2.4 DNA devices assembly

All DNA constructs were generated following the standard assembly protocol, and the BioBrick DNA parts used were obtained from the Registry of Standard Biological Parts (Table 1). Briefly, to test promoter constitutive expression, the GFP generator (part BBa_E0240 that comprises the RBS BBa_B0032, the coding sequence for GFP BBa_E0040, and the double terminator BBa_B0015) was excised from the delivery plasmid with XbaI and PstI and cloned downstream each promoter (plasmids digested with SpeI and PstI). To test the promoters’ regulated expression, the lacI, tetR, cI, luxR or araC regulators’ ORFs were cloned under regulation of the constitutive promoter P_rnpB and the RBS BBa_B0030. The ORFs were excised with XbaI and PstI and cloned in the plasmid containing the promoter, previously digested with SpeI and PstI. Subsequently, the devices containing the promoter and GFP were excised with EcoRI and SpeI and cloned upstream of the respective P_rnpB:: regulator assembly (plasmid digested with EcoRI and XbaI). For the T7 polymerase (T7pol), a similar assembly was performed.

For generation of the GG device used in the proof of concept, the ggpS ORF was amplified by PCR using Synechocystis genomic DNA as template with primers flanked by the BioBrick prefix or suffix (see Supplementary Table S1). Amplification was performed using Phusion high-fidelity DNA polymerase (Thermo Scientific), according to the manufacturer’s instructions. The PCR product was purified using the NZYGelpure kit (NZYTech), digested with XbaI and PstI and cloned downstream of P_trc.x.lacOand BBa_B0030 RBS (plasmid digested with SpeI and PstI).

All the generated constructions were transferred to pSEVA251 or pSEVA351 shuttle vectors (obtained from the ‘Standard European Vector Architecture’ repository) (³⁴) and the construct harboring the RNA polymerase from phage T7 (P_rnpB::T7pol) was cloned into the pSN15K vector (¹³). The correct assembly of the generated constructs was confirmed by PCR, restriction analysis and sequencing (STAB VIDA).