Tracking nirP1 Promoter Activity in Synechocystis

To verify nirP1 promoter activity in vivo in Synechocystis 6803, the nucleotides −70 to +30 (TSS at +1) were fused to luxAB reporter genes and integrated into the genome. Initially, cultures were set to an OD 0.7 and grown under standard BG11 or HC conditions for 2 h and subsequently transferred to NO₃⁻- or NaCO₃-free BG11 or BG11 medium supplemented with additional NH₄Cl to induce transcription. Measurements were carried out for 24 h in LC or high NH₄Cl conditions or for 7 days under N deprivation. To recover from N starvation, the bleached cells were transferred back to standard BG11 medium after 7 days and observed for 3 days. Bioluminescence was measured in vivo as total light counts per second by using a VICTOR³ multiplate reader (PerkinElmer). Prior to the measurement, cells were diluted to an OD₇₅₀ = 0.7 and 200 μL of the suspension were filled into a white 96-well plate (CulturePlate™−96, PerkinElmer). To increase the signal, 2 µL of Decanal was added to the samples. In the multiplate reader the cell suspensions were shaken for 10 s and subsequently total light emission was measured for 1 s. A strain carrying the promotorless luxAB genes served as a negative control^{51 (link)}. The raw data, the calculation, and statistical evaluations are provided in Supplementary Dataset 3.