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. 2022 Apr 21;10(3):e02562-21. doi: 10.1128/spectrum.02562-21

FIG 1.

FIG 1

Reporter gene assay to monitor transcription driven by different promoter variants. (A) General design of the plasmids used for the GFP assay. Using pVZ322 as the backbone, the coding sequence of sfgfp together with the rrnB T1 terminator sequence (51) was fused to various 5’UTR (blue) and promoter (gray) sequences. (B) The design of different constructs used for the GFP assays. In constructs 1, 4, 5 and 6, two different 5’UTRs were combined with the full-length atpT promoter PatpT328 or the PBba_J23101 promoter (iGEM Registry parts.igem.org [20]). Constructs 2 and 3 contain truncated versions of the atpT promoter. The 5’UTRs originated from atpT or the sll0639 gene encoding the oligoribonuclease/PAP phosphatase NrnA. (C) Analysis of the GFP intensity in dark/light shift experiments. Different strains cultured under continuous light were first transferred to darkness for 24 h (black bar) and then transferred to light (yellow bar). Signals measured from a simultaneously cultivated Synechocystis 6803 without any genetic modification were subtracted from the measured fluorescence values (background correction). (D) GFP assay comparing the activities of atpT promoters of three different lengths before (gray bars) and after 6 h of dark incubation (black bars). (E) Mutations introduced into the PatpT66 promoter to test the relevance of conserved sequence elements. The mutated nucleotides, putative -35 and -10 elements and the region with similarity to the HLR1 element (two direct repeats (G/T)TTACA(T/A)(T/A) separated by 2 nt [31]) is boxed and asterisks indicate matching positions. (F) GFP assay comparing the activities of PatpT66-5’UTR and the five of its mutated variants as depicted in Panel (E) before (gray bars) and after 6 h of dark incubation (black bars). The differences between groups were analyzed using t tests (Panels D and F; Tables S5 and S6 in the supplemental material) with GraphPad software. Significance was established at P < 0.05 = ** and P < 0.01 = ***. For details of the statistical analysis see Tables S5 and S6.