Eporator

Membrane-engineered mammalian cells were created by the electroinsertion of the enzyme superoxide dismutase (SOD) into the membrane of Vero cell fibroblasts following the protocol of Moschopoulou et al. [43 (link)]. Initially, cells at a density of 3 × 10⁶ mL⁻¹ were centrifuged at 1000 rpm for 2 min and the pellet was resuspended in PBS (pH 7.4). Afterwards, cells were incubated with 1500 U·mL⁻¹ CuZnSOD (EC1.15.1.1) for 20 min at 4 °C and the mixture was transferred to electroporator (Eppendorf Eporator, Eppendorf AG, Germany) cuvettes. Electroinsertion was performed by applying four pulses of an electric field at 1800 V·cm⁻¹. Then, cells were centrifuged at 1000 rpm for 2 min and resuspended in cell culture medium. Finally, the sensors were fabricated by mixing 1 volume of Vero-SOD cells with 2 volumes of 4% (w/v) sodium alginate solution and was added dropwise with the use of a 22G syringe in 0.8 M CaCl₂. Cells were immobilized in calcium alginate, forming beads containing 75 × 10³ cells per bead with an approximate diameter of 2 mm. As already reported [39 (link),43 (link)], the membrane potential of membrane-engineered Vero cell fibroblasts is affected by the interactions of electroinserted SOD molecules and superoxide anions, producing measurable changes in the membrane potential.

Plasmid extraction of synthetic gene and pGAPzαA was done with the plasmid DNA purification kit. The CaLB gene was amplified from the vector via PCR with forward primer pGAP_fw (5′-GCTGAAGCTGAATTCTTGCCATCTGGTTCTG-3′) and reverse primer pGAP_rev (5′-CACACTGGGTACCCGTTACTAGTGGATCCG-3′). The PCR product and pGAPzαA were digested using restriction enzymes EcoRI (100 U) and KpnI (100 U). After 20 min heat inactivation at 80 °C and purification of the specific DNA fragments with the PCR clean-up gel extraction kit, the digested CaLB gene and vector pGAPzαA were ligated using T4 DNA ligase (5 U) resulting in pGAP_CaLB. The plasmid construct was subsequently transformed into E. coli DH5α cells (purchased from Agilent Technologies; Santa Clara, CA, USA). The pGAP_CaLB was extracted from E. coli DH5α. About 200 ng plasmid DNA linearized by AvrII was mixed with 80 µL of competent cells, and then it was transformed into Pichia pastoris (SMD1168) cells (purchased Invitrogen GmbH, Karlsruhe, Germany) by electroporation conducted on Eppendorf Eporator (Eppendorf, Hamburg, Germany) according to the manufacturers instruction Pichia pastoris transformants via homologous recombination at the GAP promoter region between the transforming DNA and regions of homology within the Pichia genome. Positive clones were initially selected on YPDS plates containing 100 μg/mL Zeocin™ plates.