All plasmids were verified by Sanger sequencing and listed in Supplementary Table S2 . Primers used in this study were listed in Supplementary Table S3 .
Plasmid pCpf1-CT- dnaENI is constructed for making conditional mutant CT-dnaENI. To generate the repair template, a region upstream of dnaENI amplified using the primers Pall3578F940m and Pall3578R150m, a region of CT promoter (an artificial promoter with a petE promoter and a theophylline riboswitch) amplified using the primers Pall0258F475m and PV_19 from vector pCT, and a region downstream amplified using the primers Pall3578F1 and Pall3578R960 were fused by overlapping PCR using the primers Pall3578F940m and Pall3578R960. A sequence of two spacers was prepared by annealing the complementary primers cr1_all3578F23mF, cr1_all3578F23mR, cr2_all3578R32mF, and cr2_all3578R32mR. The repair template and the spacer sequence were sequentially cloned into pCpf1 at the sites of BglII/BamHI and AarI/AarI, resulting in the mutation plasmid pCpf1-CT-dnaENI.
To generate plasmid pCint2-ΔdnaA, the upstream and downstream sequences of dnaA gene as well as the kanamycin resistance cassette were amplified using specific primers listed inSupplementary Table S2 . The resulting three fragments were fused by overlapping PCR using the primers Palr2009F1255m and Palr2009R2628. This repair template was then cloned into pCint2 at the sites of BglII/BamHI, resulting in the plasmid pCint2-ΔdnaA.
To construct the CT-dnaENI conditional mutant, the mutation plasmid pCpf1-CT-dnaENI was transferred into Anabaena PCC 7120 by conjugation (Cai and Wolk, 1990 (link); Elhai et al., 1997 (link)). The exconjugants were selected on BG11 plates containing 100 μg mL–1 neomycin, 0.3 μmol CuSO4, and 1 mM theophylline and verified by PCR and Sanger sequencing.
To construct WT/pPhetR-gfp, TRS-polA/pPhetR-gfp, and CT-dnaENT/pPhetR-gfp, the replicated plasmid pPhetR-gfp was transferred into WT, TRS-polA, and CT-dnaENT, respectively, by conjugation. The positive clones were selected on BG11 plates containing 5 μg mL–1 spectinomycin and 2.5 μg mL–1 streptomycin and verified by PCR.
To construct ΔdnaA, the mutation plasmid pCint2-ΔdnaA was transferred into Anabaena PCC 7120 by conjugation, and the single-crossover was selected on BG11 plates containing 100 μg mL–1 neomycin and verified by PCR. The homologous double-crossover was selected on BG11 plates containing 8% sucrose, verified by PCR and Sanger sequencing.
To make the plasmid for transcriptional fusion of hetR, the promoter region of hetR was amplified using the primers Palr2339F915m and Palr2339R3 and subsequently cloned into pRL25N-Lgfp at the sites of BamHI/XhoI, resulting in the transcriptional fusion plasmid pPhetR-gfp. It was then moved into the cells by conjugation.
Plasmid pCpf1-CT- dnaENI is constructed for making conditional mutant CT-dnaENI. To generate the repair template, a region upstream of dnaENI amplified using the primers Pall3578F940m and Pall3578R150m, a region of CT promoter (an artificial promoter with a petE promoter and a theophylline riboswitch) amplified using the primers Pall0258F475m and PV_19 from vector pCT, and a region downstream amplified using the primers Pall3578F1 and Pall3578R960 were fused by overlapping PCR using the primers Pall3578F940m and Pall3578R960. A sequence of two spacers was prepared by annealing the complementary primers cr1_all3578F23mF, cr1_all3578F23mR, cr2_all3578R32mF, and cr2_all3578R32mR. The repair template and the spacer sequence were sequentially cloned into pCpf1 at the sites of BglII/BamHI and AarI/AarI, resulting in the mutation plasmid pCpf1-CT-dnaENI.
To generate plasmid pCint2-ΔdnaA, the upstream and downstream sequences of dnaA gene as well as the kanamycin resistance cassette were amplified using specific primers listed in
To construct the CT-dnaENI conditional mutant, the mutation plasmid pCpf1-CT-dnaENI was transferred into Anabaena PCC 7120 by conjugation (Cai and Wolk, 1990 (link); Elhai et al., 1997 (link)). The exconjugants were selected on BG11 plates containing 100 μg mL–1 neomycin, 0.3 μmol CuSO4, and 1 mM theophylline and verified by PCR and Sanger sequencing.
To construct WT/pPhetR-gfp, TRS-polA/pPhetR-gfp, and CT-dnaENT/pPhetR-gfp, the replicated plasmid pPhetR-gfp was transferred into WT, TRS-polA, and CT-dnaENT, respectively, by conjugation. The positive clones were selected on BG11 plates containing 5 μg mL–1 spectinomycin and 2.5 μg mL–1 streptomycin and verified by PCR.
To construct ΔdnaA, the mutation plasmid pCint2-ΔdnaA was transferred into Anabaena PCC 7120 by conjugation, and the single-crossover was selected on BG11 plates containing 100 μg mL–1 neomycin and verified by PCR. The homologous double-crossover was selected on BG11 plates containing 8% sucrose, verified by PCR and Sanger sequencing.
To make the plasmid for transcriptional fusion of hetR, the promoter region of hetR was amplified using the primers Palr2339F915m and Palr2339R3 and subsequently cloned into pRL25N-Lgfp at the sites of BamHI/XhoI, resulting in the transcriptional fusion plasmid pPhetR-gfp. It was then moved into the cells by conjugation.
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