iGEM 2025 — Team icjfls

{% 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 %}{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}We use the iteration of the part [BBa\_254GKC2U](https://registry.igem.org/parts/bba-254gkc2u) to show our engineering success.{% endblock %}
{% block page\_content %}

#### Index

[###### 1. Introduction](https://2025.igem.wiki/ICJFLS/engineering#heading-2)
[###### 2. The First-Generation Part pACYCDuet-1-GER-Lis](https://2025.igem.wiki/ICJFLS/engineering#heading-3)
[2.1 Design of pACYCDuet-1-GER-Lis](https://2025.igem.wiki/ICJFLS/engineering#heading-3)
[2.2 Construction of pACYCDuet-1-GER-Lis](https://2025.igem.wiki/ICJFLS/engineering#heading-5)
[2.3 Measurement of pACYCDuet-1-GER-Lis](https://2025.igem.wiki/ICJFLS/engineering#heading-6)
[###### 3. The Second-Generation Part Orthogonal combination plasmid-GER-Lis](https://2025.igem.wiki/ICJFLS/engineering#heading-7)
[3.1 Design of the orthogonal combination plasmid-GER-Lis](https://2025.igem.wiki/ICJFLS/engineering#heading-8)
[3.2 Construction of orthogonal combination plasmid-GER-Lis](https://2025.igem.wiki/ICJFLS/engineering#heading-9)
[3.3 Expression of GER and Lis](https://2025.igem.wiki/ICJFLS/engineering#heading-10)
[3.4 Measurement of orthogonal combination plasmid-GER-Lis](https://2025.igem.wiki/ICJFLS/engineering#heading-11)
[3.5 Optimization of inducer concentration and induction times](https://2025.igem.wiki/ICJFLS/engineering#heading-12)
[###### 4. Summary](https://2025.igem.wiki/ICJFLS/engineering#heading-13)

### 1. Introduction

Geraniol reductase catalyzes the conversion of geraniol to citronellol, both of which are the main componants of rose essential oil. Linalool isomerase catalyzes the conversion of geraniol to linalool, which is the main componant of lavender essential oil.

![](https://static.igem.wiki/teams/5654/results/en1.webp)

**Fig.1 The conversion of geraniol to citronellol and linalool catalyzed by geraniol reductase and linalool isomerase respectively.**

 The goal of our project is to enable *E. coli* to produce citronellol and linalool in any ratio using geraniol (synthesized by yeast), so as to meet the needs of different users for different ratios of these two essential oil components.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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### 2. The First-Generation Part pACYCDuet-1-GER-Lis

####

#### 2.1 Design of pACYCDuet-1-GER-Lis

To prevent competition among multiple plasmids in *E. coli*, a high-copy multi-gene co-expression vector pACYCDuet-1 was selected. Two genes, geraniol reductase (GER) and linalool isomerase (Lis), were designed to clone into two distinct multiple cloning sites (MCSs) of this plasmid. Both of them are expressed under the control of the strong T7/lac promoter, enabling the "one-click activation" of the synthesis of multiple essential oil components.

![](https://static.igem.wiki/teams/5654/design/fig7.webp)

**Fig.2 GER and Lis genes were constructed into the plasmid of pACYCDuet-1, followed by transformation into *E. coli.***
Geraniol Reductase, GER, Linalool isomerase, Lis.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 2.2 Construction of pACYCDuet-1-GER-Lis

Prior to gene synthesis, we optimized the gene sequences based on the codon preference of *E. coli*. Results from PCR and restriction enzyme digestion confirmed the successful construction of the pACYCDuet-1-GER-Lis plasmid.

![](https://static.igem.wiki/teams/5654/results/fig-4-construction-and-identification-of-pacycduet-1-ger-lis-recombinant-vector.webp)

**Fig.3 Construction and identification of pACYCDuet-1-GER-Lis recombinant vector using *Eco*R I, *Pst* I, *Nde* I and *Kpn* I restriction enzymes.**
 M: Marker; 1: pACYCDuet-1 Plasmid; 2: pACYCDuet-1-GER-Lis; 3: Digestion of pACYCDuet-1-GER-Lis with *Eco*R I and *Pst* I; 4: PCR product of GER using pACYCDuet-1-GER-Lis as template; 5: Digestion of pACYCDuet-1-GER-Lis with *Nde* I and *Kpn* I; 6: PCR product of GER using pACYCDuet-1-GER-Lis as template.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 2.3 Measurement of pACYCDuet-1-GER-Lis

The constructed recombinant plasmid pACYCDuet-1-GER-Lis was then transformed into competent BL21 cells. Under the induction with IPTG, the strain can efficiently express two enzymes (GER and Lis) which are used for the synthesis of citronellol and linalool respectively.

Although the "one-click activation" synthesis can be achieved since both GER and Lis genes are co-controlled by the T7 promoter and IPTG induction, it fails to meet the requirement for regulating the personalized production of different essential oil components, namely citronellol and linalool.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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### 3. The Second-Generation Part Orthogonal combination plasmid-GER-Lis

To address the problem above, we optimized the previous design using an orthogonal expression system to enable separate control over the synthesis of different essential oil components.

The core of this design lies in the use of two orthogonal promoter-inducer systems. The term "orthogonal" means there is no cross-reactivity between the two systems: inducer A only activates promoter A without affecting promoter B, and vice versa. However, there is currently no "ready-to-use" commercial single plasmid that directly integrates two such promoters on a single plasmid, so we have to construct an orthogonal combinatorial plasmid ourselves.

The T7/lac system consists of the T7 promoter and the repressor protein LacI, which is induced by IPTG to achieve high expression levels and is one of the most commonly used expression systems. The araBAD (pBAD) system comprises the pBAD promoter and the repressor protein AraC, which is induced by L-arabinose. It offers highly precise regulation, with induction levels linearly adjustable according to arabinose concentration, and is completely orthogonal to the T7/lac system. Therefore, we plan to construct an orthogonal combinatorial plasmid containing both T7/lac and pBAD promoters for expression of GER and Lis genes.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 3.1 Design of the orthogonal combination plasmid-GER-Lis

The procedure was performed in two steps: first, two recombinant plasmids, pET28a(+)-GER and pBAD-Lis, were constructed. pET28a(+) contains the T7/lac promoter and is induced for expression by IPTG; pBAD contains the araBAD promoter and is induced for expression by L-arabinose. Subsequently, the fusion of the two plasmids, namely the orthogonal combination plasmid-GER-Lis, was constructed using homologous recombination PCR. This plasmid contains KanR, GER, T7/lac promoter and LacI genes from pET28a(+)-GER, as well as AraC, pBAD promoter, Lis，ApR genes，and Ori site from pBAD-Lis, with a total length of 9926 base pairs.

![](https://static.igem.wiki/teams/5654/design/fig8new.webp)

**Fig.4 Construction of Orthogonal combination plasmid-GER-Lis using recombinant PCR method.**
Geraniol Reductase, GER, Linalool isomerase, Lis.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 3.2 Construction and identification of orthogonal combination plasmid-GER-Lis

The construction and identification of the orthogonal combination plasmid-GER-Lis were showed as follows:

![](https://static.igem.wiki/teams/5654/results/fig-7n-construction-and-identification-of-orthogonal-combination-plasmid-ger-lis.webp)

**Fig.5 Construction and identification of Orthogonal combination plasmid-GER-Lis using homologous recombination PCR method.**
 M: Marker; 1: PCR product of pET28a(+)-GER containing KanR, GER, T7/lac promoter and LacI genes, etc (4.4kb) ; 2: PCR product of pBAD-Lis containing AraC, pBAD promoter, Lis, ApR genes and Ori site (5.5kb) ; 3: Orthogonal combination plasmid-GER-Lis; 4: PCR product of GER using Orthogonal combination plasmid-GER-Lis as template; 5: PCR product of Lis using Orthogonal combination plasmid-GER-Lis as template.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 3.3 Expression of GER and Lis in *E. coli* tranformed with orthogonal combination plasmid-GER-Lis

To verify the transformation of the orthogonal combination plasmid-GER-Lis in *E. coli,*protein purification using His-tag and SDS-PAGE electrophoresis were performed on the overexpressed GER and Lis proteins. The results showed that the plasmid was well-expressed in E. coli, and the amounts of GER (46.8 kDa) and Lis (76.1 kDa) proteins were significantly higher than those in the control group.

![](https://static.igem.wiki/teams/5654/results/fig-8-purification-of-ger-and-lis.webp)

**Fig.6 SDS-PAGE indicates the expression and purification of GER and Lis expressed in *E. coli* transformed with Orthogonal combination plasmid-GER-Lis.**
 M: protein marker; 1: The supernatant obtained by centrifuging the total lysate of transformed cells; 2. The supernatant obtained by centrifuging the lysate of untransformed cells; 3. The residual liquid remaining after purifying the overexpressed proteins from the lysate of transformed cells using a His-tag column; 4. The purified overexpressed GER (46.8kD) and Lis (76.1kD) proteins.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 3.4 Measurement of orthogonal combination plasmid-GER-Lis in *E. coli*

Although we verified that the GER and Lis genes can be overexpressed in BL21 cells, further validation is required to confirm whether the expressed enzymes are active and capable of converting geraniol into citronellol and linalool. We used gas chromatography (GC) for analysis.

We are aware that overexpression of exogenous genes imposes a burden on cells, so we selected inducible plasmids. When *E. coli* grew to an OD₆₀₀ of 1.0, 100 µM IPTG and 500 µM L-arabinose were used to simultaneously induce the expression of GER and Lis. Additionally, 200 mM geraniol standard was added every two hours to a final concentration of 50 mg/L, followed by continued fermentation for 24 h. The culture was overlaid with n-dodecane to extract terpenoids, which were analyzed by GC using geraniol, citronellol, and linalool standards as controls.

![](https://static.igem.wiki/teams/5654/results/fig-9.webp)

**Fig.7 GC analysis for geraniol, citronellol and linalool production by *E. coli.*** The retention times of geraniol, citronellol, and linalool standards were 5.64 min, 5.16 min, and 3.26 min, respectively. The retention times of these components in the samples are essentially consistent with those of the standard substances..

From the GC results, we observed that in addition to the expected main peaks of geraniol, citronellol, and linalool, other miscellaneous peaks appeared. However, we are unaware of the identity of these substances, which requires further in-depth investigation.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 3.5 Optimization of inducer concentration and induction times

Our target final products are mixed essential oil components containing citronellol and linalool in different proportions, which can be produced in a single fermation process. The achievement of this goal mainly depends on the activity ratio of the key enzymes responsible for synthesis of these two substances, as they share the same substrate, geraniol. These two key enzymes (GER and Lis) are constructed under the control of two distinct promoters (pT7/lac and pBAD) on the same plasmid, with their expression induced by IPTG and L-arabinose respectively. These two promoters form a fully orthogonal regulatory system (non-interfering regulation).

However, the induction efficiencies of these two fully orthogonal regulatory systems differ, leading to variations in the expression levels of the enzymes and the catalytic efficiency of product synthesis. Additionally, there exists competition between the two enzymes for substrate utilization and carbon source consumption.

Therefore, to determine the working efficiency of the two promoters, we conducted experiments on the combined induction of expression using different concentrations of IPTG and L-arabinose. The efficiency was evaluated based on the ratio of the product concentrations of citronellol (synthesized under IPTG induction) to linalool (synthesized under L-arabinose induction).

![](https://static.igem.wiki/teams/5654/results/fig-12.webp)

**Fig.8 The measured product ratio of citronellol to linalool after 12 h of combined induction with different concentrations of IPTG and L-arabinose.**
The thermodynamic diagram (heatmap) shows the ratio of two products induced by IPTG and Arabinose at different concentrations. Different colors are used in the figure to represent different product ratios, with darker colors indicating lower ratios and lighter colors indicating higher ratios. A: Induced production ratio with higher arabinose concentration; B: Induced production ratio with lower arabinose concentration.

Comparing the heatmap of A and B in Fig.8, we figure out that after 12 h of co-induction with a relatively high concentration of arabinose (1 mM – 10 mM, Fig. 8 A) and a relatively low concentration of IPTG (0.1 mM – 1.0 mM), the product ratio (citronellol:linalool) fluctuates within the range of 1 to 10. In contrast, after 12 h of co-induction with a relatively low concentration of arabinose (0.1 mM–2.5 mM, Fig. 8 B) and a relatively low concentration of IPTG (0.1 mM – 1.0 mM), the product ratio (citronellol:linalool) fluctuates within the range of 0.1 to 3. The latter is more likely to meet the needs of most users for different proportions of these two components.

Next, we designed two additional experiments involving sequential induction with the two inducers: Experiment A: First, induce expression with 0.4 mM IPTG for 6 h then co-induce with different concentrations of arabinose for 12 h, during which the ratio of the two products was measured. Experiment B: First, induce expression with 1 mM arabinose for 6 h, then co-induce with different concentrations of IPTG for 12 h, during which the ratio of the two products was measured.

![](https://static.igem.wiki/teams/5654/results/fig-13.webp)

**Fig.9 The Effect of Different Induction Orders of IPTG and Arabinose on the Ratio of the Two Products.**
A: The thermodynamic plot (heatmap) illustrates the effects of different arabinose concentrations and induction durations on the product ratio after 6h of IPTG (0.4 mM) induction. B: The thermodynamic plot (heatmap) shows the effects of different IPTG concentrations and induction durations on the product ratio after 6 h of Arabinose (1 mM) induction. In the plot, different colors represent different product ratios: darker colors indicate lower ratios, while lighter colors indicate higher ratios.

As can be seen from Fig. 9 above, even if the two inducers have different induction timings (i.e., induced sequentially), the final desired product ratio can still be achieved by adjusting the concentrations of the inducers.

From the above figures, the optimal configuration can be identified according to the requirements for different product ratios. It is expected that these data will provide support for the personalized production of essential oil components with different proportions.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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### 4. Summary

In conclusion, through iterative design and optimization, we have successfully constructed an orthogonal expression plasmid. This plasmid contains two non-interfering promoter control systems, which can respectively induce the expression of two different genes (GER and Lis) under the regulation of two different inducers. After these two genes are expressed, they can catalyze the synthesis of citronellol and linalool with their respective efficiencies, so as to meet the needs of different users for the ratio of the two essential oil components.

In the future, we plan to integrate machine learning algorithms with real-time metabolic flux analysis to establish a dynamic mapping model between user preferences and fermentation parameters. Users only need to input their preference for the aroma ratio of rose to lavender (e.g., 7:3 or 5:5), and the system will automatically optimize culture parameters and induction parameters to precisely allocate carbon flow to target products.

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiment Protocols {% endblock %}
{% block lead %}Here is our "secret weapon," and we'd love to share it with you!
{% endblock %}
{% block page\_content %}

#### Index

* [###### Preface](https://2025.igem.wiki/ICJFLS/experiments#heading-1)
* [###### PCR protocol](https://2025.igem.wiki/ICJFLS/experiments#heading-3)
* [###### Gel electrophoresis](https://2025.igem.wiki/ICJFLS/experiments#heading-4)
* [###### Competent cell preparation](https://2025.igem.wiki/ICJFLS/experiments#heading-5)
* [###### Plasmid transforming](https://2025.igem.wiki/ICJFLS/experiments#heading-6)
* [###### Plasmid isolation](https://2025.igem.wiki/ICJFLS/experiments#heading-7)
* [###### Restriction endonuclease digestion](https://2025.igem.wiki/ICJFLS/experiments#heading-8)
* [###### Protein purification](https://2025.igem.wiki/ICJFLS/experiments#heading-10)
* [###### SDS gel electrophoresis](https://2025.igem.wiki/ICJFLS/experiments#heading-11)
* [###### Yeast Transformation](https://2025.igem.wiki/ICJFLS/experiments#heading-12)
* [###### Genome extraction from Yeast](https://2025.igem.wiki/ICJFLS/experiments#heading-13)
* [###### *E. coli* Shake-flask Fermentation](https://2025.igem.wiki/ICJFLS/experiments#heading-14)
* [###### Preparation of gas chromatography samples](https://2025.igem.wiki/ICJFLS/experiments#heading-15)
* [###### Preparation of standard geraniol sample](https://2025.igem.wiki/ICJFLS/experiments#heading-16)
* [###### Co-culture](https://2025.igem.wiki/ICJFLS/experiments#heading-17)
* [###### Yeast Fermentation](https://2025.igem.wiki/ICJFLS/experiments#heading-18)
* [###### Ingredients of main certain mediums](https://2025.igem.wiki/ICJFLS/experiments#heading-19)

## Preface

---

By modifying microbial cell factories through targeted approaches to construct a programmable biosynthetic platform in our project, we can achieve on-demand synthesis of rose essential oil, known as the "Queen of Flowers," and lavender essential oil, known as the "Universal Guardian".

## PCR protocol

---

The reaction components are assembled in the PCR protocol as described below. The final volume should be 20 µL.
1. All reagents are thawed on ice.
2. Assemble reaction mix into 20 µL volume in a thin walled 0.2 mL PCR tubes as follow:
    8 uL ddH2O
    1 µL DNA Template
    0.5 uL Primer F
    0.5 uL Primer R
    10 uL 2x Taq mixture
3. Prepare negative control reaction without template DNA. Prepare positive control reaction with template of known size and appropriate primers.
4. Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
5. Put the tube into a PCR instrument.
6. Set up the following PCR program: initial denaturing at 94°C for 4 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min, plus a final extension at 72°C for 10 min.
7. Check the PCR product by agarose gel electrophoresis.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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## Gel Electrophoresis

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1. Prepare 100mL 1xTAE buffer with 1 g agarose, and boil it three times, shake completely, and waiting for cool.
2. Pour the agarose gel into gel tray, assemble gel pouring apparatus by inserting gate into slots.
3. Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).
4. Fill the rig with 1x TAE buffer.
5. Load 8 μL of DNA maker into lane.
6. Mix 1 μL of 10x loading buffer with 5 μL DNA sample, load them into lane.
7. Run at 100 V for 30 min.;
8. Use the Gel imaging system to check the gel;and take a picture.
9. Deal with the gel carefully as medical waste.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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## The Competent Cell Preparation

---

1. Streak the *E. coli* stock on a LB-agar plate. Incubate the plate at 37°C overnight.
2. Pick a single well-isolated colony and inoculate it into 3 mL of LB broth solution. Incubate it at 37°C overnight (more than 16 h) with shaking at 220 rpm.
3. Transfer 250 uL of the saturated overnight solution to a sterile 50 mL polypropylene tube containing 25 mL of LB medium. Incubate the *E. coli* at 37°C with shaking at 220 rpm until OD600 reaching 0.6. This usually takes 2.5 h. Check the OD every 30 min after 1 h to avoid overgrowth.
4. When the medium reaches an OD600 of 0.6, chill the tube on the ice for 30 min and then centrifuge at 8000 rpm for 1 min at 4°C, discard the supernatant.
5. Re-suspend the *E. coli* in 2.5 mL of ice-cold TSS solution with gentle swirling. Incubate on ice for 10 min. Aliquot in 50 uL per tube. Now the competent cells are ready to be transformed.
Note: Transformation and Storage Solution (TSS) contains LB medium 85% 8.5 mL, PEG (wt/vol, Mw 8000) 10% 1.0 g, DMSO (vol/vol) 5% 500 μL, and MgCl2 (pH 6.5) 50 mM 500 μL.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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## Plasmid Transforming and Pick monoclonal

---

1. Thaw TSS cells on ice.
2. Add 5 uL Synthetic plasmid DNA in TSS cells, pipette gently to mix.
3. Incubate it on ice for 30 min with occasional mixing.
4. Heat shock at 42°C for 90 sec.
5. After heat shock, put it on ice for 2 min, add 0.8 mL LB medium into the cells.
6. Shake and incubate at 37°C for 60 min at 120 rpm.
7. Take out 100 uL medium and spread them on the appropriate agar plates with certain antibiotic.
8. Incubate the plates at 37°C overnight.
9. Pick monoclonal into certain antibiotic LB medium, and incubate the medium at 37°C overnight.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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## Plasmid Isolation Protocol

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1. The bacteria that contained target plasmid were cultivated at 37°C overnight.
2. Take out 1,500 uL solution of cultivated bacteria to a 2mL micro-centrifuge tube. The tube was centrifuged at 12,000 rpm for 1 min at room temperature, and then the supernatant was discarded. In this tube, repeating these steps three times to increase the concentration of the bacteria.
3. Add 250 uL Solution I buffer to suspend the precipitation. Complete suspension is vital for obtaining good plasmid yield by vortexing or pipetting up and down.
4. Add 250 uL Solution II buffer, mix gently upside down 4-6 times to make a cleared lysate. A 2-3 min incubation at room temperature may be necessary. However, this step should not be more than 5 min, and avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity.
5. Add 350 uL Solution III buffer, mix gently and fully upside down 6-8 times, centrifuge the tubes at 12,000 rpm for 10 min at room temperature.
6. Put a HiBind DNA Mini Column into a 2 mL Collection Tube.
7. The supernatant in step 4 is transferred to the Mini Column, and centrifuged at 12,000 rpm for 1 min, the filtrate is discarded.
8. Put the Column back into the Collection Tube, add 500 uL HBC Buffer, centrifuge at 12,000 rpm for 1 min, the filtrate is discarded.
9. Put the Column back into the Collection Tube, add 700 uL Washing buffer, centrifuge at 12,000 rpm for 1 min, the filtrate is discarded. Repeat the washing step once.
10. Put the Column back into the Collection Tube and centrifuge at 12,000 rpm for 2 min.
11. Put the Column into a new 1.5 mL micro-centrifuge tube and leave it for 2 min at room temperature. Then add 50 uL ddH2O to the center of the Column matrix, and leave it at room temperature for 1 min. Centrifuge at 12,000 rpm for 1 min. Discard the HiBind DNA Mini Column.
12. Measure concentration of extracted plasmid DNA by nanodrop 1000, and store plasmid DNA at -20°C.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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## Plasmid DNA Restriction endonuclease Digestion

---

DNA for downstream applications is usually digested with restriction endonucleases. Type II restriction enzymes are the most widely used in molecular biology application.
The reaction volume are carried out in 20 µL.
1. Add components to a clean tube in the order as follow:
    1 µL DNA (1 µg/µL)
    2 µL 10x buffer
    1 µL each restriction enzyme
    15 µL ddH2O
2. Mix gently and fully, and incubate the tube at certain temperature (37°C) for 1 h.
3. Stop the reaction by heat inactivation (65°C for 15 min).
4. The digested DNA is ready for use in research applications.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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## Purification of His/S-tagged protein

---

All the procedures were performed at 4 °C or on ice unless otherwise specified.
1). 10 mL BL21 cells transformed with a pET-28a-*lacZ* were cultured at 30°C until an OD660 of approx. 0.8.
2). Add IPTG to the culture (final 0.67 mM IPTG)
3). Cultivate at 30 °C for 2 h.
4). Collect cells in 5 mL tube, centrifuge at 5,000 rpm at 15 min.
5). Suspend cells in 5 mL of TBS, transfer the content to 1.5 mL tube.
6). Collect cells at 10,000 rpm at 1 min. The supernatant was discarded.
7). Add 500 µL of ice-cold lysis buffer to the cell pellet.
8). Sonicate cell (4°C).
9). Clarify lysate by centrifugation at to 10,000 rpm at 10 min.
10). After centrifugation, save 20 µL of supernatant for SDS-PAGE sample.
11). Transfer supernatant to the 1.5mL tube containing pre-balanced resin bead gel.
12). Shake gently (4°C) for 30 min.
13). Centrifuge the tube 1,200 rpm for 30 sec. The supernatant was discarded.
14). Wash resin bead gel with washing buffer, Centrifuge the tube 1,200 rpm for 30 sec. The supernatant was discarded. Repeat it once.
15). Elute the protein 3 time with 20 µL of elution buffer. It is ready to prepare the SDS-PAGE sample.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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## SDS Gel Electrophoresis

---

1). Make up 30 mL of running gel by adding components to a clean glass in the order as follow:
    12.3 mL H2O
    7.5 mL 1.5 M Tris-HCl, pH 8.8
    0.15 mL 20% (w/v) SDS
    9.9 mL Acrylamide/Bis-acrylamide(30%/0.8% w/v)
    0.15 mL 10% (w/v) ammonium persulfate (APS)
    0.02 mL TEMED
2). Mix the ingredients mentioned above.
3). Pour the solution quickly into a gel casting form, and leave about 2 centimeters below the bottom of the comb for the stacking gel. Then layer the top of the gel with water very carefully. Wait for about 30 min for the gel to polymerize completely.
4). While waiting, mix the reagents for the stacking gel (4% Acrylamide) by adding components to a clean glass in the order as follow:
    3.075 mL H2O
    1.25 mL 0.5 M Tris-HCl, pH 6.8
    0.025 mL 20% (w/v) SDS
    0.67 mL Acrylamide/Bis-acrylamide (30%/0.8% w/v)
    0.025 mL 10% (w/v) ammonium persulfate (APS)
    0.005 ml TEMED
5). Mix the ingredients mentioned above.
6). Remove the water on the top of running gel, and pour the stacking gel on top of the running gel. Insert the combs by trying not to produce bubbles stuck underneath and allow another 0.5 - 1 h for complete polymerization.
7). Prepare samples: mix the protein 4:1 with the loading buffer. Heat the sample by Boiling for 5-10 min.
8). Run gel. Clamp the gel and fill both buffer chambers with gel running buffer. Pipet the sample and molecular weight standard marker into the gel. Attach the power leads and run the gel until the blue dye reaches the bottom.
9). Visualize the proteins using Coomassie Brilliant Blue.

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## Yeast Transformation

---

1. Spread the yeast on YPD plates, incubate 2 d at 30˚C.
2. Pick a single colony and inoculate it into YPD broth, and shake for 1 d at 30˚C 200 rpm.
3. Dilute yeast at 1:1000 into 30 mL YPD until the OD value lies at 0.6-1.0. Collect yeast after 3000 rpm for 5 min.
4. Resuspend using 30 mL of ddH2O and discard the supernatant.
5. Add 1.5 mL of suspensions (TE: LiAc: H2O=1:1:8) centrifuge at 3000 rpm for 5min, discard the supernatant.
6. Resuspend yeast with 1.5 mL of suspensions, distribute to Eppendorf tubes for 50 µL each.
7. Add 5 µL of plasmid, 10 µL of Salmon sperm DNA, 300 µL of transformation reagent (TE: LiAc: H2O=1:1:8) into 50 µL of bacteria, blend gently.
8. Hot bath at 30˚C for 30 min, blend each 15 min.
9. Hot bath at 45˚C for 15 min.
10. Cold bath for 2 min.
11. Discard supernatant after 50000 rpm for 3 min then rests-end with 500 µL of ddH2O.
12. Pipette 100 µL of bacterium solution on a SC-U glucose plate, spread. Incubate for 3-4 d at 30˚C.

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## Genome extraction from Yeast

---

1. Centrifuge 1 mL of bacterium solution for 1 min, discard the supernatant.
2. Add 500 µL of GHA and 10 µL of proteinase K into the remaining and place inside a metal bath at 65˚C 15 min 800 rpm.
3. 99˚C 800 rpm 15 min.
4. After cooling, add 600 µL of GHC and well blend with vortex.
5. Pipette the solution to a spin column, 700 µL per time until all solution is added. Centrifuge with 12000 rpm for 1 min, discard the flow-through. Repeat this step until all solution is centrifuged and the supernatant discarded.
6. Add 8 µL of Buffer PD and let stand for 2 min. Centrifuge 12000 rpm for 1 min, discard the flow-through.
7. Repeat 6.
8. Add 600 µL of Buffer PW, let stand for 2 min. Centrifuge 12000 rpm for 1min then discard the flow-through.
9. Repeat 8.
10. Centrifuge at 12000 rpm for 3 min without adding anything.
11. Transport solutions to a new 1.5 mL Eppendorf tube. Dry the tube with the lid open for 5 min.
12. Add 100 µL of Buffer TB. Close the lid and let stand in 50˚C for 5 min.
13. Centrifuge at 12000 rpm for 3 min. Flow-through inside the Eppendorf tube is desired DNA.
14. Obtain concentration with a microplate reader

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## *E. coli* Shake-flask Fermentation

---

.1. Plate a preserved culture to obtain single colonies.
2. Preculture: Pick a single colony from the plate to 3 mL of culture with antibiotic (LB), grow overnight in 37˚C.
3. Add 180 µL of medium with antibiotics into each hole in a 96 well plate, then pipette 20 µL of bacterium culture into each well. Use LB medium for blanking.
4. Incubate the bacterium culture into the 50 mL medium with OD600 adjusted to 0.01 into a clean, sterilized 250 mL shake-flask.
5. Add 25 µM IPTG when OD600 value is 1.
6. 24 h after adding IPTG, transport a sample of 5 mL bacterium solution into a 15 mL centrifuge tube. Store it in a -20ºC refrigerator.

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## Preparation of gas chromatography samples from *E. coli*

---

1. Pre-warm the -20ºC bacterium culture sample.
2. Pipette 1mL organic phase (n-Hexane) to 5 mL of bacterium solution in a 15 mL centrifuge tube.
3. Vortex for 3 min.
4. Seal the 15 mL centrifuge tube with biofilm and store it in -20ºC refrigerator overnight.
5. Centrifuge 5000 rpm, 3 min to separate the organic phase from bacterium culture.
6. Transport 100 µL of organic phase to 900 µL of ethyl acetate.
7. Filter the mixture.
8. Sample is ready for gas chromatography analysis.

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## Preparation of standard geraniol sample

---

1. Dilute 97% geraniol into dodecane in the ratio of 1:1000. 100µL geraniol was added to 900 µL dodecane, noted as solution one. Then 100 µL of solution one was added to 900 µL dodecane, noted as solution two. 100 µL of solution two was added to 900 µL dodecane.

2. Dilute the solution into different concentrations with the aid of this table.

*![](https://static.igem.wiki/teams/5654/logo/protocol1.webp)*
3. With help of small injector and green organic phase filter, squeeze the solution into the GC glass tube.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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## Co-culture

---

1. Pick a free colony of *E. coli* into 5mL of LB (Amp 100 µg/mL, Kan 50 µg/mL) in each hole of a 96-well deep well plate, and grow overnight at 37˚C, 250 rpm.
2. Pick a yeast colony into each well of 5 mL LB of a 96-well deep well plate.
3. Place each of the *E. coli* and Yeast separately in a 250 mL conical flask, culture with 50 mL of corresponding broth. Initial OD value is 0.2.
4. Grow overnight culture of Yeast. *E. coli* grows until mid-log phase.
5. Put both Yeast and E. coli into a 50 mL co-culture medium, initial OD value is 0.2. The engineered *E. coli* and the engineered yeast were transferred to the synthetic fermented medium at an initial OD600 respectively and cultivated at 30˚ C, 220 rpm.

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## Yeast Fermentation

---

1. Streak Yeast on SC-Ura plate.
2. Pick a free colony into 1mL of liquid SC-Ura broth. Grow at 28˚C 250 rpm, 24 h.
3. inculcate 300 µL of bacterium solution to 30 mL of YPD broth. Grow culture for 24 h.
4. Prepare a 200 mM geraniol, pipette 40 µL each 2 h. Grow culture for 24 h at 28˚C.
5. Pipette 5 mL of fermenting liquid to 1 mL of n-hexane for extraction. Vortex mix for 2 min. Let stand overnight for extraction. Pipette 200 µL of organic phase into 800 µL of n-hexane.
6. GC characterization.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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## Ingredients of main certain mediums

---

**Materials and Concentration of LB**

    Tryptone 10 g/L
    Yeast extract 5 g/L
    NaCl 10 g/L

**Materials and Concentration of Co-culture**

    KH2PO4 13.3 g/L
    Citric acid 1.7 g/L
    (NH4)2HPO4 4 g/L
    MgSO4 pH7.0 1.3 g/L
    Yeast extract 5 g/L

**Materials and Concentration of YPD**

    Yeast extract 10 g/L
    Tryptone 10 g/L
    ddH2O 900 mL
    Agar 20 g
    20% glucose mother liquor 100 mL

**Materials and Concentration of SC-Ura**

Yeast Nitrogen Base Without Amino acids 6.7 g/L
    Yeast Synthetic drop-out without uracil 1.3 g/L
    Agar 30 g
    ddH2O 900 mL
    20% glucose 100 mL

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](https://2025.igem.wiki/ICJFLS/experiments#early)
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{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Our efforts will alleviate your anxious mood and smooth your tired body and mind.
{% endblock %}
{% block page\_content %}

#### Index

[###### Preface](https://2025.igem.wiki/ICJFLS/results#heading-1)
[###### 1. Production of Geraniol by *S. cerevisiae*](https://2025.igem.wiki/ICJFLS/results#heading-2)
[1.1 Construction and expression of pYES2-GPPS-GES](https://2025.igem.wiki/ICJFLS/results#heading-3)
[1.2 Knock down the ERG20 gene](https://2025.igem.wiki/ICJFLS/results#heading-4)
[1.3 Detection geraniol production](https://2025.igem.wiki/ICJFLS/results#heading-5)
[###### 2. Production of citronellol and linalool by *E. coli*](https://2025.igem.wiki/ICJFLS/results#heading-6)
[2.1 Construction of pACYCDuet-1-GER-Lis plasmid](https://2025.igem.wiki/ICJFLS/results#heading-6)
[2.2 Construction of orthogonal combination plasmid-GER-Lis](https://2025.igem.wiki/ICJFLS/results#heading-8)
[2.3 Expression of GER and Lis in *E. coli*](https://2025.igem.wiki/ICJFLS/results#heading-9)
[2.4 Detection the production of linalool and citronellol](https://2025.igem.wiki/ICJFLS/results#heading-10)
[###### 3. Optimization for Metablic Engineering](https://2025.igem.wiki/ICJFLS/results#heading-11)
[3.1 Reducing Product Cytotoxicity](https://2025.igem.wiki/ICJFLS/results#heading-12)
[3.2 Optimization of inducer concentration and induction times](https://2025.igem.wiki/ICJFLS/results#heading-13)
[###### 4. Summary](https://2025.igem.wiki/ICJFLS/results#heading-14)

### Preface

![](https://static.igem.wiki/teams/5654/results/r1.webp)

As shown in the figure above, the design of our project is divided into two parts: the production of geraniol in *S. cerevisiae*, and the production of citronellol and linalool in *E. coli*. Accordingly, the project results will also be presented in these two aspects, plus a third part focusing on metabolic engineering optimization, with each part elaborated separately.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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### 1.Production of Geraniol by *S. cerevisiae*

#### 1.1 Construction and expression of pYES2-GPPS-GES

The native MVA pathway in yeast cells is active. When the key enzyme GPPS is overexpressed, a relatively large amount of GPP can be synthesized. Subsequently, a substantial quantity of geraniol is produced under the catalysis of exogenous GES, and accumulates inside the cells and is later excreted into the extracellular medium. Therefore, the construction of overexpression vectors for GPPS and GES constitutes the foundation and prerequisite of this project.

We selected pYES2 (5.9 kb), the most commonly used expression vector in *S. cerevisiae*, which carries the URA3 selection marker but has only one multiple cloning site (MCS). To reduce the metabolic burden on yeast cells and enable the co-expression of GPPS and GES in cells, we linked these two genes via a ribosomal binding site (RBS) and constructed them together under the control of the galactose-inducible promoter PGAL1 (Fig. 1). This design allows the two genes to be transcribed simultaneously while being translated into two independent proteins.

![](https://static.igem.wiki/teams/5654/results/fig-1-construction-and-identification-of-pyes2-gpps-ges-recombinant-vector.webp)

**Fig. 1 Construction and identification of pYES2-GPPS-GES recombinant vector using *Bam*H I and *Eco*R I restriction enzymes.**
M: Marker; 1: pYES2 plasmid; 2: pYES2-GPPS-GES recombinant plasmid; 3: Digestion of pYES2-GPPS-GES using *Bam*H I and *Eco*R I; 4: PCR product of GPPS using pYES2-GPPS-GES as template; 5: PCR product of GES using pYES2-GPPS-GES as template.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 1.2 Knock down the ERG20 gene in the genome of *S. cerevisiae*

In yeast cells, endogenous geranyl pyrophosphate (GPP) can not only undergo pyrophosphate elimination to form geraniol, but also be converted into farnesyl pyrophosphate (FPP) under the catalysis of FPP synthase (encoded by the ERG20 gene) for the subsequent synthesis of other terpenoids. Notably, the endogenous metabolic flux of S. cerevisiae naturally favors the synthesis of FPP and sterols. To reduce this metabolic flux and enhance the conversion of GPP to geraniol, we used pUG6 plasmid to introduce a mutant FPP synthase (ERG20-F96W-N127W) into the *S. cerevisiae* genome, which replaced the wild-type ERG20 gene in situ while introducing the KanMX selection marker. The activity of the mutant FPP synthase is significantly reduced, retaining only the enzymatic activity necessary for maintaining cell viability (complete knockout of this gene is lethal).

After PCR amplification, the following fragments were ligated using the Gibson assembly method: vector backbone + upstream homology arm + KanMX expression cassette (containing the TEF1 promoter and terminator) + mutant ERG20 gene + downstream homology arm. The ligated product was transformed into *E. coli*; the extracted plasmid was verified by restriction enzyme digestion with *Eco*R I and *Xho* I (Fig. 2). Positive yeast clones were screened on plates containing G418. Additionally, the ERG20 genes from the wild-type and mutant strains were amplified separately via PCR using the same pair of homology arm primers for detection: the PCR product of the wild-type strain was approximately 2 kb (containing some homology arm sequence), while that of the mutant strain was approximately 3.7 kb (with the additional KanMX gene). The electrophoresis results are shown below:

![](https://static.igem.wiki/teams/5654/results/fig-2-erg20-gene-was-replaced-by-the-mutant-erg20-f96w-n127w.webp)

**Fig. 2 ERG20 gene was replaced by the mutant ERG20-F96W-N127W in the genome of *S. Cerevisiae.***
 M: Marker; 1: Gene knockdown plasmid digested with *Eco*R I and *Xho* I (5.0 kb and 3.0 kb) ; 2: PCR product of wild-type ERG20 gene (2kb) using homology arm primers; 3: PCR product of mutant ERG20 gene (ERG20-F96W-N127W) (3.7 kb, containing KanMX gene) using homology arm primers.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 1. 3 Detection geraniol production using gas chromatography

To investigate whether yeast cells can synthesize geraniol after overexpressing GPPS and GES, gas chromatography (GC) was employed for analysis (Fig. 3). When the yeast cells grew to a certain concentration (OD₆₀₀ = 0.8), 2% galactose was used to induce the expression of GPPS and GES, followed by continued cultivation for 24 h. A two-phase extraction method was adopted: n-dodecane was used to extract geraniol from the culture, and geraniol standard was used as a control for analysis via gas chromatography. Two samples were included in the experiment: Sample 1 was yeast (without ERG20 gene knockdown) overexpressing GPPS and GES; Sample 2 was yeast (with ERG20 gene knockdown) overexpressing GPPS and GES.

![](https://static.igem.wiki/teams/5654/results/fig-3.webp)

**Fig. 3 Gas chromatography analysis for geraniol production by *S. cerevisiae.***
The retention time of geraniol standard was 5.65 min; the retention time of geraniol produced by *S. cerevisiae* was 5.63 min.

The experimental results showed that the retention time of the geraniol standard was 5.65 min. Sample 1 contained little to no geraniol. Sample 2 exhibited a peak with a retention time (5.63 min) essentially consistent with that of the geraniol standard, indicating geraniol synthesis. The concentration of geraniol in Sample 2 was determined to be 8.58 mg/L. However, this concentration is relatively low, necessitating further optimization of the synthesis conditions.

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### 2. Production of citronellol and linalool by *E. coli*

#### 2.1 Construction of pACYCDuet-1-GER-Lis plasmid

To prevent competition among multiple plasmids in *E. coli*, a high-copy multi-gene co-expression vector pACYCDuet-1 was selected. Two genes, GER and Lis, were cloned into two distinct multiple cloning sites (MCSs) of this plasmid. Both genes, however, are expressed under the control of the strong T7/lac promoter, enabling the "one-click activation" of the synthesis of multiple essential oil components. Results from PCR and restriction enzyme digestion confirmed the successful construction of the pACYCDuet-1-GER-Lis plasmid (Fig. 4).

![](https://static.igem.wiki/teams/5654/results/fig-4-construction-and-identification-of-pacycduet-1-ger-lis-recombinant-vector.webp)

**Fig. 4 Construction and identification of pACYCDuet-1-GER-Lis recombinant vector using *Eco*R I, *Pst* I, *Nde* I and *Kpn* I restriction enzymes.**
M: Marker; 1: pACYCDuet-1 Plasmid; 2: pACYCDuet-1-GER-Lis; 3: Digestion of pACYCDuet-1-GER-Lis with *Eco*R I and *Pst* I; 4: PCR product of GER using pACYCDuet-1-GER-Lis as template; 5: Digestion of pACYCDuet-1-GER-Lis with*Nde* I and *Kpn* I; 6: PCR product of GER using pACYCDuet-1-GER-Lis as template.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 2.2 Construction and identification of orthogonal combination plasmid-GER-Lis

After the successful construction of the recombinant plasmid pACYCDuet-1-GER-Lis, it was noted that although the two genes, under the control of the same strong T7/lac promoter, enable the "one-click activation" of the synthesis of two essential oil components, namely citronellol and linalool, separate regulation cannot be achieved.

This issue can be addressed using an orthogonal expression system, which allows the expression of two promoters to be induced by two different inducers on the same plasmid. A literature search revealed no such ready-to-use commercial plasmids, necessitating de novo construction.

We first cloned the GER gene into plasmid pET28a(+) (Fig. 5), which contains a LacI-IPTG regulated promoter, and then cloned the Lis gene into plasmid pBAD (Fig. 6), which contains an AraC-arabinose regulated promoter. These two regulatory systems are fully orthogonal and do not interfere with each other. Subsequently, they were fused using homologous recombination PCR (Fig. 7).

![](https://static.igem.wiki/teams/5654/results/fig-5n-construction-and-identification-of-pet28a-ger-recombinant-vector.webp)

**Fig. 5 Construction and identification of pET28a (+)-GER recombinant vector using *Hin*d III and *Eco*R I restriction enzymes.**
M: Marker; 1: pET28a (+) Plasmid; 2: pET28a (+) – GER plasmid; 3: Digestion pET28a (+) – GER with *Hin*d III and *Eco*R I; 4: PCR product of GER using pET28a (+) – GER as template.

![](https://static.igem.wiki/teams/5654/results/fig-6nconstruction-and-identification-of-pbad-lis-recombinant-vector.webp)

**Fig. 6 Construction and identification of pBAD-Lis recombinant vector using *Pst* I and *Eco*R I restriction enzymes.**
M: Marker; 1: pBAD Plasmid; 2: pBAD-Lis plasmid; 3: Digestion of pBAD-Lis plasmid with *Pst* I and *Eco*R I; 4: PCR product of Lis using pBAD-Lis as template.

![](https://static.igem.wiki/teams/5654/results/fig-7n-construction-and-identification-of-orthogonal-combination-plasmid-ger-lis.webp)

**Fig.7 Construction and identification of Orthogonal combination plasmid-GER-Lis using homologous recombination PCR method.**
M: Marker; 1: PCR product of pET28a(+)-GER containing KanR, GER, T7/lac promoter and LacI genes, etc (4.4kb) ; 2: PCR product of pBAD-Lis containing AraC, pBAD promoter, Lis, ApR genes and Ori site (5.5kb) ; 3: Orthogonal combination plasmid-GER-Lis; 4: PCR product of GER using Orthogonal combination plasmid-GER-Lis as template; 5: PCR product of Lis using Orthogonal combination plasmid-GER-Lis as template.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 2.3 Expression of GER and Lis in *E. coli* tranformed with Orthogonal combination plasmid-GER-Lis

To verify the transformation of the orthogonal combination plasmid-GER-Lis in *E. coli*, protein purificationu using His-tag and SDS-PAGE electrophoresis were performed on the overexpressed GER and Lis proteins. The results showed that the plasmid was well-expressed in *E. coli*, and the amounts of GER (46.8 kDa) and Lis (76.1 kDa) proteins were significantly higher than those in the control group (Fig. 8).

![](https://static.igem.wiki/teams/5654/results/fig-8-purification-of-ger-and-lis.webp)

**Fig.8 SDS-PAGE indicates the expression and purification of GER and Lis expressed in *E. coli* transformed with Orthogonal combination plasmid-GER-Lis.**
 M: protein marker; 1: The supernatant obtained by centrifuging the total lysate of transformed cells; 2. The supernatant obtained by centrifuging the lysate of untransformed cells; 3. The residual liquid remaining after purifying the overexpressed proteins from the lysate of transformed cells using a His-tag column; 4. The purified overexpressed GER (46.8kD) and Lis (76.1kD) proteins.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 2.4 Detection the production of linalool and citronellol after transformation of Orthogonal combination plasmid-GER-Lis into *E. coli*

Although we verified that the GER and Lis genes can be overexpressed in BL21 cells, further validation is required to confirm whether the expressed enzymes are active and capable of converting geraniol into citronellol and linalool. We also used gas chromatography for analysis.

We are aware that overexpression of exogenous genes imposes a burden on cells, so we selected inducible plasmids. When *E. coli* grew to an OD₆₀₀ of 1.0, 100 µM IPTG and 500 µM L-arabinose were used to simultaneously induce the expression of GER and Lis. Additionally, 200 mM geraniol standard was added every two hours to a final concentration of 50 mg/L, followed by continued fermentation for 24 h. The culture was overlaid with n-dodecane to extract terpenoids, which were analyzed by gas chromatography using geraniol, citronellol, and linalool standards as controls (Fig. 9).

![](https://static.igem.wiki/teams/5654/results/fig-9.webp)

**Fig.9 Gas chromatography analysis for geraniol, citronellol and linalool production by *E. coli*.**
The retention times of geraniol, citronellol, and linalool standards were 5.64 min, 5.16 min, and 3.26 min, respectively. The retention times of these components in the samples are essentially consistent with those of the standard substances.

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### 3. Optimization for Metablic Engineering

#### 3.1 Reducing Product Cytotoxicity

The core challenge in microbial synthesis of monoterpenes is product toxicity. A common solution to this issue is the use of biphasic extraction fermentation (in-situ product removal, ISPR), which involves introducing a water-immiscible organic solvent as the second phase into the fermentation system. This allows real-time extraction of the produced terpenoids from the aqueous phase (culture medium), thereby maintaining the product concentration in the aqueous phase at a low-toxicity level and ensuring cell growth and metabolic activity.

Literature search revealed that researchers have used different organic phases for extraction, some of which are inherently toxic to microorganisms. To identify the most suitable solvent for extracting citronellol and linalool, we compared three substances: isopropyl myristate (IPM), n-dodecane, and n-hexane, each used as an organic overlay, to determine which performs best.

Induction of GER and Lis expression was initiated when the OD₆₀₀ of *E. coli* reached 0.8. Four hours after induction, 10% (v/v) of each of the three organic extraction solvents mentioned above was added for fermentation. Samples were taken every 12 h, totaling five samplings. The concentrations of citronellol and linalool in the organic phase, as well as the OD₆₀₀ and glucose concentration in the aqueous phase, were measured for each sample. The results are shown in the figure below (Fig. 10).

![](https://static.igem.wiki/teams/5654/results/fig-10.webp)

**Fig.10 Comparative analysis for the citronellol and linalool production, OD600, and glucose in the medium of *E. coli*, using different extraction solvent.**
(A): Concentration of extracted citronellol; (B): Concentration of extracted linalool; (C): OD600 value of *E. coli*; (D): Concentration of glucose in the medium.

By comparing the growth curves (OD₆₀₀) of the IPM group, n-dodecane group, n-hexane group, and the control group, it was found that the growth curves of the IPM group and n-dodecane group were similar to that of the control group, indicating good biocompatibility. In contrast, the growth rate of the n-hexane group was significantly reduced, suggesting that this solvent exerted a distinct toxic inhibitory effect on *E. coli*. Analysis revealed that n-hexane, with its small molecular size and strong hydrophobicity, tends to damage cell membranes, making it unsuitable as an extractant during fermentation. The IPM group and n-dodecane group showed similar product extraction efficiencies and good biocompatibility, making them suitable as in-situ extraction solvents for citronellol and linalool.

The characteristics of the three extractants were further analyzed by evaluating the biomass-specific productivity (= total yield/final OD₆₀₀ value) and product yield (= total mass of product formed/mass of glucose consumed) (Fig. 11).

![](https://static.igem.wiki/teams/5654/results/fig-11.webp)

**Fig.11 Comparative Analysis of Biomass-Specific Productivity and Product Yield When Using Three Extractants.**
(A): Biomass-specific productivity of citronellol; （B): Biomass-specific productivity of linalool; （C): Product yield of citronellol; （D): Product yield of linalool.

Comparative analysis revealed that n-hexane exhibited the highest biomass-specific productivity; however, further analysis showed that the product yields of the three extractants were nearly consistent. Therefore, the conclusion was drawn: IPM and n-dodecane are suitable for extracting products during fermentation, while n-hexane can be used as an extractant after fermentation.

[![](https://static.igem.wiki/teams/5654/logo/return-to-index.webp)](#early)
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#### 3.2 Optimization of inducer concentration and induction times

Our target final products are mixed essential oil components containing citronellol and linalool in different proportions, which can be produced in a single fermation process. The achievement of this goal mainly depends on the activity ratio of the key enzymes responsible for synthesis of these two substances, as they share the same substrate, geraniol. These two key enzymes (GER and Lis) are constructed under the control of two distinct promoters (pT7/lac and pBAD) on the same plasmid, with their expression induced by IPTG and L-arabinose respectively. These two promoters form a fully orthogonal regulatory system (non-interfering regulation).

However, the induction efficiencies of these two fully orthogonal regulatory systems differ, leading to variations in the expression levels of the enzymes and the catalytic efficiency of product synthesis. Additionally, there exists competition between the two enzymes for substrate utilization and carbon source consumption. Therefore, to determine the working efficiency of the two promoters, we conducted experiments on the combined induction of expression using different concentrations of IPTG and L-arabinose (Fif. 12). The efficiency was evaluated based on the ratio of the product concentrations of citronellol (synthesized under IPTG induction) to linalool (synthesized under L-arabinose induction).

![](https://static.igem.wiki/teams/5654/results/fig-12.webp)

**Fig.12 The measured product ratio of citronellol to linalool after 12 h of combined induction with different concentrations of IPTG and L-arabinose.**
The thermodynamic diagram (heatmap) shows the ratio of two products induced by IPTG and Arabinose at different concentrations. Different colors are used in the figure to represent different product ratios, with darker colors indicating lower ratios and lighter colors indicating higher ratios. A: Induced production ratio with higher arabinose concentration; B: Induced production ratio with lower arabinose concentration.

Comparing the heatmap of A and B in Fig.12, we figure out that after 12 h of co-induction with a relatively high concentration of arabinose (1 mM – 10 mM, Fig. 12 A) and a relatively low concentration of IPTG (0.1 mM – 1.0 mM), the product ratio (citronellol:linalool) fluctuates within the range of 1 to 10. In contrast, after 12 h of co-induction with a relatively low concentration of arabinose (0.1 mM – 2.5 mM, Fig. 12 B) and a relatively low concentration of IPTG (0.1 mM – 1.0 mM), the product ratio (citronellol:linalool) fluctuates within the range of 0.1 to 3. The latter is more likely to meet the needs of most users for different proportions of these two components.

Next, we designed two additional experiments involving sequential induction with the two inducers: Experiment A: First, induce expression with 0.4 mM IPTG for 6 h, then induce with different concentrations of arabinose for 12 h, during which the ratio of the two products was measured. Experiment B: First, induce expression with 1 mM arabinose for 6 h, then induce with different concentrations of IPTG for 12 h, during which the ratio of the two products was measured (Fig. 13).

![](https://static.igem.wiki/teams/5654/results/fig-13.webp)

**Fig.13 The Effect of Different Induction Orders of IPTG and Arabinose on the Ratio of the Two Products.**
A: The thermodynamic plot (heatmap) illustrates the effects of different arabinose concentrations and induction durations on the product ratio after 6 h of IPTG (0.4 mM) induction. B: The thermodynamic plot (heatmap) shows the effects of different IPTG concentrations and induction durations on the product ratio after 6 h of Arabinose (1 mM) induction. In the plot, different colors represent different product ratios: darker colors indicate lower ratios, while lighter colors indicate higher ratios.

As can be seen from Fig. 13 , even if the two inducers have different induction timings (i.e., induced sequentially), the final desired product ratio can still be achieved by adjusting the concentrations of the inducers.

From the above figures, the optimal configuration can be identified according to the requirements for different product ratios. It is expected that these data will provide support for the personalized production of essential oil components with different proportions.

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### 4. Summary

In conclusion, through iterative design and optimization, we have successfully constructed an orthogonal expression plasmid. This plasmid contains two non-interfering promoter control systems, which can respectively induce the expression of two different genes (GER and Lis) under the regulation of two different inducers. After these two genes are expressed, they can catalyze the synthesis of citronellol and linalool with their respective efficiencies, so as to meet the needs of different users for the ratio of the two essential oil components.

 In the future, we plan to integrate machine learning algorithms with real-time metabolic flux analysis to establish a dynamic mapping model between user preferences and fermentation parameters. Users only need to input their preference for the aroma ratio of rose to lavender (e.g., 7:3 or 5:5), and the system will automatically optimize culture parameters and induction parameters to precisely allocate carbon flow to target products.

![](https://static.igem.wiki/teams/5654/results/lab.webp)

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