All experiments were performed with B. subtilis NCIB 3610 (31). Laboratory strains of B. subtilis (PY79) and Escherichia coli (DH5α) were used for cloning purposes (31). Lists of bacterial strains and primers are provided in table S1 and in (31, 51, 53, 55, 61, 62, 69, 73) and table S2, respectfully.

For cloning, linearized PCR products were first transformed into PY79 (89), and then the genomic DNA of the transformed strain was transformed to NCIB 3610 (90). Linearized plasmids were transformed directly to NCIB 3610. Deletion mutations were generated by long-flanking homology PCR mutagenesis (91). All suppressor mutants were verified by whole-genome sequencing.

Plasmids for the generation of GFP reporter strains or tapA-sipW-tasA overexpression construct were constructed as follows: (i) pYC121, which contains a functional GFP gene downstream to a cloning site and a chloramphenicol resistance gene (92), was used for the construction of amyE::PcomGA-gfp and amyE::PtapA-gfp reporter strains. PCR fragments were amplified from NCIB 3610 chromosomal DNA, using primers with the suitable restriction sites for ligation into the plasmids. (ii) hag promoter (41) was amplified with primers containing EcoRI and HindIII restriction sites and integrated into pYC121 upstream to GFP. The resulted plasmid was then cut with EcoRI and BamHI and integrated into pDR183 to create a lacA::Phag-gfp(mls) reporter strain. (iii) pIK76 was created by inserting the EcoRI and BamHI fragment of pDR111, which contains the hyperspank promoter upstream to a cloning site, the lacI repressor gene (41) into the plasmid pDR183 that enables integration into the lacA locus (93). pIK76 was used for the construction of tapA-sipW-tasA overexpression strain. PCR fragment, containing the tapA-sipW-tasA operon, ribosome binding sites, and SalI and SphI restriction sites, was integrated into pIK76. Plasmid pDFR6, containing the open reading frame of TasA without the signal peptide or the stop codon cloned into pET22b (Merck), was generated as described previously (33).

The ligated plasmids were then transformed into E. coli DH5α, and ampicillin-resistant colonies were selected and confirmed by sequencing. The GFP reporter or overexpression plasmids were then integrated into the neutral amyE or lacA locus of the laboratory strain PY79 by transformation, as described above, and selected for chloramphenicol, spectinomycin, or MLS [Macrolide (Erythromycin), Lincosamide and Streptogramin] resistance. Extracted genomic DNA of the transformed strains was transformed to NCIB 3610 as described above.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.