Optimizing transformation parameters and selectable conditions to develop a synthetic biology platform in microalga Chlorella K13

The photosynthetic microalga Chlorella sp. is a promising platform for biotechnological applications due to its remarkable capacity to convert CO2 into value-added products. In this study, we aimed to optimize transformation parameters and selection conditions to establish a synthetic biology platform in Chlorella K13, a strain isolated in Thailand. The cell growth profile was analyzed to determine the logarithmic (log) phase, which represented the optimal stage for preparing competent cells for transformation. The highest growth was observed within the first 24 hours. The selectable marker system was identified. Antibiotic sensitivity tests revealed that Chlorella K13 is highly sensitive to zeocin, with a minimum inhibitory concentration (MIC) of 50 μg/mL. This result indicates that the Sh ble gene, which confers bleomycin resistance, is suitable as a positive selection marker. In addition, the strain exhibited sensitivity to potassium chlorate (KClO3) at 300 mM. Thus, this finding supports the use of the nitrate reductase (NR) gene as a negative selectable marker through NR knockout and chlorate resistance screening. To optimize DNA delivery, two electroporation buffers with different salt concentrations were determined under various field strengths. The low-salt buffer provided better results than the high-salt buffer by preventing electrical arcing under high-field strength conditions. Altogether, these results provided foundational conditions for establishing a robust synthetic biology platform in Chlorella K13.