Electroporation of Chlorella sp. with p35s-AP linearized construct

Abstract

Electroporation has been used in plants and microalgae genetic engmeenng to produce beneficial transgenic plants and microalgae. Genetic transformation by electroporation increase the level of polyunsaturated fatty acids in Ch/ore/la sp. such as m-3 C18:3 and m-6 C18:2. The objectives of this study are to transform Ch/ore/la sp. with p35S-AP construct by electroporatian and to select for the putative recombinant chlorella cells. The extracted plasmid DNA of p35S-AP was verified using PCR technique. In this step, three types of primer combination were used as follows: 35S-F/35S-R, PTE-VF1/PTE-VR2 and 35S-F/PTE-VF1. The 35S-F/35S-R primer combination successfully amplified the CaMV 35S promoter with the size of 326 bp. The combination of PTE-VF1/PTE-VR2 primer successfully amplified the antisense palmitoyl-ACP thioesterase gene fragment with the size of 617 bp while 35S-F/PTE-VF1 successfully amplified the CaMV 35S promoter and antisense palmitoyl-ACP thioesterase gene fragment with the size of 934 bp. The DNA plasmid was then digested with EcoRl restriction enzyme to produce linear plasmid followed by DNA plasmid purification.