AatII restriction enzyme

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	The kanamycin resistance cassette of pTKP2031V was deleted by digestion with XhoI and AatII (Takara Bio).

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The kanamycin resistance cassette of pTKP2031V was deleted by digestion with XhoI and AatII (Takara Bio).

Publication protocol

First, the kanamycin resistance cassette of pTKP2031V was deleted by digestion with XhoI and AatII (Takara Bio). The gentamycin resistance cassette from pVZ322 (Zinchenko et al., 1999) was amplified by PCR using KOD polymerase and the specific primers 5′-AAATTTCTCGAGTGTAAGCAGACAGTTTTA-3′ and 5′-AAACCCGACGTCTGTTAGGTGGCGGTACTT-3′, digested with XhoI and AatII, and inserted into the XhoI-AatII sites of pTKP2031V. The resultant plasmid was named pTGP2031. A region of the Synechocystis 6803 genome encoding one of two glycogen synthases, glgA(sll0945), including +1 to +1,180 bp from the translation initiation codon was amplified by PCR using KOD polymerase and the specific primers 5′-TTCCGCATGCATGAAGATTTTATTTGTGGC-3′ and 5′-TTAAGAATTCCATTGATAGGATCGTAGAA-3′. The amplified PCR fragments were digested with SphI and EcoRI (Takara Bio) and inserted into the SphI-EcoRI sites of the pUC119 vector (Takara Bio). The resultant plasmid was digested with ApaI, and the region including the gentamycin resistance cassette, psbAII promoter, and NdeI-HpaI cloning sites of pTGP2031 was amplified using KOD polymerase and the specific primers 5′-TTTGCTTCATCGCTCGAG-3′ and 5′-ATCCAATGTGAGGTTAAC-3′ and integrated into the ApaI site of the plasmid. The resultant plasmid was named pTGP0945. The sigE coding region was obtained by PCR as described previously (Osanai et al., 2011), except for the reverse primer (5′-AAAGGGGTTAACCTATAACCAACCTTTGAG-3′), which was newly synthesized and contained the HpaI site. The amplified sigE coding region digested with NdeI and HpaI was integrated into the NdeI-HpaI sites of pTGP0945 (Fig. 6A). Transformation was performed as well with pTKP2031V-rre37, except that 3 μg mL−1 gentamycin was used instead of 50 μg mL−1 kanamycin.

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