Electroporation of Chlorella sp. with pcambia 1304 linearized plasmid

Abstract

Electroporation is a widely used method in plant transformation since it is very efficient. Transformation of pCAMBIA 1304 construct into microalgae, Chlorella sp. can play an important role as a model of fundamental studies of fatty acid biosynthesis pathways of Chlorella sp. As the initial step, the pCAMBIA 1304 plasmid was successfully extracted from Escherichia coli culture. The purity of the plasmid was 1.76 and the concentration was 0.55 µg/mL. The results showed the extracted plasmid was in high quality and can be further used in subsequent steps. The pCAMBIA 1304 plasmid was successfully verified by PCR technique with primers combination 35S-F/35S-R, GG-F/GG-R and 35S-F/GG­ R. The sizes of amplified bands with were 326 bp, 676 bp and 1,426 bp respectively. PCR amplification of the CaMV 35S promoter and gfp:uidA genes fusion confirmed that the presence of pCAMBIA 1304 in extracted plasmid. The pCAMBIA 1304 plasmid was successfully linearized with EcoRI and purified by Wizard SV Gel and PCR Clean Up System Kit (Promega). The suitable voltage for electroporation of Chlorella sp. was determined with p35S-AP plasmid. The Agr mode (2.2 kV) was selected from six different electrical voltages used in electroporation of Chlorella sp.