Construction of a modular plasmid family for chromosomal integration in Bacillus subtilis

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Abstract

The investigation of molecular processes involves the generation of knockout strains, the determination of promoter strength and protein overexpression. Here, we report the construction of the multifunctional pMG expression vector family for integration into the Bacillus subtilis chromosome that allows gene expression under single copy conditions. The pMG family enables a rapid exchange of all features for integration, selection and gene expression with or without N-terminal strep-tags. This modular architecture increases the applicabilities for these plasmids tremendously, permitting the construction of pMG derivatives for chromosomal integration at versatile loci and in different Bacillus species under control of natural or heterologous constitutive or inducible promoters. Additionally, the possible replacement of the antibiotic resistance cassettes helps circumvent problems that arise when the use of more than three antibiotics is required. Furthermore, the high copy number and structural stability of the pUC19-based pMG vectors in Escherichia coli facilitates template production for target host transformation.

Highlights

► A novel vector family for chromosomal integration in B. subtilis was constructed. ► Unique restriction sites allow simple exchange of all modules. ► Untagged or strep-tagged proteins can be expressed under single-copy conditions. ► Constitutive or inducible promoters and a promoterless lacZ gene are included. ► pMG plasmids show in contrast to pBR322-based vectors no structural instability.

Introduction

The investigation of protein–protein interactions helps to understand how regulatory networks operate. For the elucidation of the mechanism of this interaction and the determination of the amino acid contact surface, the following problems have to be solved: i) various mutants of both protein-coding genes have to be constructed, ii) two mutants have to be expressed simultaneously and independently, iii) the original host has to be used for the expression and iv) stable chromosomal deletions of the wild-type genes coding for the interacting partners are required.

During our study on the interaction of glycerinaldehyde-3-phosphate dehydrogenase A (GapA) and SR1P, a 39 amino acid (aa) peptide encoded by the dual-function regulatory RNA SR1 from Bacillus subtilis (Gimpel et al., 2010), we came across the above-mentioned problems. In particular, the lack of suitable expression systems allowing the straightforward expression of gapA mutants in a ΔgapA background prompted us to construct a novel vector family. To facilitate handling, the system should have several properties such as i) cassettes for chromosomal integration, ii) a set of different antibiotic resistance genes, iii) a strep-tag sequence flanked by unique restriction sites allowing the synthesis of tagged and untagged proteins, iv) native and heterologous (constitutive or inducible) promoters, v) a high copy number in Escherichia coli and vi) a modular architecture, i.e. unique restriction sites for replacement of all cassettes.

Here we present the construction of the pMG vector family — a series of modular E. coliBacillus shuttle vectors applicable for gene expression and knockout in B. subtilis.

Section snippets

Enzymes and chemicals

Chemicals used were of the highest purity available. All chemicals were purchased from Sigma-Aldrich™. Taq-polymerases were purchased from Roche (Germany) and Solis Biodyne (Estonia). Restriction enzymes were purchased from Jena Bioscience (Germany), New England Biolabs (Germany) and Fermentas (Germany).

Strains, media and growth conditions

E. coli strain TG1 was used for cloning. B. subtilis strain MG1P was used for gapA expression. TY medium (Licht et al., 2005) served as complex medium and Spizizen medium (Anagnostopoulos and

Cassette strategy for vector construction

The ability to switch or add plasmid features facilitates the adaption of existing vectors to new individual problems. Thus we designed a multifunctional expression vector family suitable for the solution of various problems of gene expression, as gene knockout, promoter strength determination or protein expression. All plasmid family members contain amyE front and back regions for integration into the B. subtilis chromosome at the amyE locus via double crossing‐over. This approach allows gene

Acknowledgements

We thank S. Aymerich for the B. subtilis GapA deletion strain GM1501. This work was supported by grant BR1552/6-3 from Deutsche Forschungsgemeinschaft (to S. B.).

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