Electroporation of Chlorella sp. with 35 s-ap circular construct

Abstract

Electroporation has been given much attention in recent years due to its easy ability to act as a tool of genetic manipulation to generate transgenic plants. The availability of essential polyunsaturated fatty acids in Ch/ore/la sp. makes it a marketable source of omega-3 and omega-6. The 35S-AP plasmid DNA was extracted from Escherichia coli. The purity of the extracted plasmid was 1.78 while the concentration was 0. 67 µg/mL. This suggests that the plasmid obtained is of high quality. The 35S-AP plasmid was amplified with PCR technique using three primer combinations. The primer combinations 35S-F/35S-R produced a band of 326 bp. The primer combinations PTE-VF1/PTE-VR2 produced a band of 617 bp. The final primer combinations 35S-F/PTE-VF1 produced a distinct band of 943 bp. A series of voltage was tested to determine the most suitable voltage by electroporation of 35S-AP circular plasmid using the Bio-Rad Micropulser electroporation apparatus . The program Ec3 with a voltage of 3.0kV was selected to electroporate Ch/ore/la sp. with 35S-AP circular plasmid. The electroporated cells were cultured on BBM with 10 µg/mL hygromycin for selection of putative recombinant Ch/ore/la sp. Sixteen putative colonies were randomly selected and transferred to BBM grid plates with 10 µg/mL and 15 µg/mL hygromycin respectively. The putative transformed colonies grew on 10 µg/mL hygromycin but no growth was observed on 15 µg/mL hygromycin. Electroporation condition needs to be optimized to su�essfully transform Ch/ore/la sp.