Growth Measurement of Chlamydomonas reinhardtii CC-124 and UVM-4
Algal growth was monitored following the method described by Chioccioli, Hankamer, and Ross (2014) with adjustments. Optical density at 750 nm (OD₇₅₀) was measured daily using a spectrophotometer. When cultures reached an OD₇₅₀ of 0.4–0.8, corresponding approximately to the mid-logarithmic growth phase, cells were harvested for subsequent experiments. This procedure was applied to both CC-124 and UVM-4 strains.
Measurement to Determine the Optimal Antibiotic Screening Concentration
To establish the appropriate resistance screening pressure for transformants, hygromycin and paromomycin were tested at concentrations of 5, 10, and 20 μg/mL. Wild-type/negative control cells and transformed strains were cultured under identical antibiotic conditions, and their growth was compared. The results indicated: (1) all three concentrations effectively inhibited the growth of negative control cells; and (2) the growth of transformants decreased markedly as the antibiotic concentration increased.
Measurement to Assess the Target Gene Integration: PCR and qPCR
Conventional PCR was employed to verify integration of the endogenous gene CPN60C in the transformed algal cells. As CPN60C is an endogenous gene of C. reinhardtii, quantitative PCR (qPCR) was subsequently performed to determine whether expression of the target gene was specifically upregulated in transformants. Detailed procedures and parameters for both PCR and qPCR are provided in the “Experiments” section.
Measurement of Growth under Heat Stress
Due to experimental limitations, daily destructive OD measurements could not be performed to assess growth phenotypes of transgenic and control strains under heat stress. Instead, we monitored colony yellowing at a common starting growth point as a preliminary indicator of phenotypic differences between strains, which provided the basis for subsequent experiments.
For further details regarding measurement procedures, please refer to the “Experiments” and “Results” sections.
References
- Chioccioli, M., Hankamer, B., & Ross, I. L. (2014). Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris. PLOS ONE, 9(5), e97269.