1. The mediums and supplements for microalgae transformation are obtained from Kisan Bio, South Korea.

1. 1. Supplement Luria Broth with 5 mM glucose, 50 mg/L kanamycin
2. 2. Inoculate Agrobacterium. Tumefaciens (KCTC 12031) single colonies in 10mL of Luria Broth
3. 3. Under constant agitation at 200 rpm on a rotary shaker at 30°C, culture overnight in the dark
4. 4. Inoculate 5mL of overnight culture from step 3 in 50mL of same medium
5. 5. Under constant agitation at 200 rpm at 27°C, culture until OD600 = 0.8–1.2 in the dark
6. 6. Centrifuge to harvest bacterial culture
7. 7. Wash with induction medium (BG11 + 100 μM acetosyringone, pH 5.6) and dilute to OD600 = 0.5
8. 8. Pre-culture total of 5 × 106 Chlorella cells from a log-phase culture (OD600 = 0.5–1.0) on BG11 solidified with 1.5% (w/v) bacto-agar at 25°C for 5 days
9. 9. Harvest bacterial culture with induction medium
10. 10. Mix algal pellet with 200 μL bacterial suspension
11. 11. Plate on induction medium solidified with 1.5% (w/v) bacto-agar
12. 12. Co-cultivate algae with bacteria (3 days, in dark, 25°C)
13. 13. Harvest cells with 7mL of BG11 supplemented with 500 mg/L cefotaxime and 50 mg/L kanamycin to eliminate Agrobacterium (2 days, in dark, 25°C)
14. 14. Measure eGFP expression
15. 15. Plate the remaining cells on selective media with 50 mg/L kanamycin and 500 mg/L cefotaxime
16. 16. Incubate transformed cells in dark before exposure to light (2 days, 25°C)
17. 17. Grow cells on LB agar plates to detect any Agrobacterium (7 days in dark, 25°C)

1. 1. Inoculate transformed colony in 1mL BG11 selective media and incubate at 25°C under constant illumination (50 μmol photons/m2/s) and constant agitation at 100–150 rpm on a rotary shaker, culture for 7 days
2. 2. Transfer 100 μL cells to wells of a black, clear bottom 96-well plate
3. 3. Set up microplate reader (ThermoFisher Scientific, USA) as table 1
4. 4. Measure fluorescence using BG11 medium as blank and wild type cells as negative control
5. 5. Repeat steps 5-7 with supernatant in the Deep-well plate obtained by centrifugation at 3000 ×g for 10 min
6. 6. The fluorescent signal is normalized by OD600.

1. The mediums and supplements for microalgae transformation are obtained from Kisan Bio, South Korea.

1. - eGFP fluorescence was visualized by using a narrow-band filter (Olympus U-FBNA, excitation filter 470–495 nm, barrier filter 510–550 nm).
2. - Chlorophyll autofluorescence was observed using a wide-band filter (Olympus U-FBW, excitation filter 460–495 nm, barrier filter 510 nm)

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