Escherichia coli and Synechocystis cultivation protocol

Escherichia coli strains DH5α^{57 (link)} and HB101 (harbouring conjugal plasmid pRL443)^{58 (link)} were used for cloning and triparental mating, respectively. All E. coli strains were cultivated on plant-based LB-agar plates or in liquid LB medium (lysogeny broth) at 37 °C. Antibiotics were added to E. coli culture medium to the final concentrations of 20 μg mL⁻¹ spectinomycin (spec), 100 μg mL⁻¹ ampicillin (amp) and 50 μg mL⁻¹ kanamycin (kan), respectively.
Synechocystis sp. PCC 6803 strain GT-U, a derivative of the non-motile, glucose-tolerant strain GT-Kazusa^{59 (link)} was used as final host for all conjugations. For selection and strain maintenance Synechocystis was cultivated on 0.75% [w/v] agar (Type A, Sigma) plates containing TES-buffered (10 mM, pH 8.0) BG-11 mineral medium^{60 (link)}. Pre-cultures were grown in TES-buffered BG-11 liquid medium in 6-well polystyrene at 30 °C and 120 rpm. By default, CuSO₄ and Co(NO₃)₂ was not added to any basic BG11 media preparations in this study in order to: (i) minimize uncontrolled Cu²⁺ induction of P_petE, and to (ii) provide reference data for follow-up studies using Co²⁺- induction. For contamination control and plasmid maintenance, all cultures of transformed Synechocystis strains were supplied with 20 μg mL⁻¹ spectinomycin and 50 μg mL⁻¹ kanamycin.