Simple Electroporation of Chlamydomonas reinhardtii Strains With an Intact Cell Wall

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

A. Purification of the DNA cassette

1. Digest a sufficient quantity of plasmid DNA (typically 5–10 µg) with appropriate restriction enzymes (typically BbsI or BsaI for MoClo plasmids [3,9]) to isolate the desired cassette. If the size of the cassette is close to that of the vector backbone fragment, additionally cut the latter with another restriction enzyme to ensure good separation. It is also possible to linearize the plasmid without purifying the expression cassette on the gel [6].

2. Incubate the reaction for 3 h to overnight at 37 °C.

3. Separate the cassette by agarose gel electrophoresis and isolate it by gel purification.

4. Measure the DNA concentration with a NanoDrop spectrophotometer. Typical recovery yield is 60%–80%.

B. Chlamydomonas culture and harvesting

1. From this point on, all steps must be performed under sterile conditions, for example, in a laminar flow hood.

2. Inoculate Chlamydomonas reinhardtii culture in liquid TAP. Adjust the volume depending on the number of transformations; the minimum is 25 mL per transformation if cells are harvested at 2 × 10⁶ cells/mL.

3. Incubate the culture at 25 °C with shaking (~120 rpm) and constant light (50–100 μmol photons m^-2·s^-1) until it reaches a concentration of 2–5 × 10⁶ cells/mL, which takes 2–3 days. Cell concentration can be measured with a hemocytometer or a cell counter. Using optical density has not specifically been tested, but may be used following the methods developed in [12]. It is recommended to maintain the culture under 5 × 10⁶ cells/mL by dilution when necessary. This protocol works with stationary phase cultures as well, but efficiency might be slightly reduced.

C. Electroporation and recovery

1. In 50 mL tubes, harvest 50 × 10⁶ cells per transformation by centrifugation (2,500× g, room temperature, 5 min). Calculate the volume to harvest as follows: V = 5 × 10⁷/concentration. For example, if the culture concentration is 2 × 10⁶ cells/mL, the volume is V = 50 × 10⁶/2 × 10⁶ = 25 mL per transformation.

2. Remove all the supernatant and resuspend the cell pellet in CHES buffer to a final concentration of 2 × 10⁸ cells/mL.

3. In a 4-mm-gapped electroporation cuvette, mix 250 μL of the cell suspension with 50–500 ng of the DNA cassette. 200 ng of DNA is usually sufficient, but the quantity may need to be adjusted depending on the expression cassette. Several samples can be prepared at the same time, as incubation time has little effect on the transformation efficiency.

4. Right before electroporation, gently flick the cuvette to resuspend the cells. If bubbles are present, tap the cuvette on the bench to eliminate them.

5. Proceed to the electroporation at 600 V, 25 µF, and 1,000 Ω. Electroporation also works with Bio-Rad systems that lack resistance control.

Note: The measured pulse time has been between 10 and 15 ms for successful transformations.

6. Transfer the cells to a 15 mL tube filled with approximately 10 mL of TS40 and rinse the cuvette twice with 500 μL of TS40 to recover all the cells.

7. Incubate the tubes at 25 °C and a light intensity of ~50 μmol photons m^-2·s^-1 horizontally on a rotary shaker at ~120 rpm (or in a tube rotator at moderate speed) for 12–16 h.

Note: Do not skip this recovery step, as it is essential for the cells to express the resistance gene.

D. Selection of transformants

1. Harvest cells by centrifugation (2,500× g, room temperature, 5 min).

2. Pour off almost all the supernatant, leaving a volume of ~300–500 μL.

3. Resuspend the cell pellet in the remaining liquid, spread on TAP agar plates supplemented with the relevant antibiotic, and dry.

4. Seal the plates with parafilm or breathable tape and incubate at 25 °C and 50–100 μmol photons m^-2·s^-1.

5. Colonies should appear after 4–6 days (Figure 1).

Figure 1. Typical successful electroporation. Pictures were taken 0 and 6 days after plating. See Figure 2 legend for more details.

Validation of protocol

This protocol or a very similar one was successfully used in previous publications [5–8]. Here, it was further validated by comparing it with the “Crozet” protocol [4] and the MAX Efficiency commercial kit. Our protocol produced comparable results in terms of number of transformants or transgene expression, as estimated here by mVenus fluorescence (Figure 2). Transformant characterization depends on the goal of the experiment; it may include detection or localization of the insertion locus by PCR [4,13] or detection of the produced protein [6].

Figure 2. Validation of the electroporation protocol in a wild-type strain with an intact cell wall. (A) The plasmid pFCM-010 (pAR-NPTII-tRbcS2-pAβSAP-mVenus-tPsaD, Dataset S1) was built with the Chlamydomonas MoClo toolkit [3,14]. 500 ng of purified cassette were transformed into the 4A- wild type (with an intact cell wall) with our simplified protocol, the “Crozet” protocol [3,4], or the MAX Efficiency Transformation Reagent for Algae kit (Invitrogen) and selected on TAP+kanamycin plates. The transformation efficiency of our protocol was estimated at 13.3 × 10³ transformants/μg of DNA or 134 transformants/10⁶ cells; that of the “Crozet” protocol was 7.0 × 10³ transformants/μg of DNA or 70 transformants/10⁶ cells. Note that the number of transformants may depend on the expression cassette. Pictures were taken after 6 days. (B) Fluorescence of mVenus in individual transformants generated with the three protocols. 96-well plates containing 200 μL of cell culture per well were measured with a Tecan Infinite M1000 PRO plate reader (excitation: 505 nm; emission: 530 nm) and normalized with chlorophyll fluorescence (excitation: 430 nm; emission: 655 nm).

Troubleshooting

Problem: Low transformation efficiency.

Possible cause: Saturated or unhealthy culture.

Solution: Repeat the experiment and check that the preculture contains mostly single cells. If the strain is motile, cells should be swimming. Also, check that the culture is not contaminated by plating cells on Luria broth.

Supplementary information

The following supporting information can be downloaded here:

1. Dataset S1. Plasmid map of pFCM-010.

Acknowledgments

Conceptualization, M.M., F.C.; Investigation, M.M., F.C., E.L., M.H.; Data Analysis, M.M., F.C., S.W., M.S., K.K.N.; Writing—Original Draft, M.M., F.C.; Writing—Review & Editing, M.M., F.C., E.L., M.H., S.W., M.S., K.K.N.; Funding acquisition, M.S., K.K.N.; Supervision, F.C., S.W., K.K.N.

We acknowledge Dr. Masayuki Onishi for developing the original version of the protocol and Dr. Olivier D. Caspari for providing it to us. This work was supported by the Howard Hughes Medical Institute, the BioComp 4.0 program (RPTU), and UC Berkeley Sponsored Projects for Undergraduate Research (SPUR) program. K.K.N. is an investigator of the Howard Hughes Medical Institute, and S.W. is supported by the U.S. Department of Energy, Office of Science, through the Photosynthetic Systems program in the Office of Basic Energy Sciences.

Competing interests

The authors declare no conflicts of interest.

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