Development of an efficient electroporation method for iturin A-producing Bacillus subtilis ZK

Abstract

In order to efficiently introduce DNA into B. subtilis ZK, which produces iturin A at a high level, we optimized seven electroporation conditions and explored an efficient electroporation method. Using the optimal conditions, the electroporation efficiency was improved to 1.03 × 10(70 transformants/μg of DNA, an approximately 10,000-fold increase in electroporation efficiency. This efficiency is the highest electroporation efficiency for B. subtilis and enables the construction of a directed evolution library or the knockout of a gene in B. subtilis ZK for molecular genetics studies. In the optimization process, the combined effects of three types of wall-weakening agents were evaluated using a response surface methodology (RSM) design, which led to a two orders of magnitude increase in electroporation efficiency. To the best of our limited knowledge, this study provides the first demonstration of using an RSM design for optimization of the electroporation conditions for B. subtilis. To validate the electroporation efficiency, a case study was performed and a gene (rapC) was inactivated in B. subtilis ZK using a suicide plasmid pMUTIN4. Moreover, we found that the rapC mutants exhibited a marked decrease in iturin A production, suggesting that the rapC gene was closely related to the iturin A production.

Figure 1

Effects of growth phase of B. subtilis ZK on the electroporation efficiency. B. subtilis ZK was grown in LBSP medium and the electro-competent cells were prepared at different OD₆₀₀ values (0.3–1.3). One hundred nanograms of the pHT43 plasmid were used for each electroporation experiment. The field strength was 20 kV·cm⁻¹ and the electroporation buffer was TSM. The experiments were repeated three times. The error bars indicate the standard deviations from the average values.

Figure 2

Effects of the field strength on the electroporation efficiency of B. subtilis ZK. B. subtilis ZK was grown in LBSP medium and the electro-competent cells were prepared when the OD₆₀₀ value reached 0.85. The electroporation experiments were performed under a gradient of the field strength (8–26 kV·cm⁻¹). One hundred nanograms of pHT43 plasmid were used for each electroporation experiment, and the electroporation buffer was TSM. The data shown are the averages of three independent experiments, and the error bars indicate the standard deviations from the average values.

Figure 3

Effects of wall-weakening agents on the electroporation efficiency of B. subtilis ZK. Various weakening agents at different concentration gradients (0.25%–0.85% glycine (A); 0.63%–1.56% dl-threonine (B); 15–110 mg/mL Tween 80 (C) and 1–20 μg/mL ampicillin (D)) were separately added to the LBSP medium when the OD₆₀₀ value reached 0.5. After shaking for an additional 1 h, the electro-competent cells were prepared. One hundred nanograms of pHT43 were used for each electroporation experiment. The field strength was 20 kV·cm⁻¹ and the electroporation buffer was TSM. The values shown are the averages from three independent experiments and the error bars indicate the standard deviations from the average values.

Figure 4

Plots of the electroporation efficiency as a function of the DNA quantity. When the OD₆₀₀ value reached 0.5, the weakening agents (0.64% Glycine, 1.02% dl-threonine, and 0.05% Tween 80) were added to the LBSP medium and the electro-competent competent cells were prepared. B. subtilis ZK cells were transformed with various quantities of the plasmid DNA (5–500 ng). The field strength was 20 kV·cm⁻¹ and the electroporation buffer was TSMMKK. After electroporation, the cells were incubated in a water bath at 30 or 46 °C. The electroporation experiments were repeated three times, and the data shown are the means of triplicate experiments. The error bars indicate the standard deviations from the average values.

Figure 5

Inactivation of rapC gene by gene insertional mutation. (A) Construction of insertional mutation of the rapC gene; (B) Lane 1: middle encoding region of rapC (595 bp) with the P1 and P2 as the primers (C) Verification of the single crossover mutants by PCR. Lane 1–4: fragments (1069 bp) with the P3 and P4 as the primers; M: marker.

Figure 6

HPLC analysis of the fermentation of wild-type strain and mutant. (A) Absorption peak of the iturin A of the wild-type B. subtilis ZK; (B) Absorption peaks of an iturin A standard sample; (C) Absorption peak of the iturin A of the mutant; and (D) The yields of iturin A of wild-type strain, mutant, and complementary mutant. The complementary mutant was grown in LB medium in presence of IPTG (0.3 mM).

Figure 6

HPLC analysis of the fermentation of wild-type strain and mutant. (A) Absorption peak of the iturin A of the wild-type B. subtilis ZK; (B) Absorption peaks of an iturin A standard sample; (C) Absorption peak of the iturin A of the mutant; and (D) The yields of iturin A of wild-type strain, mutant, and complementary mutant. The complementary mutant was grown in LB medium in presence of IPTG (0.3 mM).