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Our wetlab team split their work across two different labs. The life science lab (LSL), where we had access to a programmable growth chamber, CO2 and our REE salts, and the Zeeman Lab where we had access to some advice from a PHD student working on C.reinhardtii and an authorisation for doing gene editing. Therefore, our GMOs stayed in the Zeeman Lab, while the investigation of growth conditions and REE absorbance happened in the LSL.

Zeeman Lab :

We tried to assemble our huge plasmid into a bacterial vector as a first step and to use for cloning. For this we first tried to assemble it without a vector to introduce it later, so that we would have the flexibility of having a reserve of an already transformation-ready vector and a backup in our bacteria. This was however very heavy on experimental steps and also caused problems about the location of the cut for the vector introduction.

Then we changed the plan and adapted the plasmid to do one big golden gate assembly that would put together the whole plasmid with a bacterial vector. The conclusion drawn was that the bacterial stock is crucial and that the vector can be cut off quite easily by exonucleases if designed properly. For this golden gate assembly to work we had to do a nuclease digestion of one of our parts to introduce it in a different part of the plasmid, reinserting the Type II S cut site by running a PCR with an overhang. This overhang allowed us to append the needed sequence on our digested parts for the following steps.

This golden gate assembly ran into multiple problems as the separation of appended and digested parts did not yield a pure product under gel electrophoresis. Gel extraction allowed us to still isolate our product which was used for the assembly. Bacterial transformation was successful, confirmed by the expression of mCherry from the bacterial backbone, however after DNA purification and isolation, the sequencing showed that the plasmid assembly was not complete and some of our parts were missing in the final product.

The whole workflow has been repeated a few times but with no successful final assembly. A final sequencing of our starting material was then done.

Zeeman Lab Protocol PDFs

Life science lab :

We made some specialised growing medium for C.reinhardtii (TAP Medium) and investigated the optimal growing conditions for growth. In a specialised growth room we tested different humidities and day/night cycles to find the sweet spot for biomass production in our medium. Then we tried to generate autolysin of our culture. To do this the algae have to be deprived of nitrogen for gamete formation, which additionally induces the production of autolysin. The cultures were subjected to the TAP-N medium to induce gamete formation. While mating the algae cells release autolysin which can be harvested by centrifugation after ensuring high mating activity under the microscope. Usually, for autolysin production, specialized strains are used that show high mating activity and lowered cell death upon nitrogen depletion. With the first trials, this was confirmed to be a source for errors, as our strains were not ideally suited to extract autolysin at a high enough rate.

In a second phase another growth medium was tested for growth with CO2 as only carbon source. The importance of this medium comes from the fact that it limits the risk of contamination, as most bacteria cannot grow in these conditions. This being the medium that will be used in our bioreactor, we need to understand how our algae behaves in these new environment.

It is also in these conditions that the wild type absorption of rare earth elements in algae was investigated. For this the phosphate precipitation of REE has to be avoided and this goes through a primary growth of our algae in the typical CO2 medium, followed by a transfer in a phosphate deprived medium that contains the REE. In addition to reducing the risk of contamination, the CO2 medium helps us to create an acidic environment that keeps the REE in solution. After a given growth time in this medium the algae is processed for analysis and the dry weight REE content is analysed. This data and the analysis of the wild type REE bioaccumulation process is crucial to understand the way our transformed organism will perform, and to highlight the advantages of our method.

Introduction

This protocol describes how to prepare Px6 Medium, a specialized culture medium designed for CO₂-dependent growth. It consists of three main stock solutions:

25× Salt Solution

500× Phosphate Solution

333× Trace Elements

The protocol includes the preparation of these stock solutions, the final medium assembly, sterilization, and handling instructions.

Materials

Component Amount (Stock) Notes/Concentration
A) 25× Salt Solution (100 mL)
Prepare 100 mL (use 40 mL per 1 L medium)
Sodium nitrate (NaNO₃) 9.56 g
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) 1.48 g
Calcium chloride dihydrate (CaCl₂·2H₂O) 0.44 g
Ultrapure water up to 100 mL
B) 500× Phosphate Solution (20 mL)
Prepare 20 mL (use 2 mL per 1 L medium)
Dipotassium hydrogen phosphate (K₂HPO₄) 7.664 g
Potassium dihydrogen phosphate (KH₂PO₄) 2.450 g
Ultrapure water up to 20 mL
C) Trace Elements (333×, 30 mL each)
Use 3 mL of each per 1 L final medium
Pre-Stocks:
- Disodium EDTA dihydrate (EDTA-Na₂·2H₂O) 4.65 g / 100 mL (Pre-1) 125 mM, adjust pH to 8 with KOH
- Potassium hydroxide (KOH) ~1.7 g For pH adjustment of Pre-1
- Sodium selenite (Na₂SeO₃) 0.173 g / 100 mL (Pre-3) 1 mM
- Sodium molybdate (Na₂MoO₄) 0.242 g (dihydrate) or 0.206 g (anhydrate) / 100 mL 10 mM
Trace Element Stocks (1000×, 10 mL each, dilute 1:3 to 333×)
TE1 - EDTA-Na₂ 2.00 mL Pre-1 + water to 10 mL Dilute 1:3 to 30 mL
TE2 - Molybdenum (Mo) 0.20 mL Na₂MoO₄ + water to 10 mL Dilute 1:3 to 30 mL
TE3 - Selenium (Se) 1.00 mL Pre-3 + water to 10 mL Dilute 1:3 to 30 mL
TE4 - Zinc·EDTA 7.2 mg Zinc sulfate heptahydrate (ZnSO₄·7H₂O) + 220 µL Pre-1 + water to 10 mL Dilute 1:3 to 30 mL
TE5 - Manganese·EDTA 11.9 mg Manganese chloride tetrahydrate (MnCl₂·4H₂O) + 480 µL Pre-1 + water to 10 mL Dilute 1:3 to 30 mL
TE6 - Iron·EDTA 82 mg EDTA-Na₂ + 23.2 mg Sodium carbonate (Na₂CO₃) + 54 mg Ferric chloride hexahydrate (FeCl₃·6H₂O) + water to 10 mL Dilute 1:3 to 30 mL; add FeCl₃ last
TE7 - Copper·EDTA 3.4 mg Copper chloride dihydrate (CuCl₂·2H₂O) + 160 µL Pre-1 + water to 10 mL Dilute 1:3 to 30 mL

Procedure

  1. A) Prepare 25× Salt Solution (100 mL)

    1. Add approximately 70 mL ultrapure water to a clean container.
    2. Dissolve the following compounds one by one:
      • 9.56 g Sodium nitrate (NaNO₃)
      • 1.48 g Magnesium sulfate heptahydrate (MgSO₄·7H₂O)
      • 0.44 g Calcium chloride dihydrate (CaCl₂·2H₂O)
    3. Adjust the volume to 100 mL with ultrapure water.
    4. Sterilize by filtering through a 0.22 µm membrane or autoclaving in a suitable container.
    5. Label and store properly.

  2. B) Prepare 500× Phosphate Solution (20 mL)

    1. Add approximately 12–15 mL ultrapure water into a container.
    2. Dissolve:
      • 7.664 g Dipotassium hydrogen phosphate (K₂HPO₄)
      • 2.450 g Potassium dihydrogen phosphate (KH₂PO₄)
    3. Adjust volume to 20 mL with ultrapure water.
    4. Sterilize by filtering through a 0.22 µm membrane.
    5. Label and store.

  3. C) Prepare Trace Elements Pre-Stocks and Stocks

    1. Prepare Pre-Stocks:
      • Pre-1 (Disodium EDTA, 125 mM): Dissolve 4.65 g EDTA-Na₂·2H₂O in ~80 mL ultrapure water, adjust pH to 8 using ~1.7 g potassium hydroxide (KOH), then fill up to 100 mL with ultrapure water.
      • Pre-3 (Sodium selenite, 1 mM): Dissolve 0.173 g sodium selenite in 100 mL ultrapure water.
      • Sodium molybdate (10 mM): Dissolve 0.242 g (dihydrate) or 0.206 g (anhydrate) in 100 mL ultrapure water.
    2. Filter sterilize all Pre-Stocks and store in a cool, dark place.
    3. Trace Element Stocks (1000×, 10 mL each, dilute 1:3 to 333×):
      • Dilute each stock 1:3 with ultrapure water to obtain 333× concentration (final volume 30 mL).
      • Store sterile, cool, and protected from light (especially the Fe·EDTA stock).

  4. D) Final Medium Preparation (1 L)

    1. In a sterile 1 L bottle, add:
      • Approximately 900 mL ultrapure water
      • 40 mL 25× Salt Solution
      • 3 mL each of the seven 333× Trace Element stocks (TE1 to TE7), total 21 mL
    2. Mix well.
    3. Adjust the volume to 1 L with ultrapure water.
    4. Sterilize by autoclaving at 121 °C for 15–20 minutes.
    5. After cooling to room temperature under sterile conditions, add 2 mL 500× Phosphate Solution.
    6. Mix gently; expect a final pH around 7.1.
    7. The medium is now ready for use.

(Reference: ScienceDirect Article - https://www.sciencedirect.com/science/article/pii/S0269749125005433?via%3Dihub)

Materials

  • Chlamydomonas reinhardtii strain CC-125
  • TAP agar plates (sterilized)
  • 6×P culture medium (with phosphate)
  • Incubator (100 rpm, 20 °C, 16 h light/8 h dark cycle)

Culture Procedure

  1. Maintain algae on sterilized TAP agar plates.
  2. Transfer a small pellet of algae into 50 mL of 6×P medium in a flask.
  3. Incubate at 20 °C, shaking at 100 rpm, with a 16 h light / 8 h dark cycle for 2–3 days.
  4. Dilute culture into 200 mL fresh 6×P medium and grow for an additional 2 days until mid-exponential phase (~2 × 10⁶ cells/mL).
  5. Harvest algae by centrifuging at 3000 rpm (1882×g) for 3 minutes.
  6. Wash pellet twice with 6×P medium without phosphate and resuspend for biouptake experiments.

Biouptake Experiment

Condition REE Present REE Concentration Description
Control None 0 mg/L No added REE
REE-Cocktail Ce + Gd + Er 3 × 100 mg/L Pure REE mixture
Red Mud Ce + Gd + Er 3 × 100 mg/L Simulated Red Mud extract
Time (Days) Time (Hours) Label
0 0 h T₀
0.5 12 h T₁₂h
1.5 36 h T₃₆h
3 72 h T₇₂h

Sample Preparation and Washing

  1. Sample Collection

    • Collect the Mass of each falcon tube to determine later the biomass
    • Transfer algal culture into falcon tube.
    • Centrifuge at 3000 × g for 10 minutes.
    • Remove supernatant

  2. Washing Steps

    • Resuspend algal pellet in 10ml of ammonium nitrate washing solution (6g/L) .
    • Centrifuge at 3000 × g for 10 minutes.
    • After centrifugation, transfer supernatant (wash solution) to a falcon tube to determine the adsorbed REE concentration

  3. Measurement

    • Resuspend pellet in 10 mL washing solution
    • Measure the Optical Density (OD₇₅₀) of resuspended algal samples (use the washing solution as blank).
    • Dilute samples if OD exceeds linear range
    • Centrifuge at 3000 × g for 10 minutes. And discard the supernatant

  4. Drying (Optional for Gravimetric Analysis)

    • Dry samples in oven (70°C)

Replication and Controls

  • Perform experiments in technical triplicates
  • Repeat experiment on at least one additional day with fresh algal cultures (biological replicates).