Microalgae Transformation (Cha et al., 2012)
-
The mediums and supplements for microalgae transformation are
obtained from Kisan Bio, South Korea.
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1. Supplement Luria Broth with 5 mM glucose, 50 mg/L kanamycin
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2. Inoculate Agrobacterium. Tumefaciens (KCTC 12031) single colonies
in 10mL of Luria Broth
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3. Under constant agitation at 200 rpm on a rotary shaker at 30°C,
culture overnight in the dark
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4. Inoculate 5mL of overnight culture from step 3 in 50mL of same
medium
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5. Under constant agitation at 200 rpm at 27°C, culture until OD600
= 0.8–1.2 in the dark
- 6. Centrifuge to harvest bacterial culture
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7. Wash with induction medium (BG11 + 100 μM acetosyringone, pH 5.6)
and dilute to OD600 = 0.5
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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. Harvest bacterial culture with induction medium
- 10. Mix algal pellet with 200 μL bacterial suspension
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11. Plate on induction medium solidified with 1.5% (w/v) bacto-agar
- 12. Co-cultivate algae with bacteria (3 days, in dark, 25°C)
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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. Measure eGFP expression
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15. Plate the remaining cells on selective media with 50 mg/L
kanamycin and 500 mg/L cefotaxime
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16. Incubate transformed cells in dark before exposure to light (2
days, 25°C)
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17. Grow cells on LB agar plates to detect any Agrobacterium (7 days
in dark, 25°C)
Fluorescence microplate reader assay (Molino et al., 2018)
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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
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2. Transfer 100 μL cells to wells of a black, clear bottom 96-well
plate
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3. Set up microplate reader (ThermoFisher Scientific, USA) as table
1
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4. Measure fluorescence using BG11 medium as blank and wild type
cells as negative control
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5. Repeat steps 5-7 with supernatant in the Deep-well plate obtained
by centrifugation at 3000 ×g for 10 min
- 6. The fluorescent signal is normalized by OD600.
Fluorescence microscopic observation of EGFP expression
(Yang et al., 2015)
-
The mediums and supplements for microalgae transformation are
obtained from Kisan Bio, South Korea.
-
- eGFP fluorescence was visualized by using a narrow-band filter
(Olympus U-FBNA, excitation filter 470–495 nm, barrier filter
510–550 nm).
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- Chlorophyll autofluorescence was observed using a wide-band filter
(Olympus U-FBW, excitation filter 460–495 nm, barrier filter 510 nm)
