3 producing the antibiotic actinorhodin.

The compound is responsible for its blue color. Figure used with kind permission John Innes Centre, Norwich UK.

Transformation of and Plasmid Purification from Bacillus subtilis

B. subtilis cells were prepared for both electroporation and chemical transformation with the E. coli-B. subtilis shuttle vector pHT08 containing the gene of interest. Chemical transformation of B. subtilis was performed using standard techniques ⁽¹⁾ . Electroporation was performed using an Eppendorf Electroporator 2510 with 2mm band gap cuvettes. One microliter of DNA was combined with 50µl of cells, and a pulse of 2300 volts applied. Cells recovered for three hours in SOC medium before plating on NB agar supplemented with the appropriate antibiotics.

To isolate DNA, single colonies of transformed B. subtilis  were inoculated into 5ml Nutrient Broth and incubated in a round-bottom culture tubes with shaking at 30°C for 18 or 22 hours. From each culture, cells were centrifuged as 4000rpm at 4°C for 10 minutes. Cell pellets were thoroughly resuspended in 250µl Cell Resuspension Solution (Promega Corporation, Madison, WI) and transferred to a sterile 1.5ml microcentrifuge tube. DNA was isolated from one-half of the samples using the standard Wizard® Plus SV Miniprep DNA Purification System protocol described in Technical Bulletin

Table 1. DNA Minipreps from B. subtillis transformed with the plasmid construct.

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