Focus: DNA Isolation

Isolation of DNA from Bacillus subtilis Using the Wizard^® Plus SV Miniprep DNA Purification System

This article describes a modified protocol that allows isolation of high-quality DNA from B. subtilis with improved yields using the Wizard^® Plus SV Miniprep DNA Purification System (Cat.# A1330).

By Benjamin Levin¹ and Joseph Sirianni²,  and Robert P. Doyle, Ph.D².
¹NSF/REU Summer Scholar 2006 (currently finishing degree at the University of Rochester), ²Department of Chemistry, Syracuse University

Published in July 2007

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(2) 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 DNA was combined with 50µl 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 innoculated 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 #TB225 (Promega Corporation, Madison, WI). One-half of the samples were treated with 100µl of lysozyme (10mg/ml) for 15 minutes at 30°C prior to adding the Cell Lysis Solution from the DNA purification system and proceeding with the protocol described in Technical Bulletin #TB225. Absorption of purified DNA samples was measured at A₂₆₀, and the DNA concentration for each sample was calculated (Table 1).

Results and Discussion

Bacillus subtilis was chosen as an expression host for the S. coelicolor gene because it is a Gram-positive, soil-dwelling bacterial species. Previously described methods for growth and transformation protocols of B. subtilis using minimal medium provided inadequate growth for this study. Culture of B. subtilis in nutrient rich Luria Broth or Nutrient Broth provided more robust growth. Additionally growing the transformed B. subtilis strains in conditions similar to those used for Streptomyces coelicolor (30°C, with shaking at 300rpm) further enhanced growth of the transformed B. subtilis.

The Wizard^® Plus SV Miniprep DNA Purification System has been used extensively with the Gram negative bacterium, E. coli. However, since B. subtilis is Gram positive, it has a high concentration of peptidoglycan in the cell wall.  We added a lysozyme step to assist in digesting the cell wall in addition to the cell lysis step described in the protocol (Technical Bulletin #TB225). Even without the additional lysozyme step the Wizard^® Plus SV Miniprep DNA Purification System was useful for isolating DNA from B. subtilis; however, with the addition of the lysozyme step, the average  plasmid concentration of the samples purified using the lysozyme step increased 1.5- to 3.0-fold. Furthermore, the  DNA was highly pure and was easily digested (Figure 2).

Figure 2. Plasmid isolated from B. subtilis using the Wizard^® Plus SV Miniprep DNA Purification System. DNA was isolated from 18- and 22-hour cultures of B. subtilis transformed with the plasmid constructs. DNA was digested with EcoRI for 1 hour at 37°C and visualized using ethidium bromide staining on an agarose gel.

References

1. Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. Third edition. Vol 1–3.
2. Anagnostopoulos, C. and Spizizen, J. (1961) J. Bacteriol. 81, 741-6.