Innova 44 44 r shaker

WT parental strain and ∆dri1 were grown in liquid BG−11 at 30 °C with shaking at 90 rpm in an Innova® 44/44 R shaker (New Brunswick). Log-phase cells were harvested and normalized to a chlorophyll concentration of 5 µg mL⁻¹ (quantified spectroscopically after methanol extraction^{103 (link)}). One milliliter of chlorophyll-normalized cells was dark acclimated for 30 min and chlorophyll fluorescence measurements were performed on a Dual-PAM 100 (Waltz), to determine chlorophyll fluorescence parameters (F₀′, F_m′_dark) in the dark. The samples were kept in the dark with a weak measuring light to determine the minimum fluorescence in the dark F₀′ for 10 s. A 600 ms saturating light pulse (peak emission at 620 nm, 5,000 μmol photons m^-2 s⁻¹) was applied to determine the dark-acclimated maximal fluorescence F_m′_dark. Samples were then exposed to actinic light at 50 μmol photons m^-2 s⁻¹ for 5 min. Saturating light pulses were triggered after 30 s, 1 min, 2 min and 5 min after actinic light was turned on. Actinic light was turned off and fluorescence measured for 2 min, with saturating pulses at 30 s, 1 min and 2 min. Data were visualized using the software Dual PAM v1.19. The abundance of major photosynthetic membrane complexes (PSI and PSII) was evaluated with BN-PAGE.

Synechocystis cells were grown in liquid BG−11 at 30 °C and shaken at 90 rpm in an Innova® 44/44 R shaker (New Brunswick) for 6 days. Exponentially growing cells (OD 750 nm ~ 0.5) were harvested at 700 x g for 10 min at 4 °C. Cell pellets were washed three times with BG−11 and finally resuspended in half of the culture volume. Overnight grown Escherichia coli DH5α cells containing either pRL443, pRL623, or pCpf1b plasmids were harvested similarly, washed three times in LB lacking antibiotics, and finally resuspended in half of the culture volume. Conjugation of editing plasmids into the different bacterial strains was carried out by mixing 100 µL of E. coli pRL443, 100 µL of E. coli pRL623, 100 µL of E. coli pCpf1b, and 300 µL of Synechocystis sp. PCC 6803. Cell mixtures were incubated at 24 °C for 2 h, spread out on a 0.45 µm MF-Millipore membrane filters (Sigma) overlaid on BG−11 agar supplemented with 5% LB, and incubated for 24 h at 24 °C. Filters were transferred onto BG−11 agar supplemented with 50 µg/mL kanamycin. Colonies were transferred to Kan50-BG−11 agar and PCR verified for gene deletion of nucleotide mutagenesis. After Sanger sequencing, correct transformants were grown for 6 days in antibiotic free BG−11 to cure the pCpf1b plasmid, and cured strains were selected on BG−11 agar supplemented with 10% sucrose (sacB counterselection gene from pCpf1b).

WT cells expressing Dri1-Strep-tag II, SdhB1-FLAG protein fusions, or untagged proteins were grown in liquid BG−11 at 30 °C and shaken at 90 rpm in an Innova® 44/44 R shaker (New Brunswick) for 6 days. Cell lysis was performed as mentioned above with the addition of 150 mM NaCl. The protein concentrations of the soluble fractions were normalized by Bradford assay (BioRad) for protein pull-downs. A total of 20 µg of soluble proteins were incubated with 10 µL of Strep-Tactin® Sepharose® resin (IBA-LifeSciences) at 24 °C for 2 hours with or without 200 µM hemin. To remove protein unspecific binding, the resin was washed five times with 100 mM Tris-HCl, 150 mM NaCl, 20% glycerol, and 1 mM EDTA pH 8.0. Proteins were eluted with 20 µL of 100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 2.5 mM desthiobiotin, pH 8.0, boiled for 5 min with SDS-sample buffer, and separated by SDS-PAGE (4-20% ExpressPlus™ PAGE Gel, Genscript), followed by immunoblotting.