iGEM 2025 — Team eth-zurich

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

## Notebook Entries

---

### 23.06–27.06

We first installed ourselves in the Pr. Zeeman lab and started by making some TAP-medium from C.reinhardtii following our protocol.

The first algal cultures were started and transferred to the LSL lab where we also made additional TAP-medium.

### 30.06–04.07

While waiting for our cassette fragments we made some additional algal cultures and started setting up the controlled temperature, light, and humidity room we had at our disposition.

We further investigated the optimal growth conditions of C.reinhardtii in this room.

### 07.07–11.07

The DNA material arrived on Monday and was first thawed and dissolved in nuclease free water.

Because the concentration measured by nanodrop was too low, we concentrated the fragment solution using a centrifugal evaporator.

The prepared samples were used to start assembling our cassette via Golden Gate assembly from the different fragments we designed.

The DNA of our Golden Gate assembly was used for transformation of E.coli.

### 14.07–18.07

The transformation failed, and the dry lab started designing a corrected version of the cassette.

A large amount of new cultures were started in the LSL lab to prepare for the autolysin extraction experiment.

Some first liquid culture media cultures were started and the autolysin extraction was attempted but failed due to a technical failure of the centrifuge.

An attempt to recycle used solid media algal culture was started.

### 21.07–25.07

We received the primers used for adding an overhang on our HDR arms in the cassette, as well as a backbone pTU1-A-RFD to replace our own designed backbone.

The Gibson assembly started by adding the overhangs to the digested fragment that contains the HDR arms. The product of that PCR was worked up by gel electrophoresis and was then further processed by gel extraction.

The extracted new HDR arms were used for a new Golden Gate assembly with our backbone and a new transformation was attempted.

The control with only the backbone was positive and some transformed bacteria were also present in the Golden Gate product transformation.

In the LSL the media recycling attempt failed and only gave very sparsely populated algae colonies. Therefore new plates were prepared for a future autolysin extraction.

### 28.07–01.08

A single colony of the transformed bacteria was prepared by miniprep and sent to sequencing for testing.

In the LSL a new autolysin extraction was performed and the sample was frozen.

### 04.08–08.08

Following the negative results of the new transformation plasmid, the HDR arms with overlap were sent to sequencing to test whether the Gibson assembly was successful.

In the LSL the activity of the extracted autolysin sample was investigated by testing its effect on healthy alga cell wall. The test was negative.

### 11.08–15.08

No major updates recorded.

### 18.08–22.08

Following the negative results of the HDR arms sequencing, our fragments were all sent to sequencing to test our starting material.

The results did not seem to show any problems with the starting material.

### 25.08–29.08

No major updates recorded.

### 01.09–05.09

No major updates recorded.

### 08.09–12.09

No major updates recorded.

### 15.09–19.09

In the LSL lab the first tests with culture media only containing CO2 as carbon source were conducted with different setups.

The trials covered automated syringe pressing, a balloon setup, and then a controlled balloon flow with clamping.

### 22.09–26.09

The first test with phosphate deprivation to test for REE salt binding was prepared. The REE salts were dissolved and a culture media was formed with them for treatment after the phosphate rich media.

### 29.09–03.10

The algae did not survive the three days in the phosphate poor buffer, so further analysis could not be done.

We repeated the transformation processes on E.coli with the new design of our cassette. Miniprep of the successfully transformed bacteria yielded a solution of our plasmid with a concentration of 198 ng/ul which was sent in for sequencing.

### 06.10–10.10

No major updates recorded.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

# Bioreactor Design

![](https://static.igem.wiki/teams/6022/engineering/cycles-hardware-v3.avif)Overview of the Bioreactor Design cycles

## Cycle 1 – Concept and Initial Design

![](https://static.igem.wiki/teams/6022/engineering/cychard-1.avif)

### Goal

Develop a modular, low-cost algal bioreactor with a working volume above 10 L. The reactor should be easily assembled
from off-the-shelf components and 3D-printed parts, with basic automation for sensing, control, and data logging.

### Design

We began by conceptualizing the overall mechanism and geometry of the reactor. The first 3D parts were designed,
including the main base and acrylic tube mounts. Early focus was placed on modularity and printability—parts that
could be printed on standard FDM printers and assembled without specialized tools.

### Build

The first 3D prints revealed several issues: slicing profiles were not optimized, and tolerances for the acrylic tubes
were too tight. The spacing had not been properly calculated, leading to poor fits.

### Learn

From this first iteration, we learned that clearance tolerances are critical for printed parts interfacing with
non-printed components. Tube slots were redesigned with an additional 2 mm of space inside and outside diameters, and
slicing settings were refined for consistent wall thickness.

![](https://static.igem.wiki/teams/6022/engineering/engineering-3d-reaktor.avif)

Initial Design of the Bioreactor Base

## Cycle 2 – Structural Redesign and Mechanism Development

![](https://static.igem.wiki/teams/6022/engineering/cychard-2.avif)

### Goal

Create a stable structure and a reliable attachment mechanism between the base and upper frame.

### Design

Following the fit improvements, we designed the tripod stand and its connectors. The first attempt used a bayonet-type
locking mechanism to connect the base and upper structure.

### Build

Due to an error in the 3D model (a misaligned layer in the CAD file), the bayonet mechanism failed to engage properly.
Instead of reprinting the large base, we adapted by designing and printing replacement nodes. The faulty parts were
removed, and the new nodes were screwed into the old base, allowing reuse of large prints while restoring
functionality.

### Learn

This cycle reinforced the importance of designing for repairability and modular fixes. The modular node approach saved
time and material while enabling rapid iteration.

![](https://static.igem.wiki/teams/6022/engineering/engineering-assembly.avif)

First Print of Reactor Base

## Cycle 3 – LED and Lid Assembly

![](https://static.igem.wiki/teams/6022/engineering/cychard-3.avif)

### Goal

Integrate lighting and finalize enclosure geometry for a functional photobioreactor.

### Design

We designed the LED module, inner lid, and outer lid. The LEDs were essential for algal growth but initially too tight
in their mounts. The module was revised with proper spacing, and a holding structure was integrated into the inner lid
for better mechanical stability.

### Build

During assembly, we discovered that the LED strips were longer than the acrylic tubes, which caused alignment and
enclosure issues. To solve this, the lid design was modified to both cover the LED extensions and secure the modules
firmly to the top.

### Learn

LED housing must allow for thermal expansion and manufacturing variances in strip length. The lid redesign improved
accessibility, safety, and alignment.

![](https://static.igem.wiki/teams/6022/engineering/engineering-screenshot.avif)

Design of LED module

## Cycle 4 – Electronics Integration and Control

![](https://static.igem.wiki/teams/6022/engineering/cychard-4.avif)

### Goal

Add sensing, control, and data-logging components to automate the system.

### Design

We integrated sensors, control wiring, and a Raspberry Pi for process automation. Initially, there was no allocated
space for the Pi or its cabling within the main cap and rod assembly.

### Build

To solve this, we designed a dedicated Pi case with a custom mounting mechanism that screws directly onto the main
cap.
The wiring was routed through the structural rods, and software was configured for temperature control.

### Test

Electronics and software were bench-tested successfully. Due to safety rules at the Grand Jamboree, full wet testing
was not conducted because the system had not yet been verified as sterilizable for biological use.

### Learn

The electronics system functioned as expected. Future work will focus on sterilization compatibility, automated
filtration, and improved cable management.

![](https://static.igem.wiki/teams/6022/engineering/engineering-print.avif)

3D printers used for producing the parts

## Cycle 5 – Future Development

### Insights and Next Steps

**Filtration:** Nylon filters were found suitable for harvesting algae. The next
step is an automated filtration and wash
module to streamline biomass collection and cleaning.

**Automation:** Expanding control to include flow rate and nutrient dosing would
move the reactor closer to continuous
operation.

# Transformation of TFD-EE in C. reinhardtii

![](https://static.igem.wiki/teams/6022/engineering/cycles-wetlab.avif)Overview of the Plasmid Design cycles

## Cycle 1 - Initial Construct

![](https://static.igem.wiki/teams/6022/engineering/wetlab-1.avif)

### Design

Our aim was to transform the green microalga *Chlamydomonas reinhardtii* to enhance the bioaccumulation
of rare earth elements (REEs) by introducing the TFD-EE gene into the nuclear genome. Although *C.
reinhardtii* is well characterized and its genome has been sequenced [11], engineering the organism
remains challenging [3]. Historically, much previous work has focused on chloroplast engineering rather
than the nucleus [1,2]. In the nuclear genome, extensive transgene silencing [3] and a very low rate of
homologous recombination have led many groups to rely on non-homologous recombination despite the drawbacks
of random insertion [4].

To address this, we implemented both approaches and compared them in terms of transformation efficiency, extent of
gene silencing, and experimental effort. The TFD-EE gene encodes a de novo–designed REE-binding protein inspired by
the calmodulin-related lanthanide-binding protein lanmodulin [5,6]. This protein binds REE cations with very high
affinity, making it an attractive candidate for our project [5,7,8]. Moreover, because it is inspired by a naturally
occurring protein ultimately related to ubiquitous calmodulin, the likelihood of adverse interference with *C.
reinhardtii* metabolism is reduced [9].

Designing a reliable plasmid for overexpression of our gene of interest in *C. reinhardtii* was one of the first steps
of our iGEM journey. This proved more involved than simply assembling documented building blocks; species-specific
constraints had to be overcome. Each design required a trade-off between the simplicity needed for rapid progress and
the complexity required for robust expression.

To monitor expression in vivo, we fused a codon-optimized TFD gene to a reporter. The GFP-derived protein mVenus,
widely used in transformation experiments, was placed at the C-terminus of TFD and separated by a short GSSSG linker
[13,14]. A C-terminal Strep-tag on mVenus was added for possible protein purification. The APHVIII gene conferred
paromomycin resistance for selection [12]. To boost expression, RBCS2i and RBCS2i2 introns were inserted at intervals
within both genes [14].

Using the commercially available m\_Paro\_mVenus plasmid as a scaffold, we placed the TFD-mVenus fusion and APHVIII
under separate promoters. For TFD-mVenus, we used the well-validated HSP70A-RBCS2 fusion promoter [14]. The expression
cassette was flanked by 500-bp homology-directed repair (HDR) arms to enable precise knock-in into the genome of
*C. reinhardtii*.

We selected the SNRK2.2 locus as the homology locus to optimize selection. Knockout of this gene induces expression of
a sulfatase that cleaves the sulfate group of an indigo-derived dye supplied in the medium, releasing the dye. As a
result, mutant colonies stain blue and are easily distinguished from non-mutant colonies or colonies with insertions
elsewhere. Combined with antibiotic selection, this readout allows us to determine transformation efficiency and the
proportion of mutants generated via non-homologous versus homologous recombination.

We also considered using an auxotrophic growth marker and inserting our gene of interest into the marker’s sequence.
This would have acted as a form of kill switch, preventing engineered algae from surviving outside a designed
environment. However, given the convenience of the blue-dye assay and the added complexity of supplying a metabolite
continuously in the bioreactor, we set this option aside and relied on containment to minimize escape risk.

Our initial plan, PCR-based amplification of the plasmid, was abandoned after our doctoral advisor highlighted the
inherent inaccuracy of cloning large DNA fragments via PCR. We pivoted to bacterial cloning and designed a sixth
fragment to enable replication in *E. coli*.

Given the construct’s size and the practical challenges of handling large DNA molecules, we opted for a minimal
bacterial backbone comprising an origin of replication and an ampicillin resistance gene under its promoter. Because
the origin of replication downstream of the ampicillin resistance gene contained a long poly-A stretch, we initially
assumed a separate terminator would be unnecessary. This auxiliary sequence was inserted into a “safety restriction
site,” which we included in our initial design for further modifications, and could be excised using BbsI.

[![Assembly overview of the V1 plasmid](https://static.igem.wiki/teams/6022/engineering/v1.svg)](https://static.igem.wiki/teams/6022/engineering/v1.svg)

Figure 1: An illustration of version 1 of our designed plasmid.

### Build

The assembled design yielded a 5,950-bp plasmid containing all elements needed for expression in *C. reinhardtii*.
Owing to the construct’s complexity, especially the repeated sequences within the introns, we split the plasmid into
five fragments for ordering. Each fragment was flanked by BsaI sites for scarless Golden Gate assembly [15]. The
ligated plasmid could be linearized at an EcoRV site positioned between the left and right homology arms prior to
transformation into *C. reinhardtii*.

### Test

We conducted the Golden Gate assembly and transformed competent *E. coli* for amplification. No antibiotic-resistant
colonies were present, and we concluded either that the Golden Gate assembly had failed, or that our design was
faulty.

### Learn

When reviewing our design and asking our advisors, the bacterial infrastructure (origin and ampicillin resistance
gene) turned out to cut down too much. We wanted to use an existing and proven unit and not tinker with it. Using an
existing bacterial backbone to insert our parts into via Golden Gate assembly seemed promising [15]. This required
rethinking our HDR strategy: our first design used two fused HDR arms to be separated during a final linearization
step; the revised approach better accommodated standard cloning workflows.

## Cycle 2 - Redesign

![](https://static.igem.wiki/teams/6022/engineering/wetlab-2.avif)

### Design

The goal of our next step was to move the right HDR arm to the downstream direction of the cassette and Golden Gate
assemble the whole cassette into the pTU1-A-RFP\_AB (kindly provided to us by Gabriel Cervera Arriaga, who is a Master
Thesis Student at the Piel Lab at ETHZ) in one step [15]. For this, we split our HDR arms-containing fragment (one of
the five) into two parts via PCR. The primers were designed such that overhangs are added to both HDR arms for the
Golden Gate assembly later on [15].

[![Assembly overview of the V2 plasmid](https://static.igem.wiki/teams/6022/engineering/v2.svg)](https://static.igem.wiki/teams/6022/engineering/v2.svg)

Figure 2: An illustration of version 2 of our designed plasmid.

### Build

We ordered the designed primers from Microsynth, conducted the PCR, and then performed the Golden Gate assembly.
Competent *E. coli* were transformed.

### Test

The transformation and selection of *E. coli* were successful. There was a clear distinction between samples transformed
with pTU1-A-RFP\_AB alone (whose colonies turned red) and the ones allegedly containing our cassette (standard brownish
color). A miniprep resulted in a plasmid that was fully sequenced at Microsynth. Sanger sequencing revealed that the
cassette had not assembled correctly, and around half of our cassette was missing. This led us to believe that
splitting our first plasmid via PCR prior to the Golden Gate did not work correctly. Sequencing the two pieces
confirmed our suspicion.

### Learn

Continuing to work with our initial DNA fragments seemed tedious and likely to create more problems. The existing
sequences did not leave room for flexible primer design to add the overhangs needed in the preceding step. Benchling
predicted the primer pairs to be prone to homodimerization and hairpin formation. That is why we decided to set aside
the existing design and develop a simplified construct.

## Cycle 3 - Minimal Construct

![](https://static.igem.wiki/teams/6022/engineering/wetlab-3.avif)

### Design

We then adopted a minimalistic redesign to reduce both construct size and sequence complexity. Most introns were
removed from TFD-EE (retaining one), and the mVenus reporter was omitted, with the Strep-tag retained to monitor
protein production. We also simplified the APHVIII promoter, replacing the fusion promoter with RBCS2p, while keeping
HSP70A-RBCS2p for TFD. The resulting 3,940-bp linear insert was directly ordered in the pTwist Amp High Copy plasmid.

[![Assembly overview of the V3 plasmid](https://static.igem.wiki/teams/6022/engineering/v3.svg)](https://static.igem.wiki/teams/6022/engineering/v3.svg)

Figure 2: An illustration of version 3 of our designed plasmid.

### Build

Competent *E. coli* cells were transformed.

### Test

Successful transformation of *E. coli* was achieved, as evidenced by bacterial growth on ampicillin-containing
medium.
The plasmid, isolated via miniprep, was fully sequenced at Microsynth.
The sequences obtained by Sanger sequencing aligned precisely with the expected sequences, confirming successful cloning of the construct.

### Learn

The amplified cassette is now ready for introduction into *C. reinhardtii*. Following autolysin treatment, the
algae can be subjected to electroporation and transformed using CRISPR-Cas.
Robust screening will be essential to identify successful transformants and to distinguish targeted homologous
recombination events from random insertions.

## Conclusion

* A robust expression cassette was designed for targeted gene integration in *Chlamydomonas reinhardtii*
* An appropriate genomic locus was identified, and the construct was adapted to facilitate site-specific insertion
* The plasmid was successfully assembled in *E. coli* and verified through iterative sequencing and systematic validation

## Key takeaways

* Iterative Design–Build–Test–Learn cycles were essential to identify and address unforeseen technical hurdles in cloning and assembly.
* Simplification of constructs improved feasibility: removing repeated introns, reporters, and fusion promoters reduced sequence complexity and facilitated cloning.
* Using well-characterized backbones and promoters reduces design risk and accelerates troubleshooting.
* A robust selection strategy is essential for reliably identifying successful transformation events.

Sources

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide
sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

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

## Golden gate (PDF)

## Miniprep (PDF)

## Monarch plasmid DNA miniprep kit (PDF)

## PCR using NEBNext High Fidelity 2X PCR Master (PDF)

## Preparing and loading protein ladders (PDF)

## Q5 high fidelity PCR (PDF)

## Single temperature double digest (PDF)

## Transformation C.reinhardtii (PDF)

## Transformation E.coli DH5 strain (PDF)

## Transformation protocol (PDF)

## 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.

## Protocol 1 : Preparation of Px6 Medium ("CO₂ Medium")

### 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.

## Protocol 2: Modified Algal Culture of Chlamydomonas reinhardtii (CC-125)

(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.

## Protocol 3: Biouptake Experiment with Chlamydomonas reinhardtii and REE Uptake

### 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).

## TAP-medium (PDF)

## Px6 Medium (CO₂ Medium) (PDF)

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{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
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## Project Overview

---

Our 2025 wet lab campaign focused on two connected objectives: enabling reliable
*Chlamydomonas reinhardtii* cultivation for future iGEM teams and demonstrating that our engineered strain
can act as an efficient rare earth element (REE) biofilter. Throughout the season we iterated on growth conditions,
nutrient media, and transformation strategies to remove major experimental bottlenecks.

During our experiments we aimed to :

1.Develop procedures and protocols to facilitate the transformation of C.reinhardtii and
its large scale production for future iGEM teams.

2. Design and construct a modular, low-cost bioreactor that allows precise control of
temperature, pH, and illumination, enabling scalable algal growth and easy replication by other teams.

3. Express our synthetic protein in C.reinhardtii and measure its effect on the chassis
metabolism and its efficiency as a biofilter.

## Refining Cultivation Protocols

---

![Hemocytometer counts used to benchmark C. reinhardtii culture density](https://static.igem.wiki/teams/6022/results/image6.avif)

Hemocytometer counts provided rapid feedback on culture density during
protocol optimisation.

For the first goal we were building upon the results of the Chlamy guide (iGEM
Kaiserslautern & iGEM Paris Sorbonne 2019). We tested and curated their best techniques to further test their
effectiveness. For example most of our experiments were done on their TAP medium, and we measured the amount of cells
in liquid media following their procedure. We then further analysed some data to test the growth of the algae
depending on additional factors such as temperature and air humidity.

## Temperature Screening

---

![Temperature-dependent growth curves for C. reinhardtii in TAP medium](https://static.igem.wiki/teams/6022/results/image4.avif)

Growth curves at 17 °C, 25 °C, and 30 °C revealed
25 °C as the most robust long-term setpoint.

As can be seen from our data the optimal growth temperature seems to be at 25°C, but 30°C was
nearly equally as good until later on during the experiment. We suspect that the additional stress of higher
temperatures adds up and only shows its deleterious effects later on during the experiment. The cell density was
measured by taking aliquots of the liquid culture, measuring the optical density on comparing this to the tabulated
values we had from counting the cells with a hemocytometer for given optical densities.

The results of this plot can be summarised by directly comparing the growth rates :

![Relative growth rate comparison across temperature regimes](https://static.igem.wiki/teams/6022/results/image1.avif)

Average growth rates highlight the performance edge at 25 °C.

## Humidity and pH Insights

---

![Specific growth rates of cultures exposed to varying humidity regimes](https://static.igem.wiki/teams/6022/results/image2.avif)

Growth remained stable across humidity regimes; the main benefit of
higher humidity was reduced medium evaporation.

However our analysis of the growth across different humidity levels did not yield any
statistically relevant results. This is not very surprising as our cells were in liquid medium (so not in urgent need
of additional H2O). The only beneficial point is that at higher humidity, the liquid medium is less volatile and will
stay full for longer. This can be interesting for CO2 media that can feed algae on longer time spans, but it of
limited use on TAP medium as it runs out of nutrients relatively quickly after 2 weeks. An additional factor is that
higher humidity facilitates fungal growth, which is a problem when growing algae, as funghi also strive very well on
TAP-medium. ([Applied Microbiology article on
TAP-medium contamination](https://www.tandfonline.com/doi/full/10.2144/000112022))

These results and those of papers investigating the optimal pH for C.reinhardtii growth. ([C. reinhardtii pH
optimisation study](https://ojs.library.ubc.ca/index.php/expedition/article/view/190221?utm_source=chatgpt.com))
Motivated our choice of building a bioreactor, to monitor the temperature and pH on large scale C.reinhardtii
cultures. To make these advances available to future iGEM teams we also made the construction of it as easy to follow
and cheap as possible (see Hardware part).

## Medium Development

---

At the recommendation of Dr. Abiusi, we prepared a slightly modified version of the 6xP Medium
(the CO₂ medium). Since CO₂ serves as a carbon source, this medium offers advantages in terms of contamination control
and tends to be slightly acidic due to dissolved CO₂.
In the absence of a direct CO₂ supply, we improvised by using
balloons, tubing, and a clamp. This setup is not ideal, as the balloon does not release CO₂ evenly or continuously
throughout the day. Nevertheless, the algae grew well, indicating that the medium likely also performs effectively in
a bioreactor environment.

![Improvised CO2 delivery using balloons, tubing, and clamps](https://static.igem.wiki/teams/6022/results/image3.avif)

Improvised CO₂ delivery with balloons kept cultures aerated while we
refined the medium.

Knowing that rare earth element (REE) phosphates have low solubility and tend to precipitate, we
conducted qualitative experiments to test this behavior. We enriched both our standard 6xP medium and a phosphate-free
variant with 100 ppm of REE salts. As expected, precipitation occurred in the phosphate-containing medium, while no
visible change was observed in the phosphate-free version. This experiment confirmed that using a phosphate-free
medium is a justified and effective choice.

![Precipitation observed in phosphate-containing media after REE enrichment](https://static.igem.wiki/teams/6022/results/image5.avif)

REE precipitation in phosphate-rich medium highlighted the need for a phosphate-free uptake step.

## Autolysin Extraction

---

We also tried a novel approach to C.reinhardtii transformation. As the chlamy guide only
investigates glass beads mediated cell wall disruption, that has a high death rate. Autolysin can be used as a
natural
way to digest the cell wall using an enzyme that C.reinhardtii produces in mating phase. This enzyme is hardly
commercially available, so the production and isolation had to be done from our own C.reinhardtii strains. This
proved
to be unsuccessful as our aliquots of extracted autolysin were not more efficient than the control to digest the
cell
wall.

We suspect this to be due to our strains. CC5415 and CC124 (mating type + and -) are strains
that
were selected for their transformation efficiency, not their mating efficiency. Usually to isolate autolysin strains
with very high mating efficiency are preferred. We did observe mating (see the cell clumps on the image below) but not
as
densely as reported in the protocol.

![Nitrogen-starved C. reinhardtii forming mating clumps](https://static.igem.wiki/teams/6022/results/algaemating.avif)

Image of the nitrogen starved *C. reinhardtii*, clumping up for
mating.

## Cassette assembly and transformations

---

Designing our cassette was quite a struggle, as C.reinhardtii can be quite picky about its
genetic sequences it wants to express (see Engineering part) but our plasmid fragments successfully went through
assembly simulations.

Although in the Lab we saw something else, as all our sequencing showed defects on our plasmid.
The first Golden Gate assemblies failed, and after additional fine tuning of our sequence failed to yield results. We
decided to try to make the problematic fragments bind first in a Gibbson assembly. For this we designed primers with
overhangs to the backbone on our homologous arms for C.reinhardtii transformation. But even this somehow led to very
dirty gel electrophoresis that forced us to proceed with gel extraction.

![Gel electrophoresis highlighting fragments selected for Gibson assembly](https://static.igem.wiki/teams/6022/results/gelscan.avif)

Gel electrophoresis of the preparation of our Gibbson assembly parts.
The ticked bands were used for gel extraction.

The following golden gate assembly did yield some transformed bacteria, but sequencing showed
that it was always only fragments of our cassette that were integrated and even after multiple attempts no clean
product could be isolated, and no advisor knew how to solve the problem.

In an attempt to do more trouble shooting, we tried to send to sequencing our starting material
and some intermediates.

The sequencing shows that the PCR gel extraction to introduce the overhangs for Gibbson assembly
yielded quite unspecific DNA material which probably interfered with our different experimental steps.

The starting material however seemed to be fine.

The origin of the problem is unsure but it the most probable case would be the large amount of
introns that are necessary for an efficient protein production in our transformed algae, that display a very high GC
content and therefore also have large repercussions on the efficiency of most synthetic steps. For example the melting
temperature was so high that we had to go up to 72°C for our PCR with high fidelity Q5, which is at the very edge of
its working range.

In general these high GC content regions that are very common in the C.reinhardtii genome are
known to cause problems. Special additives to buffer to increase PCR efficiency in their presence exist, but we did
not manage to get one as they are not widely available.

In our last engineering cycle we decided to sacrifice the introns and some additional genes (see
cycle 3 in Engineering), these simplification allowed us to get a small enough plasmid for it to be ordered already
assembled. The transformation of it happened without a problem on the 3rd of october and the sequencing results
confirming these results arrived on the 6th of october. We therefore had no time anymore to purify our plasmid for
C.reinhardtii transformation. But this would have been the very next step.

## Bioreactor Hardware Integration

---

![Assembly of the algal bioreactor](https://static.igem.wiki/teams/6022/hardware/asse-big.avif)

Assembly of the algal bioreactor.

In parallel with our biological experiments, we developed a modular, low-cost bioreactor
designed to enable scalable and controlled cultivation of *Chlamydomonas reinhardtii*.
The system was engineered to precisely monitor and regulate both temperature and pH.

The reactor is primarily composed of off-the-shelf components and 3D-printed parts, and features the following elements:

* Transparent **acrylic cultivation chamber** (>10 L) for optimal light transmission and easy cleaning
* **LED illumination modules** tuned to the spectral range optimal for algal growth
* **Integrated sensors** (pH and temperature) connected to a **Raspberry Pi** controller
* **Custom software** for data logging and automated regulation
* **Modular, tripod-mounted design** for easy scalability and maintenance

According to the Grand Jamboree safety regulations, no wet testing with non-sterilizable equipment was permitted.
Therefore, only **dry and functional tests** were conducted so far, confirming stable electronic control
and reliable sensor feedback.

The bioreactor is now ready for **biological integration** and future optimization
with fully sterilizable components.

This hardware system directly supports both main objectives of our project:

* It provides an **open, modular platform** for cultivating *C. reinhardtii* for future iGEM teams.
* It lays the foundation for **quantitative bioaccumulation and bioremediation studies**
  using our genetically engineered algae.

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