Materials and reagents

Biological materials

1. Chlamydomonas reinhardtii, tested strains: CC-4051 (4A-), CC-5101 (T222⁺), CC-4425 (D66), CC-124, CC-4533, CC-5325 (available at the Chlamydomonas Resource Center, www.chlamycollection.org)

Note: This protocol does not work with true cell-wall-less strains like UVM4, for which glass bead transformation is recommended [9].

2. Expression plasmid for Chlamydomonas reinhardtii

Note: This protocol does not cover plasmid design and cloning, but we recommend using MoClo vectors [3,9] (available at the Chlamydomonas Resource Center, www.chlamycollection.org/product/moclo-toolkit).

Reagents

1. Tris base (tris(hydroxymethyl)aminomethane) (Fisher Scientific, catalog number: BP152-1)

2. Potassium phosphate dibasic (K₂HPO₄) (Fisher Scientific, catalog number: BP363-500)

3. Potassium phosphate monobasic (KH₂PO₄) (VWR, catalog number: EMD-PX1565-1)

4. Ammonium chloride (NH₄Cl) (VWR, catalog number: BDH9208-500G)

5. Magnesium sulphate heptahydrate (MgSO₄·7H₂O) (Fisher Scientific, catalog number: M63-500)

6. Calcium chloride dihydrate (CaCl₂·2H₂O) (Fisher Scientific, catalog number: C79-500)

7. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-Na₂) (Sigma-Aldrich, catalog number: E4884)

8. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P6310)

9. Ammonium molybdate tetrahydrate [(NH₄)₆Mo₇O₂₄] (Fisher Scientific, catalog number: A674-500)

10. Zinc sulfate heptahydrate (ZnSO₄·7H₂O) (Sigma-Aldrich, catalog number: Z4750)

11. Manganese(II) chloride tetrahydrate (MnCl₂·4H₂O) (Sigma-Aldrich, catalog number: M3634)

12. Iron(III) chloride hexahydrate (FeCl₃·6H₂O) (Sigma-Aldrich, catalog number: F2877)

13. Sodium carbonate (Na₂CO₃) (Sigma-Aldrich, catalog number: BP357-1)

14. Copper(II) chloride dihydrate (CuCl₂·2H₂O) (Sigma-Aldrich, catalog number: 307483)

15. Glacial acetic acid (CH₃COOH) (VWR, catalog number: EMD-AX0073-9)

16. Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144SI-212)

17. Agar (VWR, catalog number: 90000-762)

18. Blasticidin S hydrochloride (Fisher Scientific, catalog number: 50-712-729)

19. Zeocin (Thermo Fisher Scientific, catalog number: R25001)

20. Hygromycin B (Thermo Fisher Scientific, catalog number: 10687010)

21. Kanamycin monosulfate (TCI, catalog number: K0047)

22. Paromomycin sulfate (Sigma-Aldrich, catalog number: P5057)

23. Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S4014)

24. Nourseothricin sulfate (GoldBio, catalog number: N-500)

25. N-cyclohexyl-2-aminoethanesulfonic acid (CHES) (Sigma-Aldrich, catalog number: C2885)

26. Sucrose (Millipore, catalog number: 573113)

27. Sorbitol (Thermo Fisher Scientific, catalog number: 036404.36)

Solutions

1. 1 M Tris base (see Recipes)

2. Phosphate buffer II (see Recipes)

3. Solution A (see Recipes)

4. Kropat’s stock solutions (see Recipes)

a. 125 mM EDTA-Na₂ stock solution

b. 285 μM (NH₄)₆Mo₇O₂₄

5. Kropat’s trace elements solutions (1,000×-concentrated) (see Recipes)

a. 25 mM EDTA-Na₂

b. 28.5 μM (NH₄)₆Mo₇O₂₄

c. Zn-EDTA (2.5 mM ZnSO₄, 2.75 mM EDTA-Na₂)

d. Mn-EDTA (6 mM MnCl₂, 6 mM EDTA-Na₂)

e. Fe-EDTA (20 mL FeCl₃, 22 mM EDTA-Na₂, 22 mM Na₂CO₃)

f. Cu-EDTA (2 mM CuCl₂, 2 mM EDTA-Na₂)

6. Liquid TAP (see Recipes)

7. TAP plates with antibiotics (see Recipes)

8. CHES buffer (see Recipes)

9. TS40 (40 mM sucrose in TAP) (see Recipes)

Recipes

1. 1 M Tris base

a. Weigh 121.14 g of Tris.

b. Dissolve in 800 mL of distilled water.

c. Adjust the volume to 1,000 mL with distilled water.

2. Phosphate buffer II

a. Weigh 10.8 g of K₂HPO₄ and 5.6 g of KH₂PO₄.

b. Dissolve one by one in 80 mL of distilled water.

c. Adjust the volume to 100 mL with distilled water.

3. Solution A

a. Weigh 20.0 g of NH₄Cl, 5.0 g of MgSO₄·7H₂O, and 2.5 g of CaCl₂·2H₂O.

b. Dissolve one by one in 400 mL of distilled water.

c. Adjust the volume to 500 mL with distilled water.

4. Kropat’s stock solutions

Kropat’s trace elements solutions preparation is based on the following protocol www.chlamycollection.org/content/uploads/2022/05/Kropats-Trace-Elements-safety-update.pdf [10]. Selenite is omitted as it is not necessary for this protocol. If sodium selenite is used, it should be handled with care, as it is toxic.

a. 125 mM EDTA-Na₂ stock solution

i. Dissolve 11.63 g of EDTA-Na₂ in 250 mL of distilled water.

ii. Adjust to pH 8.0 with KOH pellets.

iii. Adjust the volume to 300 mL with distilled water.

b. 285 μM (NH₄)₆Mo₇O₂₄

i. Dissolve 88.0 mg of (NH₄)₆Mo₇O₂₄ in 200 mL of distilled water.

ii. Adjust the volume to 250 mL with distilled water.

5. Kropat’s trace elements solutions (1,000×-concentrated)

a. 25 mM EDTA-Na₂

Mix 50 mL of 125 mM EDTA-Na₂ and 200 mL of distilled water.

b. 28.5 μM (NH₄)₆Mo₇O₂₄

Mix 25 mL of 285 μM (NH₄)₆Mo₇O₂₄ and 225 mL of distilled water.

c. Zn-EDTA (2.5 mM ZnSO₄, 2.75 mM EDTA-Na₂)

i. Dissolve 180.0 mg of ZnSO₄.7H₂O in 200 mL of distilled water.

ii. Add 5.5 mL of 125 mM EDTA-Na₂.

iii. Adjust the volume to 250 mL with distilled water.

d. Mn-EDTA (6 mM MnCl₂, 6 mM EDTA-Na₂)

i. Dissolve 297.0 mg of MnCl₂.4H₂O in 200 mL of distilled water.

ii. Add 12 mL of 125 mM EDTA-Na₂.

iii. Adjust the volume to 250 mL with distilled water.

e. Fe-EDTA (20 mL FeCl₃, 22 mM EDTA-Na₂, 22 mM Na₂CO₃)

i. Dissolve 2.05 g of EDTA-Na₂ and 0.58 g of Na₂CO₃ in 200 mL of distilled water.

ii. Add and dissolve 1.35 g of FeCl₃.6H₂O.

iii. Adjust the volume to 250 mL with distilled water.

f. Cu-EDTA (2 mM CuCl₂, 2 mM EDTA-Na₂)

i. Dissolve 85.0 mg of CuCl₂.2H₂O in 200 mL of distilled water.

ii. Add 4 mL of 125 mM EDTA-Na₂.

iii. Adjust the volume to 250 mL with distilled water.

6. Liquid TAP

a. Add the following solutions to 900 mL of distilled water: 20 mL of 1 M Tris base, 1 mL of phosphate buffer II, 10 mL of solution A, 1 mL each of Kropat’s trace elements solutions [25 mM EDTA-Na₂, 28.5 μM (NH₄)₆Mo₇O₂₄, Zn-EDTA, Mn-EDTA, Fe-EDTA, and Cu-EDTA], and 1 mL of glacial acetic acid.

b. Adjust pH to 7.0 with concentrated HCl.

c. Adjust the volume to 1,000 mL with distilled water.

d. Autoclave for 30–40 min.

7. TAP plates with antibiotics

a. Prepare TAP as in Recipe 6 (without the autoclaving step).

b. Add 15 g of agar per liter.

c. Autoclave for 30–40 min.

d. Mix well and let the solution reach a temperature of 40–50 °C.

e. Add relevant antibiotic at the following final concentration: blasticidin S (50 mg/L), zeocin (15 mg/L), hygromycin B (20 mg/L), kanamycin (50 mg/L), paromomycin (20 mg/L), spectinomycin (100 mg/L), or nourseothricin (10 mg/L) [4,11].

f. Mix well and pour plates.

g. Store at 4–8 °C for up to 2 months.

8. CHES buffer (10 mM CHES pH 9.25, 40 mM sucrose, 10 mM sorbitol)

a. Weigh 1 g of CHES, 6.84 g of sucrose, and 0.91 g of sorbitol.

b. Dissolve one by one in 400 mL of distilled water.

c. Adjust pH to 9.25.

d. Bring to 500 mL with distilled water.

e. Filter sterilize.

9. TS40 (40 mM sucrose in TAP)

a. Dissolve 6.84 g of sucrose in 500 mL of TAP.

b. Filter sterilize.

Laboratory supplies

1. Gel extraction kit (e.g., Monarch Spin DNA, New England Biolabs, catalog number: T1120S)

2. Bottle top 0.45 μm filers (e.g., Nalgene Rapid flow, Thermo Scientific, catalog number: 565-0010)

3. 4-mm-gapped electroporation cuvettes (USA Scientific, catalog number: 9104-6050)

4. Parafilm or breathable tape (3M, catalog number: Vent Tape 394)

Equipment

1. Gene Pulser with Pulse Controller (Bio-Rad, catalog number: 1652662)

2. NanoDrop spectrophotometer (ThermoFisher Scientific, model: NanoDrop Lite Plus)

3. Hemocytometer or cell counter (e.g., Beckman Coulter, model: Multisizer 4e Coulter Counter)

4. Growth chamber with light and temperature control (e.g., Percival, Conviron, BioChamber) and a rotary shaker

Note: A simpler culture setup, such as hanging fluorescent bulbs over a shaker at room temperature, has yielded similar results.

5. Standard laboratory equipment (e.g., pipettes, centrifuge, microcentrifuge, laminar flow hood, glass beakers, 15- and 50-mL sterile tubes)

Procedure