Fig. 4: Designing and testing synthetic chloroplast promoters and library chloroplast transformation.
a, Synthetic promoter characterization. Promoters were characterized by measuring the NanoLuc activity of transplastomic strains containing different constructs, which differ in their promoters. The NanoLuc signals of transplastomic strains containing a NanoLuc expression cassette (green) are plotted as arbitrary units normalized to OD750 and compared with a dummy sequence (grey). The dashed line demarcates synthetic promoters (left, light green) from endogenous ones (right, dark green). For all measurements, nbiological = 5, ntechnical = 3. In each box plot, the central line indicates the median, the box indicates the lower and upper quartiles, and the whiskers represent the data points that fall within 1.5 times the interquartile range from the lower and upper quartiles. Any data point outside this range is considered an outlier. b, Conceptual workflow for transforming construct libraries into the chloroplast. Sequencing analysis showed that 82% of transformants contained a single promoter variant, and for 18% multiple sequence variants could be detected, c, Fluorescence measurement of the transplastomic promoter library. mScarlet-I fluorescence of the transplastomic strains containing different promoter variants was measured (excitation, 569 nm; emission, 605 nm). Fluorescence is plotted as arbitrary units normalized to OD 750. A total of 768 transplastomic strains were measured, and a range of different mScarlet-I signals were detected. The grey bars represent examples that were selected in d for sequencing. d, Sequence analysis of sequenced transplastomic strains. Pairwise Hamming distance is plotted for the promoter sequence variant, which was found in a set of 32 sequenced transplastomic strains.
