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. 2023 Aug 31;14:1230723. doi: 10.3389/fpls.2023.1230723

Figure 3.

Figure 3

New designs to achieve higher PRK expression. (A) Design of the pCMM-25 and pCMM-26 constructs. (B) Pipeline for the identification of strains with high PRK content by selection of transformants of HSM plates, followed by growth to saturation in 96-well plates in TAP medium, and re-plating on HSM agar plates. Thestrains with the fastest growth on the plate were selected for further analysis. (C) Anti-PRK western blot on total soluble protein extracts of the ΔPRK, CC-4533, and complemented strains obtained by transforming the ΔPRK strain with th pCMM-24 (α strains), pCMM-26 (β strains) or pCMMM-25 (D5 strain) constructs. 100% corresponds to 12 µg of total protein. A gradient of the total protein extract of the strain CC-4533 was used for the quantification of the PRK content in the different complemented strains, indicated as PRK level (%). (D) growth profile of CC-4533 and complemented strains. Cultures were inoculated at 105 cells/mL and incubated in HSM at 25°C under continuous light (100 µmol photons m-2 s-1). (E) Lag phase measured for CC-4533 and complemented strains, calculated from the data in (D, F) Spot test on HSM minimal medium after 5 days of growth at 25°C in the light (50 µmol photons m-2 s-1). The number of cells spotted is indicated on the left and the strain is indicated below each test line.