Fig. 3: Characterization of regulatory parts for chloroplast gene expression.
a, 5′UTR characterization. 5′UTRs were characterized by measuring NanoLuc activity of transplastomic strains containing different constructs, which differ in their 5′UTR. The NanoLuc signals of transplastomic strains containing a NanoLuc expression cassette (green) are plotted as arbitrary units normalized to OD750 and compared with the WT strains (grey). For all measurements, nbiological = 5, ntechnical = 3. b, 5′ characterization under four different conditions. The heat map shows the NanoLuc signal of transplastomic strains under light, dark, TAP and HSM conditions. Similar expression patterns can be observed under all four conditions. c, 3′UTR characterization. 3′UTRs were characterized by measuring NanoLuc activity of transplastomic strains containing different constructs, which differ in their 3′UTR. The NanoLuc signals of transplastomic strains containing a NanoLuc expression cassette (green) are plotted as arbitrary units normalized to OD750 and compared with the WT strains (grey). For all measurements, nbiological = 5, ntechnical = 3. d, Function of IEEs. Two cistrons are connected via an IEE. After processing via imported cis-factors, both cistrons are translated separately. e, IEE characterization. IEEs were characterized by measuring NanoLuc and Fluc activity of transplastomic strains containing different constructs, which differ in their IEE. The luciferase signals of transplastomic strains containing a NanoLuc expression cassette (green) are plotted as arbitrary units normalized to OD750 and compared with Fluc luminescence (blue). The dashed line differentiates IEEs with insufficient support for robust expression (left) from those facilitating strong expression (right). For all measurements, nbiological = 5, ntechnical = 3. For each measurement, we included a dummy part that lacks any secondary structure, promoter elements, ribosome-binding motifs or RNA-binding protein sites, serving as a negative control. 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.
