A step-by-step approach to establishing an efficient genetic transformation protocol for Chlorella vulgaris using...

Chlorella vulgaris, a unicellular green microalga, has shown diverse applications in biotechnology. Particularly in microalgae genetic engineering, the genetic transformation of C. vulgaris offers a sustainable and cost-effective approach to producing recombinant proteins. Despite this, the thick cell wall of C. vulgaris poses challenges for efficient genetic transformation. This current study presents a detailed electrotransformation protocol of C. vulgaris using the pCAMBIA1303 vector, with validated transgene stability across generations.

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. Stability of the transformed C. vulgaris was also assessed across five generations.

Positive amplifications of the pCAMBIA1303 in the PCR and RT-PCR assays were indicative of the success of the transformation procedure of C. vulgaris. DNA sequencing was then performed to validate the identity of these amplicons. Furthermore, the stability assessment demonstrated persistent maintenance of transformed C. vulgaris cultures across five generations, as evidenced by reproducible PCR amplification.

The 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.