Bamhi and xhoi restriction enzymes

The genomic region of Synechocystis 6803 including the fumC (slr0018) open reading frame (ORF) with the BamHI-XhoI fragment was amplified by PCR using the KOD -plus- neo DNA polymerase (Toyobo, Osaka, Japan) and the following primer set: forward, 5′-GAAGGTCGTGGGATCATGGTAAATTCCCACCGC-3′ and reverse, 5′-GATGCGGCCGCTCGAGCTAGTCAGCAATCGGGG-3′; the BamHI and XhoI restriction enzymes were obtained from TakaraBio (Shiga, Japan). The resultant fragment was cloned into the BamHI-XhoI sites of pGEX5X -1 (GE Healthcare Japan, Tokyo, Japan). Amino acid substitution was performed commercially by TakaraBio (Shiga, Japan). For SyFumC_A314E, the region +940–942 from the start codon in the fumC ORF was changed from GCC to GAA.
These vectors were transformed into E. coli DH5α cells (TakaraBio) and 5 L of transformed E. coli were cultivated in LB medium at 30 °C with shaking (150 rpm); protein expression was induced overnight in the presence of 0.01 mM isopropyl β-D-1-thiogalactopyranoside (Wako Chemicals, Osaka, Japan).

The amino acid sequence of Sbp5-2 was confirmed by rapid-amplification of cDNA ends (RACE). rTRM7, rTRM6, and rTRM7 mutation (all Cys to Lys) genes were cloned from Sbp5-2 by PCR. The target genes were digested by BamHI and XhoI restriction enzymes (Takara) at 25 °C for 30 min, and inserted into pET32a by T4 Ligase at 37 °C for 30 min. The recombinant plasmids were then transformed into DH5α Escherichia coli competent cells (Novagen) respectively. All constructed plasmids were sequenced and verified by Sangon. Primers utilized in plasmids construction are listed in Supplementary Table 4.