A Step-by-Step Approach to Establishing an Efficient Genetic Transformation Protocol for Chlorella vulgaris Using Electroporation

Abstract Chlorella vulgaris, a unicellular green microalgae, has shown diverse applications in biotechnology. Particularly in microalgae genetic engineering, genetic transformation of C. vulgaris offers a sustainable and cost-effective approach to producing recombinant protein. Despite this, the thick cell wall of C. vulgaris poses a challenge, often necessitating optimization of the transformation parameters to overcome the challenge. This current study presents a detailed methodology for efficient electroporation-mediated transformation of C. vulgaris. The electroporation procedure described here includes C. vulgaris cell preparation and electroporation parameters such as pulse voltage, capacitance, and resistance. Prior to the cultivation of the transformed cells in a selective BG11 medium, recovery conditions of the newly transformed cells in a regular BG11 medium were also described. Through optimization of these parameters, an improved transformation efficiency while maintaining cell viability was achieved. Positive amplifications of the pCambia1303 t-DNA region in the PCR and RT-PCR assays were indicative of the success of the electroporation-mediated transformation of C. vulgaris. DNA sequencing was then used to confirm the identity of these amplicons, thus validating the successful transformation procedure. The electroporation-mediated transformation procedure established here provides a reliable and reproducible method for carrying out genetic modification on C. vulgaris to facilitate its potential use as a bio-factory in numerous biotechnological applications.