NheI-HF®

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	After the amplification, the DNA fragment and the pDR111 vector were digested with NheI-HF and SalI-HF restriction enzymes (NewEngland BioLabs), followed by ligation.

Protocol tips
After the amplification, the DNA fragment and the pDR111 vector were digested with NheI-HF and SalI-HF restriction enzymes (NewEngland BioLabs), followed by ligation.

Publication protocol

All bacterial strains used are listed in Table 1. Table 2 lists all the primers used in this study. In order to construct new B. subtilis strains, standard transformation and Spp1 transduction protocols were used for genomic integration and plasmid transformation [31]. To generate the amyE::Phs-comP construct, the open reading frame (ORF), with its native ribosome binding site (RBS), was amplified from the PY79 strain using the ComP-NheI-R & ComP-native-RBS-F primer pair. After the amplification, the DNA fragment and the pDR111 vector were digested with NheI-HF and SalI-HF restriction enzymes (NewEngland BioLabs), followed by ligation. The final construct has the insert downstream of a hyperspank inducible promoter found in the pDR111 vector. To generate sacA::PcomQXP-comQX and sacA::PcomQXP-comQ constructs, the native promoter of the comQXP operon with the ORF of comQX or comQ only, were amplified from the PY79 strain, using the forward primer ComQ-into-ECE174-F-[BamHI], either with ComQX-into-ECE174-R-[EcoRI] or with ComQ-into-ECE174-R-[EcoRI], respectively, and cloned into plasmid ECE174 using the designated restriction enzymes. The sacA::PcomQXP-comX construct was generated by amplifying the whole ECE174::PcomQXP-ComQX (AEC840) plasmid without the comQ ORF, using the dcomQ-R and dcomQ-F primer pair. The dcomQ-R primer exists at the end of comQ in the forward direction, and dcomQ-F primer exists at the beginning of comQ in the reverse direction. The amplified fragment was treated with DpnI and then T4 Polynucleotide Kinase (NewEngland BioLabs), followed by self-ligation. To generate the pDL30::PcomQXP-rapPT236N construct, the ORF, with its native RBS, was amplified from the ECE174::PrapP-rapPT236N (AEC735) plasmid using the RapP-SphI-R & hsRapP-F primer pair. In addition, the DNA fragment pDL30::PcomQXP was amplified from the pDL30::PcomQXP-3xYFP (AEC962) plasmid using the PQXP-NheI-R & pDL30-SphI-F primer pair. After the amplification, the DNA fragments were digested with NheI-HF and SphI-HF restriction enzymes, followed by ligation. To generate the pDR111::Phs-phrP construct, the ORF, with its native RBS, was amplified from the NCIB3610 strain using the PhrP-SalI-F & PhrP-NheI-R primer pair. After the amplification, the DNA fragment and the pDR111 vector were digested with NheI-HF and SalI-HF restriction enzymes, followed by ligation. To generate pDR111::Phs-phrPint, the entire pDR111::Phs-phrP (AEC1272) plasmid was amplified using PhrP-NO-signal-seq-R & PhrP-NO-signal-seq-F primer pair. The purified DNA was treated with DpnI and then T4 Polynucleotide Kinase, followed by self-ligationA

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