iGEM 2025 — Team sfls-shenzhen

# Lab Notebook

Detailed laboratory protocols and procedures

## Laboratory Protocols

This page will contain detailed lab notebook entries, protocols, and experimental procedures.

### Coming Soon

This section is currently under development. Check back later for detailed lab notebook documentation.

# Engineering

Our engineering approach and design principles

## Engineering Yeast for 1,8-Cineole Biosynthesis: An Engineering Cycle Practice

This project aims to construct an engineered strain of *Saccharomyces cerevisiae* capable of efficiently synthesizing **1,8-cineole (eucalyptol)**, a monoterpenoid widely used in pharmaceuticals, fragrances, and insecticides. We selected BY4741 as the host strain due to its well-defined auxotrophic background (His, Leu, Met, Ura), which facilitates multiple rounds of screening and plasmid co-transformation.

Design → Build → Test → Learn

### 1. Design

**Key Design Strategies Included:**

#### A. Introduction of a Cineole Synthase

The high-efficiency cineole synthase **CinS1** (UniProt: A6XH05) from *Salvia fruticosa* was selected. It utilizes GPP as a substrate and achieves a cineole yield of up to **72.4%**.

#### B. Enhancement of Precursor Supply

* Overexpression of **truncated HMG1 (tHMG1)** to strengthen the MVA pathway.
* Overexpression of **IDI** to balance the IPP/DMAPP ratio.
* Replacement of the native **ERG20 promoter** with the glucose-sensitive **PHXT1 promoter** to regulate the GPP/FPP ratio.
* Introduction of **ScCK and AtIPK** to enhance isoprenol utilization.

#### C. Construction of a Regulatory System

Utilization of **GAL1 and GAL10 promoters**, activated upon glucose depletion, to express heterologous pathways and avoid substrate toxicity. We chose the **pYES2 plasmid** as the expression vector, featuring the URA3 selection marker and AmpR resistance, suitable for manipulation in both yeast and *E. coli*.

### 2. Build

#### A. Plasmid Construction

* **pYES2** was double-digested with BamHI and EcoRI, and the synthesized CinS1 gene fragment was inserted.
* Expression cassettes containing **GAL1-ScCK** and **GAL10-AtIPK** were constructed.
* The **CRISPR-Cas9 system** was used to knock out GAL80 and replace the native ERG20 promoter with PHXT1.
* All constructs were verified by **colony PCR** and **Western blot**.

#### B. Yeast Transformation

* Competent cells were prepared using the **LiAc method**.
* **Electroporation** was performed, followed by plating on SD/-Ura plates containing G418.
* Positive clones were selected via **dual screening** (auxotrophy and antibiotic resistance).

### 3. Test

#### A. Phenotypic Validation

* Positive transformants showing robust growth were selected on SD/-Ura plates.
* **Colony PCR** confirmed the integration of target genes.
* **Western blot** verified the expression of the cineole synthase and other key enzymes.

#### B. Metabolite Analysis

* **LC-MS/MS** was used to detect IPP/DMAPP and GPP levels.
* **GC-MS** was used to analyze 1,8-cineole production.
* **OD600** and substrate consumption were monitored during fermentation.

#### C. Preliminary Results

* The engineered strain successfully **activated the GAL promoters** after glucose depletion.
* **1,8-Cineole production** was significantly higher than in the control strain.
* Replacement of the ERG20 promoter led to increased **GPP accumulation**, favoring monoterpene synthesis.

### 4. Learn and Next Iteration Design

#### A. Key Findings

* The cineole synthase **CinS1 was expressed well in yeast**, but production might be limited by endogenous GPP supply.
* The **ERG20 promoter replacement was effective** but incomplete; further attenuation of its activity might be necessary.
* The initial design did not fully account for the impact of **byproduct accumulation** on cell growth.

#### B. Design Improvements for the Next Cycle

* **Construct an erg20 mutant plasmid:** To further reduce ERG20 activity and enhance GPP availability.
* **Introduce a Dynamic Regulation System:** Implement a two-stage fermentation strategy (glucose/galactose) for more precise pathway control.
* **Optimize Promoter Combinations:** Experiment with stronger or more sensitive promoters to drive IDI and tHMG1.
* **Build a Multi-Gene Co-expression Vector:** Modularize the cineole synthesis pathway to facilitate future debugging and standardization.

### Conclusion

We successfully completed a full engineering cycle: starting from rational design, we constructed an engineered yeast strain capable of synthesizing 1,8-cineole and experimentally verified its feasibility. The data obtained provides clear directions for the next round of engineering optimization.

All constructed plasmids and strains have been documented in the **iGEM Registry** for reference and use by future teams.

# Experiments

Detailed experimental protocols and methodologies for our synthetic biology research.

## 1. Preparation of Sterilized Reagents

#### 1.1 Media without glucose

LB media, etc., sterilized at **121℃ for 20 min**

#### 1.2 Media with glucose

YPD, SD-Ura media, etc., sterilized at **115℃ for 15 min**

#### 1.3 Antibiotics

**Filtration sterilization**

## 2. Plasmid Extraction using TIANprep Mini Plasmid Kit

1. Use a permanent marker to label the top of a 2 ml centrifuge tube with your number.
2. Transfer 1 ml of the *E. coli* overnight culture into the tube.
3. Place it into a balanced centrifuge and spin for 1 min at **12000 rpm**, pour off the supernatant. Be careful not to disturb the pellets.
4. Invert your tube and tap gently on the surface of a clean paper towel to drain thoroughly.
5. Add **250 μL of Buffer P1** to your tube. Vortex to resuspend the pellet. Check that no cell clumps should be visible.
6. Add **250 μL of Buffer P2** and mix gently by inverting the tube 6-8 times. Solutions will become relatively clear. Do not allow the lysis reaction to proceed for more than 5 mins.
7. Add **350 μL of Buffer P3** and invert the tube immediately but gently 6-8 times. The solution should become cloudy. Shake the tube gently to disperse the precipitate.
8. Spin for **10 mins at 12000 rpm** to pellet the precipitate thoroughly.
9. At the same time, label a spin column on the top and by the side. Balancing the column by applying 500 μL of Buffer BL, spin 1min at 12000 rpm. Discard the flow-through.
10. Apply 600 μL of the supernatant from step 9 to the spin column by pipetting, spin 1min at 12000 rpm. Discard the flow-through.
11. Add **600 μL of Buffer PW** to the column, sit for 1min. Spin 1 min at 12000 rpm. Discard the flow-through.
12. Repeat step 11. Discard the flow-through. Put the column back, spin 3mins at 12000 rpm to remove the residual wash buffer.
13. Get a clean 1.5 mL centrifuge tube and label the top. Place the column into this tube, add **50 μL of Buffer EB** to the center of the column, let stand at **50℃ for 5 mins**. Spin 3 mins at 12000 rpm.

## 3. Plasmid Construction

#### 3.1 PCR Mix Preparation

| Component | Volume |
| --- | --- |
| 10x T4 buffer | 1 μL |
| Promoter | 1 μL |
| Gene | 1 μL |
| Terminator | 1 μL |
| Plasmid | 1 μL |
| Restriction Enzyme | 0.5 μL |
| ddH2O | 4.5 μL |

#### 3.2 Initial Incubation

Place the tube into PCR machine, set **55°C for 1 hr**.

#### 3.3 Ligation Program

Add **0.5 μL T4 ligase** into the tube. Place the tube into PCR machine and run the following program:

1. 25℃ → 1 hr

2. 50℃ → 15 min

3. 80℃ → 15 min

4. 10℃ → hold

## 4. *E. coli* Transformation

1. Fill the ice box with ice, and put *E. coli* Competent Cells (50μL per tube) to make it melt on ice.
2. Add **2μL plasmid** to one tube of 50μL *E. coli* Competent Cells. Incubate the tube on ice for at least **30 min**.
3. Set the water bath to **42℃** and place the tube with *E. coli* into the water bath for **30-90 seconds** (Heat shock).
4. Immediately place the tube back on ice and incubate for **5 min**.
5. In an ultra-clean table, add **250μL pre-warmed LB culture medium** to the *E. coli* centrifuge tube and tap the bottom of the tube to mix well.
6. Incubate at **37℃ for at least 45 min** with shaking to allow expression of resistance genes.
7. Using a pipette, take 100μL shaken bacteria from the centrifuge tube and add it to the LB plate marked with Antibiotics. Use sterilized glass beads and apply evenly.
8. Discard the glass beads and incubate those plates **upside down overnight at 37℃** in a constant temperature incubator.

## 5. Yeast Transformation using Frozen-EZ Yeast Transformation II™

### 5.1 Preparation of Competent Cells

Grow yeast cells (BY4741) at **30°C in 10 ml YPD broth** until mid-log phase.

1. Pellet the cells at **500 x g for 4 minutes** and discard the supernatant.
2. Add 10 ml Frozen-EZ Yeast Solution 1 to wash the pellet. Repellet the cells and discard the supernatant.
3. Add 1 ml Frozen-EZ Yeast Solution 2 to resuspend the pellet.

### 5.2 Transformation

1. Mix **50 μl of competent cells** with **0.2-1 μg DNA** (in less than 5 μl volume); add 500 μl Frozen-EZ Yeast Solution 3 and mix thoroughly.
2. Incubate at **30°C for 45 minutes**. Mix vigorously by flicking with finger or vortexing 2-3 times during this incubation.
3. Spread **50-150 μl** of the transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading.
4. Incubate the plates at **30°C for 2-4 days** to allow for growth of transformants.

## 6. Colony PCR

#### 6.1 Pick and Incubate Single Colony Bacteria

In the laminar flow hood, add 1 ml of LB liquid medium containing the appropriate antibiotics into a 1.5 ml EP tube, and use the pipette tip to pick (white) single colonies and discard them into the centrifuge tube. (Pick **10-15 single colonies** from each plate.)

#### 6.2 Incubation

Incubate at **37°C with 220 rpm** shaking for 3 hours on a shaker. Proceed with single colony PCR validation.

#### 6.3 PCR Mix

| Component | Volume |
| --- | --- |
| 2× Taq PCR mix | 5 μL |
| Primer F | 0.4 μL |
| Primer R | 0.4 μL |
| ddH2O | 3.2 μL |
| Template DNA | 1 μL |

#### 6.4 PCR Program

Mix the PCR system using a vortex mixer, and centrifuge shortly. Then place the PCR tube in the thermal cycler and run:

| Step | Temperature | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 94℃ | 3 min | 1x |
| Denaturation | 94℃ | 20 s | 30 cycles |
| Annealing | 56℃ | 30 s |
| Extension | 72℃ | 90 s |
| Final Extension | 72℃ | 5 min | 1x |
| Hold | 12℃ | ∞ | - |

## 7. Electrophoresis

1. Measure **1g of agarose**, mix it with 100mL of TAE.
2. Microwave for **1-3 minutes** until agarose dissolves.
3. Pour the mixture into a gel tray with well comb in place.
4. Let the mixture cool down for **10-15 minutes**.
5. Once solidified, place the agarose gel into a gel box.
6. Load samples and marker into the wells of the gel.
7. Put the gel into the electrophoretic chamber.
8. Start the electrophoretic chamber for **20 minutes, 130V**.
9. Remove the gel afterwards and place it in a UV light source.
10. Compare the sample markings to the marker.

# Results

Biosynthesis and Optimization of 1,8-Cineole Production in Yeast

## Verifying Transformation by Yeast Colony PCR and Gene Sequencing

![Colony PCR verification](https://static.igem.wiki/teams/5970/images/result/screenshot-2025-10-08-at-01-43-background-removed-14.webp)

![Gene sequencing results](https://static.igem.wiki/teams/5970/images/result/screenshot-2025-10-08-at-01-39-background-removed-03.webp)

## Step 1: Synthesis of 1,8-Cineole

The gene of **1,8-cineole synthase** was transferred into *Escherichia coli*, and then *E. coli* was transformed into yeast and colony culture was carried out.

![Electrophoresis gel](https://static.igem.wiki/teams/5970/images/result/result-img3.webp)

Markers: 1000kb plus | 100bp

#### Electrophoresis Analysis

Our electrophoresis map shows the **1,8-cineole synthase gene** with a gene fragment size of **1116bp**. This is because it is placed between the **HindⅢ and EcoR1 sites of pYES2**, with connectors added at both ends. The total length is **1128bp**, corresponding to the positions of our two markers (100bp and 1kb plus) above the 1000 band.

![Yeast colony culture](https://static.igem.wiki/teams/5970/images/result/result-img4-bg-removed.webp)

![Colony selection](https://static.igem.wiki/teams/5970/images/result/result-img5-background-removed.webp)

## Step 2: Increasing the Production of 1,8-Cineole

We increased the yield of **1,8-cineole** by replacing ordinary yeast cells with **ERG20ww mutants** and conducting **SD-Ura screening**.

## Conclusion

We successfully obtained **1,8-cineole** through biosynthesis and increased its yield using **ERG20ww mutants**.