iGEM 2025 — Team anatolia-college-gr

{% extends "layout.html" %}
{% block styles %}
{% endblock %}
{% block page\_content %}

## Notebook

---

### Prepare Agar Plates with Nutrient Agar

1. Place agar bottles in a water bath at 90°C until the agar liquifies.
2. Leave at room temperature to cool down to approximately 50°C so it can be handled.
3. Pour 10 mL of the liquified agar onto 5 cm petri dishes and leave them at room temperature to cool down.
4. Transfer the plates to the refrigerator till the agar solidifies (overnight).

### Inoculation with *E. coli*

1. Place agar plates and bacterial stab inside the incubator at 30°C.
2. Once warmed, transfer them to the hood to start the inoculation.
3. Scrape the culture from the bacterial stab with a wire loop and inoculate each plate (negative control).
4. Poured 50 μL of ampicillin sensitive *E. coli* and swab onto the plates (positive control).
5. Place 3 ampicillin disks on each plate once the inoculation was complete.
6. Transfer the plates into the incubator and let them grow overnight.

### Plate Quality Control

1. Once your plates have solidified and dried, test them to make sure the antibiotic functions properly.
2. Take out two plates.
3. On the first plate, streak out a strain that you know to be resistant to the antibiotic.
4. On the second plate, streak out a strain that’s not resistant to the antibiotic.
5. Incubate both plates overnight at the appropriate growth temperature and check for growth.

![Plate quality control](https://static.igem.wiki/teams/5868/notebook/ntb1.avif)

### Bacterial Inoculation

1. Using a sterile pipette tip or toothpick, select a single colony from your agar plate.
2. Drop the tip or toothpick into the bottle with the nutrient liquid and swirl.
3. Loosely unscrew the cap.
4. Incubate bacterial culture at 30°C overnight in the incubator.
5. After incubation, check for growth, which is characterized by a cloudy haze in the media.

![Bacterial growth 1](https://static.igem.wiki/teams/5868/notebook/ntb2.avif)
![Bacterial growth 2](https://static.igem.wiki/teams/5868/notebook/ntb3.avif)
![Bacterial growth 3](https://static.igem.wiki/teams/5868/notebook/ntb4.avif)

### Maxiprep

#### Buffer Preparation

1. Add 88 ml of 95-100% ethanol to the 23 ml ZymoPURE™ Wash 2 (Concentrate) before use.
2. If ZymoPURE™ P2 or Binding Buffer has precipitated, dissolve by incubating at 30-37°C for 10-20 minutes and mix by inversion. Do not microwave.

#### Before Starting

Centrifuge up to 150 ml of bacterial culture at ≥ 4.4 RPM for 25 minutes to pellet the cells (wet pellet ~5 ml). Discard supernatant.

#### Plasmid DNA Purification

1. Add 4 ml of cold ZymoPURE™ P1 (Red) to the bacterial cell pellet and resuspend completely by vortexing or pipetting.

![P1 buffer resuspension 1](https://static.igem.wiki/teams/5868/notebook/ntb5.avif)
![P1 buffer resuspension 2](https://static.igem.wiki/teams/5868/notebook/ntb6.avif)

2. Add 4 ml of ZymoPURE™ P2 (Blue) and gently invert 6 times. Let sit 2-3 min at room temperature. Cells are lysed when solution appears clear, purple, and viscous.

![P2 lysis](https://static.igem.wiki/teams/5868/notebook/ntb7.avif)

3. Add 4 ml of ZymoPURE™ P3 (Yellow) and invert gently 5 more times after it turns completely yellow. A yellowish precipitate will form.

![P3 neutralization](https://static.igem.wiki/teams/5868/notebook/ntb8.avif)

4. Attach plug to Luer Lock at bottom of ZymoPURE™ Syringe Filter-X. Place the syringe upright and load lysate. Wait 5-8 minutes for precipitate to float.
5. Remove Luer Lock plug and push solution through filter until ~10 ml cleared lysate is recovered. Save cleared lysate.

![Syringe filter 1](https://static.igem.wiki/teams/5868/notebook/ntb9.avif)
![Syringe filter 2](https://static.igem.wiki/teams/5868/notebook/ntb10.avif)

7. Remove and discard 15 ml Reservoir-X from Zymo-Spin™ V-PX Column. Centrifuge 13.5 RPM for 1 min to remove residual wash buffer.

![Centrifuge column](https://static.igem.wiki/teams/5868/notebook/ntb11.avif)

#### Elution & Storage

13. Transfer column into clean 1.5 ml tube, add 400 μl ZymoPURE™ Elution Buffer, incubate 2 min, centrifuge ≥16,000 × g for 1 min.
14. Optional: Remove endotoxins with EndoZero™ Spin-Column.
15. Dilute eluate 1:10 before spectrophotometer reading.
16. Store eluted plasmid DNA at ≤ -20°C.

### Plasmid Transformation Confirmation - Agarose Gel Electrophoresis

1. Weight 0.5 g agarose powder.
2. Mix with 50 ml 1x TAE buffer in a microwavable flask.

![Agarose in TAE](https://static.igem.wiki/teams/5868/notebook/ntb12.avif)

3. Microwave for 1 min until agarose dissolves.
4. Cool to ~50°C.
5. Pour agarose into gel tray with comb in place.
6. Add 3 μL Midori Green dye to agarose solution.

![Add dye](https://static.igem.wiki/teams/5868/notebook/ntb13.avif)

7. Let gel solidify (4°C 10-15 min or room temp 20-30 min).

![Add TAE over gel](https://static.igem.wiki/teams/5868/notebook/ntb14.avif)

8. Add loading buffer to DNA samples (4 μL per 15 μL) and pipette ~5 μg DNA into wells.

![Add loading buffer](https://static.igem.wiki/teams/5868/notebook/ntb15.avif)

9. Place gel into electrophoresis box, cover with 1x TAE or TBE.
10. Load molecular weight ladder in first lane.
11. Load samples into remaining wells.
12. Run at 80-150 V until dye is 75-80% down (~1–1.5 h).

![Running gel](https://static.igem.wiki/teams/5868/notebook/ntb16.avif)

13. Visualize DNA fragments using a fluorescence imaging device.

![Visualize gel](https://static.igem.wiki/teams/5868/notebook/ntb17.avif)

**Note:** The experiment was not continued due to insufficient yeast culture density for transformation.

{% endblock %}

{% extends "layout.html" %}
{% block styles %}
{% endblock %}
{% block page\_content %}

## Transformation Preparation Cycle 1

---

##### Design

Initially, we believed that we had to grow the leucine-deficient yeast strain (BY4742) in the same conditions necessary for successful transformation (plasmid uptake) — meaning we thought the yeast could stabilize even under leucine-deficient conditions and grow to reach required density.

##### Build & Test

1. Plated 10μL of the yeast in YND CSM-Leu agar plates (prepared with 100 mL dH₂O, 0.67 g YNB, 2 g glucose, 0.83 g CSM-Leu, and 1.5 g agar powder).
2. Incubated plates overnight at 37°C.
3. Checked plate for growth the next day and found insufficient density to complete transformation.

![Cycle 1 build step 1](https://static.igem.wiki/teams/5868/engineering/eng1.avif)

![Cycle 1 build step 2](https://static.igem.wiki/teams/5868/engineering/eng2.avif)

##### Learn

We learned that the yeast strain requires nutrient-rich medium (including leucine) and cannot reproduce on its own without leucine.

## Transformation Preparation Cycle 2

---

##### Design

1. Took into account results from Cycle 1 and searched for nutrient-rich medium.
2. Decided to opt for YPD to facilitate growth.
3. Increased the yeast volume plated to improve chances of higher yield.

![Cycle 2 design step](https://static.igem.wiki/teams/5868/engineering/eng3.avif)

##### Build & Test

1. Prepared 1% YPD powder mix solution as recommended by the manufacturer.
2. Added the YPD mix to agar plates instead of the CSM-Leu.
3. Observed that the desired growth was still not achieved.

##### Learn

We recalled that our yeast strain had been spilled when we opened the packaging. This suggested it was thawed during delivery and refrozen, which could have significantly compromised yeast viability and affected density yield during fermentation.

![Cycle 2 learn step](https://static.igem.wiki/teams/5868/engineering/eng4.avif)

{% endblock %}

{% extends "layout.html" %}
{% block styles %}
{% endblock %}
{% block page\_content %}

## Plasmid Selection and Expression System

---

### 1. Plasmid Selection Criteria

The plasmid vector type must be *Saccharomyces cerevisiae* since it is the only organism besides natural hosts (lavender and kiwi) that includes part of the MVA pathway.

The promoter must be constitutive in order for our solution to be scalable in the long run.

The plasmid must be high copy to obtain the respective yield of DNA to increase the volume of alcohol production.

The plasmid must contain a LEU2 promoter in order to be uptaken by genetically modified, leucine deficient yeast.

![](https://static.igem.wiki/teams/5868/wikiassets/exp-pic.avif)

### 2. Our Plasmid and Expression System

The plasmid pJT1 (Addgene #98299) is designed for heterologous gene expression in *Saccharomyces cerevisiae*. Built on the pRS425 backbone, it supports stable replication and maintenance within yeast cells, while also containing a bacterial origin of replication and an ampicillin resistance marker for propagation in *E. coli* during cloning.

The plasmid expresses target genes under the control of strong, constitutive yeast promoters TEF1 and TEF2, which drive continuous transcription without the need for induction. This simplifies experimental workflows and ensures consistent gene expression under standard growth conditions.

Transcription in yeast is mediated by RNA polymerase II, with mRNAs undergoing typical eukaryotic processing steps such as capping, splicing, and polyadenylation. Each expression cassette includes appropriate yeast transcription terminators, such as TRPL3 or TRPL41B, to promote mRNA stability and efficient termination.

The expressed coding sequences are translated by the host’s ribosomes, producing functional proteins within the yeast cytoplasm. Because the promoters are constitutive and the plasmid maintains a relatively high copy number in yeast, pJT1 enables strong and reliable expression levels, making it well suited for metabolic pathway engineering and functional studies.

Selective pressure, typically via an auxotrophic marker encoded by the pRS425 backbone, ensures that only yeast cells retaining the plasmid continue to grow, maintaining stability of the construct across generations.

{% endblock %}

{% extends "layout.html" %}
{% block styles %}
{% endblock %}
{% block page\_content %}

## To conclude our iGEM Journey

---

We have successfully propagated the pJT1 plasmid in E.coli and maxiprepped it to extract its DNA. Although concentration was high, which will allow us to use a mini-prep kit next time we conduct this experiment, it is possible that the extracted plasmid is highly contaminated with protein and/or lipids and other substances, which could be theoretically optimized through Proteinase K digestion and chloroform washes.

We faced challenges when growing leucine-deficient yeast likely due to faulty handling during transportation, leading to spillage. We will repeat this experiment when we receive a new sample from Euroscarf and continue up to extraction of alcohols with the proposed protocols.

## Future Studies

---

Considering future success of our experimental protocol three distinct areas of focus for future research should follow: increasing production, creating prototypes, and testing practical applications.

Increasing the yield of linalool is a top priority in order to produce enough of it for consumer use. Targeted metabolic engineering, such as overexpressing the mevalonate pathway enzymes and deleting competing pathways, or CRISPR multiplex editing, which applies multiple genetic modifications simultaneously, can accomplish this. Stress-induced yeast adaptation may also result in variations that are more resilient to elevated terpene levels, boosting stability and productivity. In addition to strain engineering, scaling into bioreactors with regulated pH and oxygen will enable higher, more reliable production, advancing the process toward industrial viability.

Future teams should start creating real diffuser prototypes at the same time. One approach is to create a closed-cartridge system in which linalool is continuously produced in situ by yeast, resulting in a live and renewable source of the alcohols. Linalool could also be extracted and purified into stable oils that could be vaporized with heat or ultrasonic waves. Wearable micro-diffusers that offer individual stress relief throughout the day, like pendants or pins, are an example of more creative approaches. It will be possible to determine which design best strikes a balance between usability, safety, and practicality by testing these various systems.

Just as important, we see a need to demonstrate the human well-being benefits of our diffuser. Partnerships with psychologists or medical researchers could allow stress trials that measure heart rate or cortisol biomarkers before and after exposure to linalool. It would also be possible to use user studies to assess optimum concentrations and release rates and preferred product form presentations. Safety tests to verify that yeast-derived linalool is as pure and benign as the plant-produced varieties are necessary in order to trust that we can begin deploying these scents out in the open world.

Last but not least, different scenarios in which our platform may apply should also be investigated in future endeavors. Not only are linalool and nerolidol stress-reducing, but they also play a part in fragrances as well as consumer products. Building yeast that can produce more than one terpene at a time could create blends made for relaxation, focus, or well being. Additionally, a comparison of life-cycle analyses for yeast fermentation versus conventional plant extraction will demonstrate the sustainability advantage of this process and highlights how the project can be environmentally friendly as well as scalable.

In summary, our proof-of-concept lays the groundwork upon which this work has future potential to develop into a practical, nontoxic and ecological diffuser enabling biotechnology in daily life. By integrating strain optimization, product prototyping, human-centered testing and entry into new applications, our project has the ability to transition from lab bench to patient bedside, transforming a synthetic biology concept into a real stress-reduction device is a promising foundation for iGEM studies in the future.

{% endblock %}