Fig. 1
From: CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16
Construction and characterization of the electroporation vectors with and without the RP4 partitioning system. (A) Depictions of plasmids optimized for electroporation containing either the pBBR1 (pKERep) or the pSa (pKESa) replicon, which contain Rep and oriV or ResP, RepA and oriV respectively. To increase plasmid stability the RP4 partitioning system was added to create pKERepPar and pKESaPar. (B) Electroporation efficiencies for the four constructed plasmids (n = 3), obtained for C. necator H16 on TSBKan agar plates after 2 days at 28 °C. Presence of the plasmids was controlled by the applied selection and an eGFP derived green appearance of colonies. Error bars, SEM. (C) Segregational stability determination on antibiotic free TSB media at 28 °C. Cells were cultivated in 24 h intervals started by inoculation with the prior culture (n = 3). Loss of plasmid was determined by plating on TSB agar followed by a transfer of colonies to TSBKan plates and monitoring of the eGFP dependent green appearance of colonies. (D) Measurement of growth rates and (E) eGFP derived fluorescence during a 12 h cultivation of C. necator H16 carrying the four constructed plasmids (n = 3). Wild type C. necator H16 carrying no plasmid (WT) was used as a control. Cultures grew on antibiotic free TSB media at 28 °C as described in Materials and Methods. Error bars, SEM