iGEM 2025 — Team uprize-ii

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# 1. Experiment Phase 1: Construction of engineered strains containing GPS, CsTPS1, and GeDH.

## Plasmid Recombination and Transformation Experiment 1

**Objective:** Using pET28a, pET21a, and pBAD33 as vectors, the amino acid sequences of the three enzymes, GPS, CsTPS1, and GeDH, were designed into three recombinant plasmids respectively to facilitate subsequent strain screening and target protein expression.

### Design of High-Copy Recombinant Protein Plasmids 01/07/2025 Tuesday

**Objective:** Optimize the sequences of the three enzymes (GPS, CsTPS1, and GeDH) and insert these gene sequences into the vector plasmid pUC19 using restriction enzyme digestion and ligation methods to obtain high-copy recombinant protein plasmids. These sequences were designed in SnapGene.

**Results:**

![Fig.1 Recombinant plasmid designs for pUC19-GPS, pUC19-CsTPS1 and pUC19-GeDH](https://static.igem.wiki/teams/5938/notebook/fig1.webp)

Fig.1 Recombinant plasmid designs for pUC19-GPS, pUC19-CsTPS1 and pUC19-GeDH

**Participants:** Ethan Wu, Boyan Zhang

### Design of High-Expression Recombinant Protein Plasmids 01/07/2025 Tuesday

**Objective:** Using pET28a, pET21a, and pBAD33, three high-expression plasmids, as vectors, the amino acid sequences of the enzymes GPS, CsTPS1, and GeDH were utilized to construct recombinant protein expression systems via seamless cloning techniques, aiming to achieve higher target protein expression levels.These sequences were designed in SnapGene.

**Results:**

![Recombinant plasmid designs for pET28a-GPS, pET21a-CsTPS1 and pBAD23-GeDH](https://static.igem.wiki/teams/5938/notebook/fig2.webp)

Fig.1 Recombinant plasmid designs for pUC19-GPS, pUC19-CsTPS1 and pUC19-GeDH

**Participants:** Ethan Wu, Boyan Zhang

### Obtaining the High-Copy Recombinant Protein Plasmids 08/07/2025 Tuesday

**Objective:** The high-copy protein plasmid we designed is stored in *E. coli*. To obtain the target gene fragments, it needs to be extracted.

**Results:** The name and concentration of the plasmids are shown in Table 1.

Table 1. The name and concentration of the plasmids.

| Name | Concentration (ng/μl) |
| --- | --- |
| pUC19-GPS | 342.05 |
| pUC19-CsTPS1 | 281.00 |
| pUC19-GeDH | 294.83 |

**Participants:** Ruiyang Wen, Yanzhao Liu

### Obtaining the target gene fragments 08/07/2025 Tuesday

**Objective:** The fragments of GPS, CsTPS1, and GeDH genes were obtained through restriction enzyme digestion from the pUC19 plasmid designed in the previous experiment. Subsequently, PCR amplification was performed for the subsequent Gibson cloning.

**Results:** Through the experiment, we expect to have obtained fragments of the three target genes. The results will be further confirmed through agarose gel electrophoresis and sequencing. The concentrations of the three target gene fragments after PCR amplification are shown in Table 2.

Table 2. The name and concentration of the target gene fragments.

| Name | Concentration (ng/μl) |
| --- | --- |
| GPS | 205.18 |
| CsTPS1 | 199.72 |
| GeDH | 186.13 |

**Participants:** Ruiyang Wen, Yanzhao Liu

### Obtaining the vectors 08/07/2025 Tuesday

**Objective:** In order to assemble the recombinant protein plasmids, we need to design primers targeting appropriate sites on the three plasmids, pET28a, pET21a, and pBAD23, and perform PCR amplification to obtain sufficient vector for Gibson cloning.

**Results:** The concentrations of the three plasmids after PCR amplification are shown in Table 3.

Table 3. The name and concentration of the plasmids.

| Name | Concentration (ng/μl) |
| --- | --- |
| pET28a | 172.66 |
| pET21a | 169.45 |
| pBAD23 | 177.53 |

**Participants:** Ruiyang Wen, Yanzhao Liu

### Validation of vector and target gene 07/08/2025 Tuesday

**Objective:** Preliminary confirmation of vector and target gene acquisition using DNA agarose gel electrophoresis. Preparing for the next step of Gibson cloning.

**Results:** Preliminary validation of the obtained vector and target gene fragments was performed using DNA agarose gel electrophoresis. The sizes of GeDH, CsTPS1, and GPS fragments are 1266 bp, 1665 bp, and 1158 bp, respectively. The sizes of the pET28a, pET21a, and pBAD23 vectors are 5206 bp, 5340 bp, and 5352 bp, respectively. The specific results are shown in Fig. 3.

![Fig.3 M: Marker; 1:GeDH; 2:CsTPS1; 3:GPS; 4: pET28a; 5: pET21a; 6: pBAD23](https://static.igem.wiki/teams/5938/notebook/fig3.webp)

Fig.3 M: Marker; 1:GeDH; 2:CsTPS1; 3:GPS; 4: pET28a; 5: pET21a; 6: pBAD23

**Notes:** 1% Agarose gel, running time: 25min

**Analysis:** The obtained fragment sizes are consistent with our expectations, allowing us to preliminarily conclude that the fragments are correct.

**Participants:** Hanying Yu, Yilin Qian

### Assembly and transformation 07/09/2025 Wednesday

**Objective:** The vector and target gene fragments obtained in the previous experiment were joined together to form a complete recombinant protein plasmid by Gibson cloning. Then transform the BL21 strain with plasmids carrying the target gene sequences to produce bacterial strains for subsequent protein expression and purification.

**Notes:** Since the three plasmids carry Kanamycin, Ampicillin, and Chloramphenicol resistance genes, the ability of the strains to grow on LB plates containing these antibiotics can be used as a preliminary indication that the three plasmids have been successfully introduced into the BL21 strains.

**Results:** Out of the 4 LB plates, 3 did not show any transformed colonies, and 1 was contaminated with unwanted bacteria.

![Fig.4 First transformation results](https://static.igem.wiki/teams/5938/notebook/fig4.webp)

Fig.4 First transformation results

**Analysis:** The failure may have been caused by improper handling during the transformation process. Most of the team members are not very familiar with the experimental procedures, and some details, as well as aseptic techniques, might not have been properly followed during the experiment.

**Participants:** Hanying Yu, Yilin Qian

### Second transformation 07/12/2025 Saturday

**Objective:** The first transformation was unsuccessful, but one contaminated plate still showed colony growth. A second transformation was attempted, and the engineered strain was successfully constructed.

**Results:** One LB plate grew the desired colonies in this experiment. Whether the transformation was successful still requires further validation.

![Fig.5 Second transformation results](https://static.igem.wiki/teams/5938/notebook/fig5.webp)

Fig.5 Second transformation results

**Participants:** Hanying Yu, Yilin Qian

## The validation of transformation Experiment 2

**Objective:** Using colony PCR and gene sequencing to confirm the presence of the three recombinant protein plasmids in the engineered strains.

### Design of primers for colony PCR 07/16/2025/ Wednesday

**Objective:** Primers were designed upstream and downstream of the target gene to perform PCR amplification on the engineered strains. The size of the bands was used to determine whether the target gene fragment was successfully assembled.

**Results:** Three primers were designed to perform PCR amplification on the three plasmids. The upstream and downstream regions of the primers were designed to avoid the target gene, so that if the transformation is successful, an ideal-length fragment can be amplified.

Table 4. The primer sequences for GPS, CsTPS1 and GeDH genes

| Gene types | Forward Primer | Reverse Primer |
| --- | --- | --- |
| GPS | 5'-CTGAATTGACTCTCTTCCG-3' | 5'-CCGGGCCGCGGACGCCAGC-3' |
| CsTPS1 | 5'-ACAGCAATGGCATCCTGGTC-3' | 5'-CTCTTCCTTTTCTCGCCACG-3' |
| GeDH | 5'-CGCTGATGTACTGACAAGCC-3' | 5'-TTAACTGCCCCGTCCTGCCG-3' |

**Participants:** Ethan Wu, Zibin Tang

### Colony PCR 07/20/2025 Sunday

**Objective:** Using the three designed primers, two colonies from the second transformation plate were selected for PCR. The size of the amplified bands was observed to determine the success of the amplification.

**Results:** The results of DNA agarose gel electrophoresis showed that the band sizes matched the expected ones(2754bp, 3234bp and 1911bp), as shown in Fig.6. This suggests that the transformation was successfully achieved.

![Fig.6 M: Marker; 1,2:GeDH; 3,4:CsTPS1; 5,6:GPS](https://static.igem.wiki/teams/5938/notebook/fig6.webp)

Fig.6 M: Marker; 1,2:GeDH; 3,4:CsTPS1; 5,6:GPS

**Analysis:** The results of DNA agarose gel electrophoresis are consistent with the expectations. The electrophoresis image shows a small amount of primer dimer. To present the results more clearly, only the electrophoresis image of the target band was included.

**Notes:** 1% Agarose gel, running time: 23min

**Participants:** Ethan Wu, Zibin Tang

### DNA sequencing 07/21/2025 Monday

**Objective:** Two strains verified by colony PCR were subjected to Sanger sequencing to obtain definitive results, facilitating subsequent experiments.

**Results:** The sequencing results indicate that all three plasmids were successfully introduced into the BL21 strain, confirming that the engineered strain has been successfully constructed.

**Analysis:** The sequencing results were identical to the known sequences, indicating that the plasmid had been successfully introduced.

**Participants:** Ethan Wu, Zibin Tang

### Bacterial Stock Preparation 07/26/2025 Friday

**Objective:** Prepare bacterial stocks from two colonies grown on LB plates, verified by colony PCR and sequencing, for long-term storage and use .

**Results:** oSix bacterial stocks were obtained and stored at -80oC.

**Notes:** Before bacterial stock preservation, the cultures should be incubated for a sufficient duration to ensure an adequate bacterial concentration , facilitating subsequent revival .

**Participants:** Zibin Tang, Yilin Qian

### Validation of Growth Capacity of Engineered Strains 07/26/2025 Saturday

**Objective:** By tracking the OD600 during the growth of engineered *E. coli*, wild-type *E. coli*, and *E. coli* with empty vector, we verified whether the growth capacity of the engineered strains was affected.

**Results:** Take 9 x 50 mL Erlenmeyer flasks and divide them into 3 groups:

Group 1: Engineered bacteria + LB + antibiotics

Group 2: Wild-type bacteria + LB

Group 3: Empty vector bacteria + LB + antibiotics

Ensure the initial OD600 is 0.2 for each group during inoculation. Cultivate in LB medium for 24 hours. According to the analysis of the growth curve results, there was no significant difference in growth capacity among the engineered strain, the empty vector strain, and the wild-type strain. Therefore, it can be concluded that the growth capacity of the engineered strain did not significantly decrease(Fig.7).

![Fig.7 Group 1: Engineered bacteria + LB + antibiotics](https://static.igem.wiki/teams/5938/notebook/fig7.webp)

Fig.7 Group 1: Engineered bacteria + LB + antibiotics; Group 2: Wild-type bacteria + LB;
 Group 3: Empty vector bacteria + LB + antibiotics

**Participants:** Zibin Tang, Yilin Qian

# 2. Experiment Phase 2: Validation of transcription and translation.

## Validation at the transcriptional level Experiment 1

**Objective:** At the transcriptional level, the bacteria were validated to confirm whether the target gene was successfully transcribed in BL21. The analysis of the target gene's mRNA levels provides guidance for optimizing expression conditions and ensures a solid foundation for subsequent experiments.

### Bacterial Strain Activation 07/27/2025 Sunday

**Objective:** Revive the BL21-GPS, CsTPS1, and GeDH strains stored at -800C to restore their optimal growth conditions and accelerate proliferation.

**Results:** After cultivation, the bacterial strains in the shake flasks have been revived.

**Participants:** Zibin Tang, Yilin Qian

### Scale-up cultivation 07/27/2025 Sunday

**Objective:** Cultivate the engineered strains in a larger culture system to obtain bacterial strains in the logarithmic growth phase.

**Results:** The LB liquid medium became obviously turbid, and after 6 hours of cultivation, the OD600 reached 0.698.

**Analysis:** When OD600 is approximately 0.6-0.8, the bacteria are in the actively growing logarithmic phase, which is suitable for inducing the transcription of the target gene .

**Participants:** Ruiyang Wen, Yanzhao Liu

### Induction and cell harvesting 07/28/2025 Monday

**Objective:** Use IPTG and L-arabinose as inducers to initiate the transcription of the GPS, CsTPS1, and GeDH genes, followed by cultivation and bacterial collection.

**Results:** After induction, the OD600 reached 1.953. Ten milliliters of the bacterial culture were taken to collect the cells.

**Participants:** Ruiyang Wen, Yanzhao Liu

### Total RNA extraction 07/29/2025 Tuesday

**Objective:** Verify whether the GPS, CsTPS1, and GeDH genes have been successfully transcribed into mRNA. Provide a template for subsequent reverse transcription and qPCR analysis.

**Results:** The A260/A280 ratio was 1.915 , indicating good purity and integrity with no significant DNA contamination. The concentration was 315.12 ng/µl, making it suitable for subsequent transcription-level validation experiments.

**Participants:** Ruiyang Wen, Yanzhao Liu

### Reverse transcription to cDNA 07/29/2025 Tuesday

**Objective:** Reverse transcribe total RNA into cDNA to prepare for subsequent qPCR analysis.

**Results:** After reverse transcription, the cDNA concentration was 78.23 ng/µL, which is suitable for use as a template for qPCR.

**Analysis:** The theoretical concentration of cDNA is 100 ng/µL. The actual concentration is lower, likely due to losses during the experimental process.

**Participants:** Ruiyang Wen, Yanzhao Liu

### Design of qPCR primers 07/29/2025 Tuesday

**Objective:** In the sequences of GPS, CsTPS1, and GeDH, select about 200 bp conserved and non-repetitive regions for amplification. Design three primers of approximately 20 bp each to prepare for the qPCR experiment.

**Results:** The designed primer sequences are shown in Table 5.

Table 5. The qPCR primer sequences for GPS, CsTPS1 and GeDH.

| cDNA types | Forward Primer | Reverse Primer |
| --- | --- | --- |
| GPS | 5'-ATGATCTTTTCCAAAGGCCT-3' | 5'-GCGTGGACGCGGATCATCAC-3' |
| CsTPS1 | 5'-ATGAGCTCCGGTGAAACCTT-3' | 5'-CTCCTTTTCTAGCCTGACGA-3' |
| GeDH | 5'-ATGGTTCAGAACCCGGGTGC-3' | 5'-CACCTGGCAGCAGTTAGCCT-3' |

### qPCR 08/01/2025 Friday

**Objective:** : By analyzing the data obtained from qPCR, the transcription levels of the three target genes, GPS, CsTPS1, and GeDH, were validated.

**Results:** We selected the bacteria before induction as the control group, while the bacteria after induction were used as the treated group. Detailed results are shown below.

Table 6. Average of Ct values obtained from qPCR.

| Gene types | Sample | Average of Ct values (Target genes) | Average of Ct values (Reference gene: 16S rRNA) |
| --- | --- | --- | --- |
| GPS | Treated group  Control group | 22.04 26.51 | 19.56 20.02 |
| CsTPS1 | Treated group  Control group | 23.09 27.53 | 19.73 20.00 |
| GeDH | Treated group  Control group | 21.40 25.56 | 19.57 20.07 |

Table 7. ??Ct and relative expression level

| Gene types | Sample | ??Ct | Relative expression level |
| --- | --- | --- | --- |
| GPS | Control group  Treated group | 0 -4.01 | 1 16.11 |
| CsTPS1 | Control group  Treated group | 0 -4.17 | 1 18.00 |
| GeDH | Control group  Treated group | 0 -3.66 | 1 12.64 |

![Fig.8  Relative expression level of GPS, CsTPS1 and GeDH](https://static.igem.wiki/teams/5938/notebook/fig8.webp)

Fig.8 Relative expression level of GPS, CsTPS1 and GeDH

**Analysis:** From the qPCR results, we can conclude that the transcription levels of the three target genes have significantly increased after induction. Prove that all three genes can be extensively transcribed in BL21

**Participants:** Ethan Wu, Boyan Zhang

## Validation at the Translation level Experiment 2

**Objective:** At the translation level, bacteria were validated to confirm whether the target gene was successfully translated into protein in BL21. This part is of great significance for the normal expression of exogenous genes in BL21.

### Bacterial Strain Activation 08/02/2025 Saturday

**Objective:** Revive the BL21-GPS, CsTPS1, and GeDH strains stored at -80oC to restore their optimal growth conditions and accelerate proliferation.

**Results:** After cultivation, the bacterial strains in the shake flasks have been revived.

**Participants:** Yilin Qian, Ruiyang Wen

### Protein Extraction and Quantification 08/04/2025 Monday

**Objective:** Extract total protein from the revived BL21-GPS, CsTPS1, and GeDH strains and quantify the protein concentration using the Nanodrop .

**Results:** Protein extraction was successful, and the concentration of all samples was adjusted to 1 µg/µL for downstream Western Blot analysis.

**Participants:** Yanzhao Liu, Hanying Yu

### Protein Extraction and Quantification 08/04/2025 Monday

**Objective:** Extract total protein from the revived BL21-GPS, CsTPS1, and GeDH strains and quantify the protein concentration using the Nanodrop.

**Results:** Protein extraction was successful, and the concentration of all samples was adjusted to 1 µg/µL for downstream Western Blot analysis.

**Participants:** Yanzhao Liu, Hanying Yu

### SDS-PAGE and Western Blot 08/05/2025 Tuesday

**Objective:** Separate the extracted proteins by SDS-PAGE and transfer them to a PVDF membrane for subsequent antibody detection of GPS, CsTPS1, and GeDH proteins.

**Results:** Proteins were successfully separated by SDS-PAGE, and transfer to the PVDF membrane was confirmed by Ponceau S staining.

**Participants:** Yanzhao Liu, Hanying Yu

### Antibody Incubation and Detection 08/07/2025 Thursday

**Objective:**Incubate the membrane with primary and secondary antibodies to detect GPS, CsTPS1, and GeDH proteins, followed by ECL-based visualization.

**Results:** : Specific bands corresponding to GPS (46.9 kDa), CsTPS1 (63.7 kDa), and GeDH (41.6 kDa) were detected, as illustrated in Fig.9, confirming successful protein expression.

Ensure the initial OD600 is 0.2 for each group during inoculation. Cultivate in LB medium for 24 hours. According to the analysis of the growth curve results, there was no significant difference in growth capacity among the engineered strain, the empty vector strain, and the wild-type strain. Therefore, it can be concluded that the growth capacity of the engineered strain did not significantly decrease(Fig.7).

![Fig.9  The Western blot results of GPS, CsTPS1, and GeDH.](https://static.igem.wiki/teams/5938/notebook/fig9.webp)

Fig.9 The Western blot results of GPS, CsTPS1, and GeDH.

**Analysis:** The SDS-PAGE results reveal only two bands , which do not align with the anticipated outcome. The lower band appears unusually thick, suggesting that due to the minimal difference in molecular weight between GPS and GeDH, the two bands have merged during electrophoresis. This observation allows us to conclude that all three proteins are successfully expressed in BL21.

**Participants:** Yanzhao Liu, Boyan Zhang

# 3. Experiment Phase 3: Analysis of products of engineered strains.

## Fermentation Experiment 1

**Objective:** The engineered bacteria are fermented and cultured in TB medium to obtain citronellal products, and explorations are carried ou t to optimize the subsequent conditions.

### The activation of the strain 08/08/2025 Friday

**Objective:** Restore the activity and growth and reproduction ability of the strain in a dormant state , ensure the stability of its physiological characteristics, and provide viable and consistent bacteria for subsequent fermentation production and experimental research.

**Results:** The activation of *Escherichia coli* was stopped when the OD600 value reached 0.523. At this time , the bacterial cells were in the logarithmic growth phase, with relatively good vitality and consistent physiological states, making them suitable for being transferred into the TB medium for further cultivation.

**Participants:** Ethan Wu, Boyan Zhang

### Scale-up cultivation 07/27/2025 Sunday

**Objective:** Transfer the bacteria to the TB medium and conduct enlarged cultivation at 37oC and 200 rpm to obtain a larger number of bacteria, so that more proteins can be produced in the subsequent induction.

**Results:** ftAer culturing for 12 hours , the 1L of TB medium became obviously turbid, with distinct flocculent phenomena visible in the medium . The OD600 value reached 0.588, which is suitable for induction.

**Participants:** Ethan Wu, Boyan Zhang

### Induction by IPTG and arabinose 08/09/2025 Saturday

**Objective:** Induce the BL-21-GPS-CsTPS1-GeDH strain with IPTG and arabinose at 22oC and 200 rpm to make it express proteins in large quantities .

**Results:** Stop the fermentation 72 hours after adding the inducer, collect the fermentation broth, and measure the OD600 value at this time, which reaches 5.213.

**Participants:** Yilin Qian, Yanzhao Liu

## Yield analysis Experiment 2

### Analyze the components of the products by HPLC 08/13/2025 Wednesday

**Objective:** The product components were analyzed by high-performance liquid chromatography (HPLC), and the content of citronellal in the fermentation supernatant and cell lysate was quantitatively analyzed . Meanwhile, the differences between the two were compared.

**Results:** Due to the limitations of laboratory conditions , we were unable to independently carry out the HPLC measurement. Therefore, we sent the samples to Beijing TD Company for analysis. Only the results are presented below.

![Fig.10  The standard curve of the citronellal sample.](https://static.igem.wiki/teams/5938/notebook/fig10.webp)

Fig.10 The standard curve of the citronellal sample.

![Fig.11  TB fermentation supernatant HPLC](https://static.igem.wiki/teams/5938/notebook/fig11.webp)

Fig.11 TB fermentation supernatant HPLC

![Fig.12  HPLC of cell disruption solution](https://static.igem.wiki/teams/5938/notebook/fig12.webp)

Fig.12 HPLC of cell disruption solution

![Fig.13  Significance Analysis of Concentration Differences between Cell Lysate and Fermentation Supernatant.](https://static.igem.wiki/teams/5938/notebook/fig13.webp)

Fig.13 Significance Analysis of Concentration Differences between Cell Lysate and Fermentation Supernatant.

**Analysis:** As shown in Fig. 10 , we established a standard curve using citronellal standards with concentrations of 0.4 g/L, 0.7 g/L, 1 g/L, 1.3 g/L, and 1.6 g/L. Subsequently, we analyzed the supernatant sample of the fermentation broth, and the calculated concentration reached 0.89 g/L(Fig. 11 ). The concentration of the cell lysate was analyzed, and it reached 0.91 g/L(Fig. 12). After performing a significance analysis on the results, no significant difference was found between the two, as shown in Fig. 13. There was no significant difference between the two. To reduce the operational difficulty, the fermentation supernatant will be directly used for analysis in the follow-up.

**Participants:** Hanying Yu, Zibin Tang

### UV absorption analysis 8/16/2025 Saturday

**Objective:** To verify the accuracy of the HPLC analysis results, we also used UV absorption method to detect the concentration of the fermentation supernatant . We selected the same wavelength as that in liquid chromatography to measure the absorbance, and compared the results with those of HPLC.

**Results:** The concentration measured by ultraviolet absorption method is basically the same as that of HPLC , so the result can be judged to be correct.

**Participants:** Hanying Yu, Zibin Tang

# 4. Experiment Phase 4: The optimization of production conditions.

## Response surface design experiment Experiment 1

**Objective:** Use the Design-Expert 13 software to design the response surface , determine the maximum and minimum values of the experimental conditions , and provide guidance for subsequent experiments.

### Response Surface Methodology (RSM) Experimental Design 08/17/2025 Sunday

**Objective:** Restore the activity and growth and reproduction ability of the strain in a dormant state , ensure the stability of its physiological characteristics, and provide viable and consistent bacteria for subsequent fermentation production and experimental research.

**Results:**

Table 8. Variables and levels in central composite design

| Factors | Levels | | |
| --- | --- | --- | --- |
| Temperature(oC) | -1 | 0 | 1 |
| IPTG (mM) | 0.10 | 0.50 | 0.90 |
| l-Arabinose (% w/v) | 0.020 | 0.110 | 0.200 |

**Participants:** Ruiyang Wen, Yanzhao Liu

### RSM Experimental 08/18/2025 Monday

**Objective:** Conduct experiments according to the conditions provided by Design-Expert 13 to obtain the yield of citronellal under different fermentation conditions, providing data support for the response surface.

**Results:**

Table 9. Box-Behnken Response Surface Experimental Design and Results

| Std | Run | A:Temperature | B:IPTG | C:l-Arabinose | Citronellal(g/L) |
| --- | --- | --- | --- | --- | --- |
| 12 | 1 | 28 | 0.9 | 0.2 | 0.68 |
| 4 | 2 | 36 | 0.9 | 0.11 | 0.77 |
| 5 | 3 | 20 | 0.5 | 0.02 | 0.82 |
| 15 | 4 | 28 | 0.5 | 0.11 | 0.84 |
| 3 | 5 | 20 | 0.9 | 0.11 | 0.90 |
| 10 | 6 | 28 | 0.9 | 0.02 | 0.88 |
| 13 | 7 | 28 | 0.5 | 0.11 | 0.90 |
| 9 | 8 | 28 | 0.1 | 0.02 | 0.54 |
| 11 | 9 | 28 | 0.1 | 0.2 | 0.81 |
| 8 | 10 | 36 | 0.5 | 0.2 | 0.60 |
| 6 | 11 | 36 | 0.5 | 0.02 | 0.65 |
| 2 | 12 | 36 | 0.1 | 0.11 | 0.43 |
| 14 | 13 | 28 | 0.5 | 0.91 | 0.60 |
| 1 | 14 | 20 | 0.1 | 0.11 | 0.84 |
| 7 | 15 | 20 | 0.5 | 0.2 | 0.96 |

Multivariate quadratic response surface regression model:

Citronellal=-0.4275A2-1.18B2+0.01C2-0.145AB-0.4325AC-0.6975BC-1.18A+0.3137B+0.32C+2.84

Table 10. ANOVA for Quadratic model

| Source | Sum of Squares | df | Mean Square | F-value | p-value |  |
| --- | --- | --- | --- | --- | --- | --- |
| **Model** | 21.19 | 9 | 2.35 | 8.90 | 0.0134 | significant |
| A-Temperature | 11.16 | 1 | 11.16 | 42.18 | 0.0013 |  |
| B-IPTG | 0.7875 | 1 | 0.7875 | 2.98 | 0.1451 |  |
| C-l-Arabinose | 0.8192 | 1 | 0.8192 | 3.10 | 0.1388 |  |
| AB | 0.0841 | 1 | 0.0841 | 0.3178 | 0.5973 |  |
| AC | 0.7482 | 1 | 0.7482 | 2.83 | 0.1535 |  |
| BC | 1.95 | 1 | 1.95 | 7.35 | 0.0422 |  |
| A2 | 0.6748 | 1 | 0.6748 | 2.55 | 0.1712 |  |
| B2 | 5.16 | 1 | 5.16 | 19.51 | 0.0069 |  |
| C2 | 0.0004 | 1 | 0.0004 | 0.0014 | 0.9717 |  |
| **Residual** | 1.32 | 5 | 0.2647 |  |  |  |
| Lack of Fit | 1.28 | 3 | 0.4272 | 11.01 | 0.0868 | not significant |
| Pure Error | 0.0416 | 2 | 0.0208 |  |  |  |
| **Cor Total** | 22.51 | 14 |  |  |  |  |

**Analysis:**We established a standard curve using citronellal standards with conFactor coding is Coded. Sum of squares is Type III - Partial. The Model F-value of 8.90 implies the model is significant. There is only a 1.34% chance that an F-value this large could occur due to noise. P-values less than 0.0500 indicate model terms are significant. In this case A, BC, B² are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. If there are many insignificant model terms (not counting those required to support hierarchy), model reduction may improve your model. The Lack of Fit F-value of 11.01 implies the Lack of Fit is not significant. There is a 8.68% chance that a Lack of Fit F-value this large could occur due to noise.

**Participants:** Ruiyang Wen, Yanzhao Liu

### RSM Analysis 08/25/2025 Monday

**Objective:**: Create a response surface to analyze the influence of temperature (A), IPTG (B), and l-Arabinose (C) on the yield of citronellal . The results are shown in Fig.11-13.

**Results:**

![Fig.14  Response surface of A and B on citronellal concentration](https://static.igem.wiki/teams/5938/notebook/fig14.webp)

Fig.14 Response surface of A and B on citronellal concentration

![Fig.15  Response surface of A and C on citronellal concentration](https://static.igem.wiki/teams/5938/notebook/fig15.webp)

Fig.15 Response surface of A and C on citronellal concentration

![Fig.16  Response surface of B and C on citronellal concentration](https://static.igem.wiki/teams/5938/notebook/fig16.webp)

Fig.16 Response surface of B and C on citronellal concentration

**Analysis:** From the analysis of the response surface, contour plot, and regression equation, the optimal fermentation conditions for citronellal are as follows: a temperature of 20oC, an IPTG concentration of 0.46 mM, and an l-Arabinose concentration of 0.2% w/v. A verification experiment was conducted under the above optimized conditions.

**Participants:** Hanying Yu, Boyan Zhang

### Analyze the components of the products by HPLC 08/26/2025 Tuesday

**Objective:** Analyze the product components of the fermentation supernatant after the optimization of conditions through HPLC technology, and conduct a quantitative analysis of the content of citronellal in the fermentation supernatant.

**Results:** We analyzed the supernatant sample of the fermentation broth after the optimization of conditions. Through calculation, the concentration of the sample reached 1.01 g/L(Fig.17).

![Fig.17 Optimal fermentation conditions predicted by response surface HPLC](https://static.igem.wiki/teams/5938/notebook/fig17.webp)

Fig.17 Optimal fermentation conditions predicted by response surface HPLC

**Analysis:** From the analysis of the response surface, contour plot, and regression equation, the optimal fermentation conditions for citronellal are as follows: a temperature of 20oC, an IPTG concentration of 0.46 mM, and an l-Arabinose concentration of 0.2% w/v. A verification experiment was conducted under the above optimized conditions.

### Construction of Mutant Protein 08/28/2025 Thursday

**Objective:** Based on the analysis of dry lab, we constructed a mutant protein（GeDH） that theoretically has the strongest binding ability to citronellol (the precursor of citronellal), and mutated four amino acids, namely Q60R, L125P, F147Y, and A300D. We hope to further increase the yield of citronellal through mutation.

**Results:** We analyzed the supernatant sample of the fermentation broth after the optimization of conditions. Through calculation, the concentration of the sample reached 1.01 g/L(Fig.17).

![Fig.18  The conformation of the last frame in molecular dynamics simulation](https://static.igem.wiki/teams/5938/notebook/fig18.webp)

Fig.18 The conformation of the last frame in molecular dynamics simulation

In the picture, the ligand is colored blue. In 54883, the wild type is shown in blue, and the mutant is presented as sticks in dark purple. The two fit well together. We used the amino acid sequence of the protein to reconstruct the plasmid, and constructed the engineered strain using the same methods as in Experiment Phase 1 and Experiment Phase 2. The plasmids carrying GPS and CsTPS1 in the new strain are the same as the original ones, while GeDH uses the mutated sequence. Since the process is rather tedious and repetitive, the relevant procedures are not described in detail here.

### Fermentation of Engineered Bacteria with Mutant Protein 09/24/2025 Wednesday

**Objective:** To conduct a fermentation experiment for citronellal production using the engineered strain with mutant protein, and verify whether the mutated GeDH can enhance the yield of citronellal. Due to time constraints, we commissioned Beijing TD Company to construct the engineered strain. After obtaining the engineered strain, we fermented.

**Results:** Culturing was carried out under the optimal culture conditions determined by response surface analysis. The results showed that the citronellal yield of the mutant strain reached 1.36 g/L(Fig.19).

![Fig.19  Fermentation HPLC under optimal conditions of mutants](https://static.igem.wiki/teams/5938/notebook/fig19.webp)

Fig.19 Fermentation HPLC under optimal conditions of mutants

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# 1. Overview

Our project aims to establish a green mosquito-repellent technology route for use in campuses and communities. It addresses the growing global and local challenges of mosquito nuisance and disease risks, as well as the shortcomings of traditional chemical repellents and fumigation products in terms of safety, odor, and environmental impact in enclosed spaces. We selected citronellal—a naturally derived, human- and animal-friendly compound—as the core active ingredient. Using synthetic biology, we constructed a sustainable and scalable biosynthetic pathway in engineered *E. coli*. Through statistical optimization and enzyme engineering, we transformed the "capable of production" into "high-yield production," laying the foundation for subsequent slow-release formulations and real-world applications.

From an engineering framework perspective, our project strictly adhered to the iGEM DBTL (Design–Build–Test–Learn) cycle and completed four rounds of iteration:

The first DBTL cycle focused on **Chassis and Pathway Construction**. We established a three-enzyme cascade system (GPS–CsTPS1–GeDH) using multi-plasmid co-expression (pET28a / pET21a / pBAD23) and dual induction with IPTG and L-arabinose, achieving stable and regulatable expression of the pathway in the BL21 background.

The second DBTL cycle emphasized **Validation of Growth Capacity, Transcription, and Translation**. We measured the growth curves of engineered and wild-type strains to assess changes in growth capability. At the transcriptional level, qPCR was used to quantify the fold increase in mRNA before and after induction. At the protein level, Western blot detected three specific bands of expected molecular weights.

The third DBTL cycle centered on **Product Fermentation**. We preliminarily determined fermentation parameters and successfully produced citronellal. We established a quantification protocol for citronellal using both HPLC and UV absorption methods, and compared intracellular and extracellular citronellal concentrations.

The fourth DBTL cycle involved **Process Optimization**. Using the Box–Behnken response surface methodology, we systematically investigated three factors: induction temperature, IPTG, and L-arabinose. The optimal combination, primarily involving low-temperature induction, was identified and validated in confirmation batches, providing statistical certainty for fermentation parameters. Additionally, we employed computational rational design to screen key sites in GeDH based on molecular docking and dynamics simulations, and constructed combinatorial mutants. This improved the catalytic efficiency of the terminal conversion step without altering the upstream design, leading to a further increase in yield.

Our project has successfully transitioned from "capable of producing citronellal" to "producing citronellal more stably and at higher yields." The engineered strain achieved citronellal titers approaching gram-per-liter levels in shake-flask cultures. Dry lab work provided crucial guidance for the project's success and reduced the wet lab search space, while wet lab data (qPCR, Western blot, HPLC, etc.) continuously refined models and hypotheses, driving further DBTL cycles.

![Project Design Diagram](https://static.igem.wiki/teams/5938/engineering/projectdesigndiagram.webp)

Figure 1.1 Project Design Diagram

# 2. DBTL Cycle 1: Chassis and Pathway Construction

## 2.1 Design

The goal of our first cycle was to select appropriate enzymes and a chassis cell for the biosynthesis of citronellal. We chose to reconstruct a three-enzyme cascade pathway—"geranyl pyrophosphate (GPP) → geraniol → citronellal"—in an *E. coli* BL21 background. The genes GPS, CsTPS1, and GeDH were cloned into pET28a, pET21a, and pBAD23 vectors, respectively, forming a compatible multi-plasmid co-expression system with distinct antibiotic resistances. This design facilitates the selection of transformants and enables dual induction and dose regulation via IPTG and L-arabinose. The strategy emphasizes modular, replaceable expression cassettes to allow for flux distribution and subsequent optimization (e.g., induction strength, temperature).

![plasmid design](https://static.igem.wiki/teams/5938/engineering/plasmiddesign.webp)

Fig. 2.1 Plasmid Design

* **pET28a-GPS**: Contains the geranyl pyrophosphate synthase gene (GPS) under the T7 promoter (IPTG-inducible). pET28a provides a high-expression T7 system and kanamycin resistance for selection. This plasmid was designed to produce high levels of GPS in *E. coli*.
* **pET21a-CsTPS1**: Contains the tea tree CsTPS1 geraniol synthase gene under the T7 promoter (IPTG-inducible, ampicillin resistance). This plasmid drives the conversion of GPP to geraniol. CsTPS1 was placed on a separate plasmid to balance expression levels.
* **pBAD23-GeDH**: Contains the geraniol dehydrogenase gene (GeDH) under the L-arabinose-inducible araBAD promoter (chloramphenicol resistance). This allows independent induction of the final oxidation step using arabinose.

## 2.2 Build

We first obtained the sequences of the three target genes and codon-optimized them for *E. coli*. The optimized sequences were synthesized by Sangon Biotech (Shanghai) and delivered as fragments cloned into pUC19 plasmids. Target fragments and backbone vectors (from the lab stock) were amplified via PCR and verified by agarose gel electrophoresis (Fig. 2.2).

![2.2 Marker](https://static.igem.wiki/teams/5938/engineering/marker.webp)

Fig. 2.2 M: Marker; 1: GeDH; 2: CsTPS1; 3: GPS; 4: pET28a; 5: pET21a; 6: pBAD33

The fragments were then assembled into the vectors using Gibson assembly and transformed into BL21 cells. Successful transformants were obtained (Fig. 2.3).

![bacterial transformants](https://static.igem.wiki/teams/5938/engineering/23a.webp)

Fig. 2.3 Bacterial Transformants

## 2.3 Test

To verify the correct construction of the engineered strain, we designed specific primer pairs for each exogenous gene (GPS, CsTPS1, GeDH) for genotypic identification. The primers targeted internal conserved regions and vector-flanking regions to enhance specificity and directional accuracy. Two independent single colonies were picked from the transformation plate and directly subjected to colony PCR. Gel electrophoresis showed amplification bands at the expected sizes for all three genes, with no non-specific bands or primer dimers (Fig. 2.4). These results provided initial evidence that all three genes were correctly inserted and oriented in the host strain, indicating successful transformation.

![M:marker](https://static.igem.wiki/teams/5938/engineering/24marker.webp)

Fig. 2.4 M: Marker; 1,2: GeDH; 3,4: CsTPS1; 5,6: GPS

To mitigate false positives from colony PCR (e.g., due to template contamination or homologous amplification), the two positive clones were sent to Sangon Biotech for Sanger sequencing. Forward and reverse reads covered the insertions and vector junctions. The sequences matched the designed sequences exactly, with intact reading frames, correct start/stop codons, and no frameshifts or unintended mutations. All three genes were confirmed to be correctly oriented, without deletions or inversions. This validated the accuracy of the engineering beyond PCR size matching. For consistency in subsequent experiments, the doubly verified clones were preserved as glycerol stocks at -80°C, serving as standardized starting materials for expression and fermentation tests.

## 2.4 Learn

*E. coli*'s endogenous MEP pathway continuously supplies IPP/DMAPP, making it a natural metabolic chassis for monoterpene precursor synthesis. BL21(DE3) offers advantages in cost, ease of operation, and laboratory reproducibility. We chose GPP as the nodal point: GPS condenses IPP and DMAPP into GPP, CsTPS1 converts GPP to geraniol, and GeDH oxidizes geraniol to citronellal. This modular "GPS → CsTPS1 → GeDH" pathway aligns with host metabolism and allows decoupled optimization at multiple levels (expression strength, enzyme activity, induction strategy). We not only obtained an engineered strain theoretically capable of producing citronellal but also established a verifiable checklist and quality thresholds for the next cycle, ensuring that subsequent assessments of growth capacity, transcription/translation levels, and product formation are evidence-based, reproducible, and iterative.

# 3. DBTL Cycle 2: Validation of Growth Capacity, Transcription, and Translation

## 3.1 Design

Colony PCR and Sanger sequencing provided solid evidence that the "three enzymes are correctly inserted and oriented." However, the actual product flux depends on the expression levels of the three expression cassettes within the cells. Therefore, the focus of the second DBTL cycle shifted to "three-layer validation":

1. Measuring the growth curve of the engineered strain to analyze its growth capacity.
2. Using qPCR to quantify the fold change in transcription before and after induction.
3. Using WB/SDS-PAGE to confirm stable expression of the three enzymes at their expected molecular weights.

The multi-plasmid dual induction system offers flexibility but also introduces potential noise from resource competition and copy number variations. To address this, we fixed the inoculation amount, induction timing, and antibiotic pressure in subsequent validations, comparing differences between the engineered strain and empty vector/wild-type controls.

Only after confirming normal transcription and translation can we proceed with process optimization and protein engineering; otherwise, we must return to fine-tuning expression balance and induction strategies.

At the transcriptional level, qPCR primers covering approximately 200 bp were designed, with 16S rRNA as the internal reference, to assess mRNA upregulation before and after induction. At the translation level, SDS-PAGE/Western Blot was used to detect the three target bands.

![growth curve](https://static.igem.wiki/teams/5938/engineering/growthcurve.webp)

Fig. 3.1 Growth curve, qPCR, and WB

## 3.2 Build

### 3.2.1 Growth Curve

Nine 250 mL conical flasks were divided into three groups, each with a 50 mL system:

* Group 1: Engineered strain + LB + antibiotics
* Group 2: Wild-type strain + LB
* Group 3: Empty vector strain + LB + antibiotics

The initial OD600 of all groups was adjusted to 0.2, and they were cultured in LB medium at 37°C and 220 rpm for 24 hours.

### 3.2.2 qPCR

Primers were designed for GPS, CsTPS1, GeDH, and the internal reference (16S rRNA) to amplify 100–200 bp fragments with a Tm of ~60°C and GC content of 40–60%. RNA extraction was performed using a Meiji kit, and reverse transcription was done with a Genstar kit. The qPCR reaction system had a final volume of 20 µL, using 2× SYBR Green Master Mix with a single primer concentration starting at 0.2 µM. The cycling program consisted of 2–3 min pre-denaturation at 95°C, followed by 40 cycles of 10–15 s at 95°C and 30 s at 60°C.

### 3.2.3 Western Blot

Before translation-level validation, the engineered strain stored at -80°C was revived. The revived culture was subjected to total protein lysis and concentration measurement using NanoDrop for rapid assessment. After sample standardization, SDS-PAGE separation was performed following conventional procedures: equal loading, constant voltage electrophoresis to fully separate target proteins by molecular weight. After electrophoresis, a PVDF membrane was used for wet transfer. After confirming successful transfer, immunodetection was carried out: membrane blocking to reduce non-specific binding, incubation with His-tag primary antibody, followed by HRP-labeled secondary antibody for signal amplification, and finally ECL chemiluminescence for development and recording.

## 3.3 Test

### 3.3.1 Growth Curve

To evaluate whether the "three-plasmid dual induction" design imposed a basal growth burden on the host, standardized growth curve tests were conducted under identical culture conditions and selection pressures using the engineered strain, empty vector strain, and wild-type strain as controls. All groups entered the logarithmic phase with the same initial inoculation amount, with fixed shaking speed and temperature, and OD600 was recorded at regular intervals. Means and standard deviations were obtained from at least three biological replicates. Figure 3.2 shows the overall growth trajectories of the three groups: the lag phase length, logarithmic phase slope, and final OD600 upon entering the stationary phase highly overlapped, with no abnormal fluctuations or significant tailing.

The results clearly indicate that the engineered strain's basal growth capacity in the uninduced state is comparable to that of the empty vector and wild-type strains, suggesting that multi-plasmid coexistence and selection pressure did not cause measurable growth impairment. In other words, the genetic construction did not introduce significant host burden or toxic phenotypes, and the engineered strain can proliferate at the same rate as the controls. This conclusion provides a reliable starting point for subsequent expression induction and fermentation experiments: when comparing transcription/translation levels and product formation in the next cycle, "growth defects" can be excluded as a confounding factor, and observed differences can be primarily attributed to pathway expression and catalytic efficiency itself.

![group 1](https://static.igem.wiki/teams/5938/engineering/32.webp)

Fig. 3.2 Group 1: Engineered bacteria + LB + antibiotics; Group 2: Wild-type bacteria + LB; Group 3: Empty vector bacteria + LB + antibiotics

### 3.3.2 qPCR

To assess whether the exogenous pathway was effectively activated at the transcriptional level after induction, qPCR analysis was performed on GPS, CsTPS1, and GeDH using 16S rRNA as the internal reference and the 2^-ΔΔCt relative quantification framework. The results showed that under simultaneous induction with IPTG (T7 system) and L-arabinose (PBAD system), the relative transcription levels of all three genes were significantly upregulated: GPS ~16.1×, CsTPS1 ~18.0×, and GeDH ~12.6× (Fig. 3.3). This evidence indicates that our dual induction design can stably and strongly drive the expression of the three enzymes at the transcriptional level.

In terms of expression patterns, the upregulation fold changes of CsTPS1 and GPS were slightly higher than that of GeDH, suggesting that the terminal oxidation step (where GeDH is located) is relatively conservative in transcriptional activation amplitude. This difference may stem from the kinetic characteristics of different promoters/regulatory elements (T7 and PBAD have different response strengths and saturation behaviors) or reflect the "upper limit" of induction concentrations set to avoid cellular burden. From an engineering perspective, this provides direction for subsequent optimization: on one hand, pathway balance can be improved by fine-tuning induction strength/timing (e.g., inducing upstream first, then GeDH); on the other hand, while maintaining mRNA quality and stability, exploring RBS strength grading or GeDH protein engineering could enhance the flux of the terminal step. In summary, the qPCR results not only verify that the "pathway is successfully activated" but also provide quantitative basis and priority clues for optimization at the translation and catalytic levels in the next round.

![relative expression](https://static.igem.wiki/teams/5938/engineering/relativeexpression.webp)

Fig. 3.3 Relative expression level of GPS, CsTPS1, and GeDH

### 3.3.3 Western Blot

To verify the expression of the three exogenous enzymes at the translation level, Western Blot was performed on the induced strain. The results showed clear specific bands at the expected molecular weight windows: GPS 46.9 kDa, CsTPS1 63.7 kDa, and GeDH 41.6 kDa (see Fig. 3.4).

It is important to note that GPS and GeDH have similar molecular weights and both carry the same epitope tag, appearing as a slightly thicker merged band on the membrane. This phenomenon is common in multi-target membrane detection and does not affect the determination of "presence/absence of expression."

![western blot](https://static.igem.wiki/teams/5938/engineering/westernblot.webp)

Fig. 3.4 The Western blot results of GPS, CsTPS1, and GeDH.

## 3.4 Learn

After comparing standardized growth curves of the engineered strain, empty vector strain, and wild-type strain under uninduced conditions, we observed that the three groups highly overlapped in lag phase duration, logarithmic phase slope, and stationary phase final OD600, showing no significant differences. Thus, it can be concluded that multi-plasmid coexistence did not impose a measurable basal growth burden, and the metabolic pressure of the engineered strain is comparable to that of the wild-type, allowing it to safely proceed to subsequent induction expression and fermentation stages without additional chassis "burden reduction" measures.

At the transcriptional level, we quantified the three exogenous genes using 16S rRNA as the internal reference. After induction, the relative transcription levels of GPS, CsTPS1, and GeDH were significantly upregulated, indicating that the plasmids can be stably transcribed within the host, providing direct evidence for subsequent protein expression and catalysis of the pathway. At the translation level, we detected the three target proteins within the expected molecular weight windows.

The three types of evidence—growth curve, qPCR, and WB—form a complete closed loop from "chassis health → transcriptional activation → protein expression": the engineered strain showed no growth impairment, and the three genes were stably and efficiently transcribed and translated after induction, marking the successful "activation" of the metabolic pathway and have the conditions to advance to the downstream product level. These results provide reliable assurance for subsequent fermentation experiments and parameter optimization, laying a solid foundation for achieving citronellal biomanufacturing.

# 4. DBTL Cycle 3: Product Fermentation

## 4.1 Design

The goal of this cycle was to use fermentation + analytical chemistry to prove that the metabolic pathway is "product-visible" and to establish a reproducible quantification workflow. The fermentation strategy used TB medium as the substrate, with simultaneous induction by IPTG + L-arabinose during the stable logarithmic phase. The analytical strategy used HPLC as the main method to establish an external standard curve for citronellal, while UV-Vis was used for rapid verification to achieve "dual-channel consistency" and enhance data credibility. To determine product distribution, the fermentation supernatant and cell lysate were quantified separately, and significance analysis was conducted to determine whether it is feasible to only test supernatant in the future.

## 4.2 Build

At the process level, after revival and activation in LB, the culture was scaled up in TB. Induction expression was initiated with IPTG and L-arabinose at OD600 ≈ 0.6. TB medium was chosen for three reasons: glycerol metabolism alleviates catabolite repression and reduces acetate accumulation; high nitrogen and buffering systems support late-stage activity and steady-state; our previous growth curves indicated that the engineered strain's metabolic burden was close to that of the wild-type, making it suitable for scale-up. During sample collection, both supernatant and lysate were taken for subsequent detection.

At the analytical level, citronellal standards at 0.4, 0.7, 1.0, 1.3, and 1.6 g/L were prepared according to the external standard method to establish an HPLC standard curve; quantification was done using a UV detector for peak integration. To enhance evidence strength, UV-Vis was performed in parallel. Additionally, both cell lysate and fermentation supernatant were quantified to analyze whether there was a statistical difference between them.

## 4.3 Test

The standard curve for citronellal concentration is shown in Fig. 4.1. The retention time for citronellal was approximately 14 minutes.

![standard curve](https://static.igem.wiki/teams/5938/engineering/41.webp)

Fig. 4.1 The standard curve of the citronellal sample.

Next, we used HPLC to detect the citronellal concentration in the TB medium fermentation supernatant (Fig. 4.2). The analysis showed a citronellal concentration of 0.89 g/L, successfully achieving citronellal production through fermentation.

![hplc](https://static.igem.wiki/teams/5938/engineering/42.webp)

Fig. 4.2 HPLC of TB fermentation supernatant.

Furthermore, considering that citronellal might also exist intracellularly, we also detected the citronellal concentration in the cell lysate (Fig. 4.3). The analysis showed a citronellal concentration of 0.91 g/L.

![HPLC4.3](https://static.igem.wiki/teams/5938/engineering/43.webp)

Fig. 4.3 HPLC of cell lysate.

Significance analysis revealed no significant difference between the two (Fig. 4.4). Therefore, subsequent experiments directly used the fermentation supernatant for detection.

![significance](https://static.igem.wiki/teams/5938/engineering/44significance.webp)

Fig. 4.4 Significance Analysis of Concentration Differences between Cell Lysate and Fermentation Supernatant.

Although liquid chromatography is generally highly accurate, to make our results more credible, we performed UV-Vis in parallel on the samples. The results showed little difference, confirming the quantification conclusion from a second technical path.

## 4.4 Learn

This round yielded three clear conclusions and next steps:

1. The metabolic pathway has been confirmed at the "product level," with the engineered strain capable of producing citronellal close to g/L levels.
2. The intra-/extracellular product distribution is similar, so "direct supernatant measurement" can represent the overall level, significantly simplifying subsequent batch screening.
3. The consistency between HPLC and UV-Vis provides a robust quantification foundation for subsequent parameter optimization and comparative experiments.

Based on these conclusions, we will expand the factors in the next round to include induction temperature, IPTG, and L-arabinose levels, using response surface methodology for statistical optimization. In the meantime, dry lab rational protein design will be combined to achieve higher yields.

# 5. DBTL Cycle 4: Process Optimization

## 5.1 Design

The goal of this cycle was to transform citronellal production from "capable" to "high-yield." On the process side, we used the Box-Behnken three-factor three-level design of response surface methodology (RSM), with citronellal concentration as the response, to systematically investigate induction temperature, IPTG concentration, L-arabinose concentration, and their interactions and quadratic effects, avoiding the pitfalls of single-factor methods that ignore synergy and nonlinearity. This design has evenly distributed points and avoids extreme conditions, allowing 15 experiments to fit a model including main effects, interactions, and quadratic terms.

In parallel with "dry lab" work, rational design was focused on the pathway's rate-limiting step, GeDH (Fig. 5.1): using Chai1 to predict protein structure, Funclib to predict mutation networks and provide thermostability scores, and Rosetta for molecular docking, with GROMACS molecular dynamics to assess complex stability and calculate binding free energy, we filtered out superior sequences for wet lab validation.

![dry lab](https://static.igem.wiki/teams/5938/engineering/drylab.webp)

Fig. 5.1 Dry lab roadmap.

## 5.2 Build

According to the Box-Behnken design (Table 5.1), 15 shake flask fermentations were completed, covering combinations of 20/28/36°C, 0.10/0.50/0.90 mM IPTG, and 0.02/0.11/0.20% (w/v) L-arabinose. The corresponding citronellal concentration data matrix was obtained experimentally.

Table 5.1. Variables and levels in central composite design

| Factors | Levels | | |
| --- | --- | --- | --- |
| -1 | 0 | 1 |
| Temperature(℃) | 20 | 28 | 36 |
| IPTG（mM） | 0.10 | 0.50 | 0.90 |
| l-Arabinose (% w/v) | 0.020 | 0.110 | 0.200 |

ANOVA was performed on the results: the overall model was significant (F=8.90, p=0.0134), with temperature A as the most significant main effect (F=42.18, p=0.0013); the IPTG×L-arabinose (BC) interaction was significant (p=0.0422), indicating that dual induction requires setting; the quadratic term of IPTG (B²) was highly significant (p=0.0069), indicating an optimal concentration range; lack of fit was not significant (p=0.0868), indicating reliable model fitting. The model provided the optimal conditions: 20°C, IPTG 0.46 mM, L-arabinose 0.20% (w/v), as shown in Table 5.2.

Table 5.2. Box-Behnken Response Surface Experimental Design and Results

| Std | Run | A:Temperature | B:IPTG | C:l-Arabinose | Citronellal(g/L) |
| --- | --- | --- | --- | --- | --- |
| 12 | 1 | 28 | 0.9 | 0.2 | 0.68 |
| 4 | 2 | 36 | 0.9 | 0.11 | 0.77 |
| 5 | 3 | 20 | 0.5 | 0.02 | 0.82 |
| 15 | 4 | 28 | 0.5 | 0.11 | 0.84 |
| 3 | 5 | 20 | 0.9 | 0.11 | 0.90 |
| 10 | 6 | 28 | 0.9 | 0.02 | 0.88 |
| 13 | 7 | 28 | 0.5 | 0.11 | 0.90 |
| 9 | 8 | 28 | 0.1 | 0.02 | 0.54 |
| 11 | 9 | 28 | 0.1 | 0.2 | 0.81 |
| 8 | 10 | 36 | 0.5 | 0.2 | 0.60 |
| 6 | 11 | 36 | 0.5 | 0.02 | 0.65 |
| 2 | 12 | 36 | 0.1 | 0.11 | 0.43 |
| 14 | 13 | 28 | 0.5 | 0.11 | 0.91 |
| 1 | 14 | 20 | 0.1 | 0.11 | 0.84 |
| 7 | 15 | 20 | 0.5 | 0.2 | 0.96 |

Simultaneously, based on dry lab work, protein stability was scored (Fig. 5.2).

![protein](https://static.igem.wiki/teams/5938/engineering/52protein.webp)

Fig. 5.2 Protein stability score distribution.

Docking results were also scored (Fig. 5.3).

![docking](https://static.igem.wiki/teams/5938/engineering/53docking.webp)

Fig. 5.3 Docking score distribution.

We found that quadruple mutants generally scored better than triple and double mutants. Based on comprehensive results, four candidate proteins and the wild-type were selected for molecular dynamics simulation (Table 5.3).

Table 5.3 Protein stability and molecular docking scoring results

| GeDH | Protein\_score | Dock\_score | Score |
| --- | --- | --- | --- |
| 64117 | -1187.136 | -1344.596 | -1281.6121 |
| 63928 | -1182.999 | -1344.791 | -1280.0742 |
| 54883 | -1184.333 | -1343.538 | -1279.8561 |
| 54456 | -1186.282 | -1342.141 | -1279.7974 |
| WT | -1161.051 | -863.977 | -982.8066 |

The dominant conformations during dynamics simulation are shown in Fig. 5.4. The ligand is shown in blue. In the figure 54883, blue represents the wild-type, and dark purple represents the mutant stick representation, showing good fit. 54456 also binds at the correct position with a high binding free energy.

![dominant](https://static.igem.wiki/teams/5938/engineering/54dominant.webp)

Fig. 5.4 Dominant conformations during dynamics simulation.

Based on dry lab analysis of molecular docking/dynamics, combination mutation was performed on GeDH at sites Q60R, L125P, F147Y, and A300D, aiming to enhance affinity and catalytic efficiency for the intermediate (citronellol/related aldehyde alcohol state). To isolate variable effects, only the GeDH expression cassette was replaced while keeping the chassis and upstream enzymes unchanged (details see model section).

## 5.3 Test

Fermentation was conducted under the optimal conditions predicted by the response surface. HPLC quantification of the supernatant showed a citronellal concentration of 1.01 g/L (Fig. 5.5).

![55hplc](https://static.igem.wiki/teams/5938/engineering/55.webp)

Fig. 5.5 HPLC of fermentation under response surface-predicted optimal conditions.

Under the same conditions, rapid quantification of the GeDH mutant supernatant showed a concentration of 1.36 g/L, indicating that the rationally designed variant significantly increased flux at the terminal oxidation step (Fig. 5.6).

![56hplc](https://static.igem.wiki/teams/5938/engineering/56.webp)

Fig. 5.6 HPLC of mutant fermentation under optimal conditions.

## 5.4 Learn

This round yielded three actionable engineering insights:

1. Lowering the induction temperature is the primary lever for increasing yield.
2. Dual induction requires coordination, and IPTG has an optimal window—higher doses do not always yield benefits.
3. Enzymatic reconstruction of the terminal step is effective, and together with the process optimization can yield significant gains.

We solidified "20°C, IPTG 0.46 mM, L-arabinose 0.20%" as the current standard induction conditions; confirmed the quantification strategy of "direct supernatant measurement + HPLC external standard as the main method, UV as the auxiliary method; and extended the GeDH mutation strategy to saturation mutagenesis/combination mutagenesis of upstream enzymes. We plan to include dissolved oxygen, pH, and induction timing in the next round of RSM factor sets during scale-up trials to further statistically improve volumetric yield and batch-to-batch consistency. These improvements collectively provide both process and molecular handles for further reducing production costs and advancing toward pilot-scale amplification.

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# 1. Preparation of Experimental Reagents and Media

Each group will prepare the corresponding volume/quantity of reagents/media.

## 1.1 Experimental Objective

To prepare reagents needed for subsequent experiments.

##### Genetic Engineering:

* LB Liquid Medium: 20 ml/tube \* 5
* LB Liquid Medium: 20 ml/bottle \* 10
* CaCl2 0.1M: 50 ml
* LB Solid Medium (1.5% Agar): 200 ml
* TB Liquid Medium: 50 ml/bottle \* 70

## 1.2 Experimental Procedure: Preparation of Reagents for Genetic Engineering

### 1.2.1 Preparation and Sterilization of Solutions

**LB Medium**

A total of 300 ml is needed, but 400 ml will be prepared to ensure sufficient volume during aliquoting.

Preparation Method: [Tryptone: 4g, Yeast Extract: 2g, NaCl: 4g] + [400 ml pure water], stir well in a beaker, aliquot 20 ml into 10 conical flasks, and the remainder into a blue-cap bottle, and autoclave (loosen the cap for the blue-cap bottle).

**LB Solid Medium (1.5%)**

[Tryptone: 2g, Yeast Extract: 1g, NaCl: 4g] + [200 ml pure water] + [Agar powder: 3g], prepare directly in a 500 ml conical flask, do not stir, cover, and autoclave (loosen the cap for the blue-cap bottle).

**Calcium Chloride (for competent cell preparation)**

[0.55g CaCl2] + [50 ml pure water], prepare directly in a 100 ml blue-cap bottle, and autoclave.

**TB Medium**

A total of 3500 ml is needed, but 4000 ml will be prepared to ensure sufficient volume during aliquoting.

Preparation Method: [47.2 g tryptone, 94.4 g/L yeast extract, 37.6 g K2HPO4, 8.8 g KH2PO4, 16 ml glycerin] + [4000 ml pure water], stir well in a beaker, aliquot 50 ml into 70 conical flasks, and the remainder into a blue-cap bottle, and autoclave (loosen the cap for the blue-cap bottle).

**Plastic Consumables**

1.5 ml centrifuge tubes, 20 pcs, packaged in a plastic bag, and autoclaved.

### 1.2.2 Pouring Plates

Add Ampicillin (1 ml + 1 μl, final concentration 50 μg/ml) to the sterilized and cooled LB solid medium below 60°C, mix well, and pour plates into 90 mm plastic petri dishes, approximately 10-15 ml per plate, at least 10 plates needed.

# 2. Inoculation (Preparation for Plasmid Extraction)

Add 15 ml of autoclaved LB medium from 1.2.1 to a sterile 50 ml centrifuge tube, add 15 μl of Ampicillin stock solution (1 ml + 1 μl, final concentration 50 μg/ml), mix well. Add 150 μl of DH5α bacterial culture.

Cap and remove from the biosafety cabinet, incubate in a shaker at 220 rpm, 37°C, overnight.

# 3. Fundamental Experiments in Genetic Engineering/Molecular Biology

## 3.1 Objective

The molecular biology/genetic engineering experiments on the 11th are conducted individually. Each student must perform the basic molecular biology/genetic engineering experiments. The experiments on the 12th will be conducted in groups.

## 3.2 Experimental Procedures

### 3.2.1 Plasmid Miniprep and Concentration Measurement

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-1-1.webp)

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-1-3.webp)

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-1-4.webp)

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-1-5new.webp)

### 3.2.2 PCR

#### 3.2.2.1 Fragment Preparation

PCR Conditions for Fragment

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-2-1-1.webp)

Estimated Time: 1 hour (Morning Session)

PCR Conditions for pet28a Fragment

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-2-1-2.webp)

Estimated Time: 1 hour (Noon Break)

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-2-1-3.webp)

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-2-1-4.webp)

#### 3.2.2.2 Purification of PCR Products

Due to the complexity of the PCR system, which contains templates, primers, enzymes, and other impurities that may hinder seamless cloning, purification is necessary to obtain the target gene fragment.

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-2-2-1new.webp)

After purification, measure DNA concentration again and calculate the yield of the purification process.

### 3.2.3 Agarose Gel Electrophoresis

Prepare the gel during the PCR amplification of the fragment. In the afternoon, add the loading buffer and then run the gel electrophoresis.

(1) Prepare the gel (do this during the morning PCR session; due to the limited number of gel trays, prepare two small gels per group).

**Agarose Gel Electrophoresis**

* Purpose: Used for the separation, identification, and extraction of nucleic acid fragments.
* Principle: Fragments of nucleic acids (DNA or RNA) are separated based on their size. After applying an electric field, the negatively charged DNA or RNA fragments migrate through the agarose gel.Smaller nucleic acid fragments migrate faster. Higher agarose concentration in the gel reduces pore size, affecting the separation. Higher agarose concentrations result in smaller pores, making it suitable for separating smaller fragments.

![](https://static.igem.wiki/teams/5938/experiments/fig3-2-2-2-3.webp)

Our target gene and vector backbone are approximately 700 bp and 6000 bp, respectively, so prepare a 1% agarose gel.

Dissolve 1 g of agarose in 100 ml TAE buffer (not pure water!) in a 250 ml Erlenmeyer flask, cover with foil, and heat in a microwave until dissolved. Cool to approximately 60°C, add nucleic acid dye, mix, and pour into the gel tray. Cover with foil and allow to solidify in the dark.

(2) Preparation of Electrophoresis Samples

Mix a small amount of the purified PCR product with loading buffer (6X), in a 1:5 ratio.
Suggested: Take 10 µl each of the fragments in a 0.2 ml EP tube, add 2-3 µl loading buffer, and mix well.

(3) Loading Samples

(4) Electrophoresis and Analysis

Run at 90V. After verifying the molecular weight, store the samples at -20°C for use on the 12th.

# 4. Competent Cells, Seamless Cloning, and Transformation

## 4.1 Experimental Procedures

### 4.1.1 Preparation of Competent Cells

Upon arriving at the laboratory, first, pre-cool the calcium chloride solution prepared and sterilized on the 10th in an ice bath.

Transfer 1.25 ml of the bacterial culture into 1.5 ml centrifuge tubes (distributed into 8 tubes) and place them on ice for 10 minutes to cool to 0°C. Centrifuge the bacterial culture at 4°C, 4000 rpm for 10 minutes, and discard the supernatant (invert the tubes to drain the medium thoroughly).

Add 150 μL of ice-cold 0.1 M CaCl₂ to resuspend, combine two tubes into one, and place on ice for 30 minutes (resulting in 4 tubes total). Centrifuge again at 4°C, 4000 rpm for 10 minutes, discard the supernatant, and add 100 μL of ice-cold 0.1 M CaCl₂ to resuspend to obtain BL21 competent cells for immediate transformation. If not used immediately, add an equal volume of 50% glycerol, rapidly freeze in liquid nitrogen, and store at -80°C.

### 4.1.2 Seamless Cloning

Design the seamless cloning system according to the manual (classroom exercise; concentrations vary per individual, so each person's table will differ).

![](https://static.igem.wiki/teams/5938/experiments/fig4-1-2-1.webp)

**1. Concentration Determination of Linearized Vectors and Insert Fragments**

If the linearized vector and insert fragments have been purified through a gel recovery kit, and the absorbance reading shows no apparent bands or smears remaining, you can use Nanodrop or similar spectrophotometers to measure concentration. However, the A260/A280 ratio should only be considered reliable between 1.8 and 2.0. Instruments like Nanodrop, Qubit, and PicoGreen are recommended for concentration measurement. When sample concentrations are below 10 ng/μl, different instrument models may yield significantly varying readings based on A260.

**2. Calculation of Vector and Insert Fragment Usage:**

For single-fragment source recombination, the minimum recommended vector usage is 0.03 pmol, and the minimum recommended insert fragment usage is 0.06 pmol (with a vector-to-insert molar ratio of 1:2).

For multi-fragment source recombination, the minimum DNA usage for each fragment is 0.03 pmol (with a vector-to-insert molar ratio of 1:1).

The amount of DNA used in these calculations can be approximated using the following formulas:

* For Single-Fragment Source Recombination:

+ Optimal Cloning Vector Usage=[0.02\*Number of Base Pairs in the Cloning Vector]ng(0.03pmol)
+ The optimal amount of insert fragment=[0.04\* The number of base pairs in the insert fragment]ng(0.06pmol)

* For Multi-Fragment Source Recombination:

+ Optimal Cloning Vector Usage=[0.02\*Number of Base Pairs in the Cloning Vector]ng(0.03pmol)
+ The optimal usage amount for each fragment=[0.02\* The number of base pairs in each fragment]ng(0.03pmol)

* For single-fragment source recombination, the insert fragment usage should not exceed 20 ng. When the insert fragment is larger than the vector, it is advisable to calculate the appropriate mass conversion, treating the insert fragment as the vector and vice versa.
* For multi-fragment source recombination, each fragment should not exceed 10 ng. If the minimum recommended usage is below this value, directly use 10 ng/μl.
* In single-fragment source recombination, to avoid non-specific bands in PCR, do not use direct linearized DNA. Add no more than 1/5 of the total reaction volume (2 μl), or directly clone after gel extraction.

**3. Prepare the Following Reaction Systems on Ice:**

![](https://static.igem.wiki/teams/5938/experiments/fig4-1-2-2.webp)

1. X/Y is calculated based on the calculated vector and insert fragment amounts. To ensure amplification accuracy, vectors and insert fragments should be concentrated before reaction setup and used in no less than 1 μl volumes.
2. Positive Controls-1 and -2 employ plasmids as templates, with amplification lengths <3 kb. Primer Tm values should be >60°C.
3. c.Positive Control-3 uses genomic DNA as a template. Use high-fidelity polymerases for amplification.
4. d.Fragment length, GC content, and sequence complexity affect reaction time. High GC content in insert fragments can hinder the reaction; consider adding DMSO or betaine to enhance amplification.

**4. Gently mix using a pipette (avoid vortex mixing) and briefly centrifuge the reaction mixture to the bottom of the collection tube.**

**5. Recombination Reaction Conditions:**

Single-Fragment Recombination: Incubate at 50°C for 5 minutes; cool to 4°C or place on ice immediately.

2-3 Fragment Recombination: Incubate at 50°C for 15 minutes; cool to 4°C or place on ice immediately.

4-5 Fragment Recombination: Incubate at 50°C for 30 minutes; cool to 4°C or place on ice immediately.

* It is recommended to perform the reaction on instruments such as a PCR machine that offers precise temperature control.
* This protocol accommodates 0.01 - 0.25 pmol input of vectors and fragments. When the total vector and insert volume exceeds 5 μl, a lower input amount is acceptable, but avoid exceeding the recommended reaction time.
* Store the recombination product at -20°C for up to a week. Purify only when necessary for further transformation.

### 4.1.3 Transformation

**Recombinant Product Transformation**

1. Thaw chemically competent cells on ice (e.g., Fast-T1 Competent Cell, Vazyme #C505).
2. Add 5-10 μl of the recombinant product to 100 μl of competent cells. Gently flick the tube to mix (avoid vortexing) and incubate on ice for 30 minutes.

* The volume of recombinant product should not exceed 1/10 of the volume of competent cells used.

3. Heat shock the cells at 42°C for 30 seconds, then immediately place them back on ice for 2-3 minutes.
4. Add 900 μl of SOC or LB medium (without antibiotics) and incubate at 37°C with shaking for 1 hour (rotation speed 200-250 rpm).
5. Pre-warm the required LB agar plates containing the appropriate antibiotic at 37°C in an incubator.
6. Centrifuge at 5,000 rpm (2,500 x g) for 5 minutes and discard 900 μl of the supernatant. Resuspend the bacterial pellet in the remaining medium and spread it gently on the pre-warmed agar plate with the antibiotic.
7. Invert the agar plates and incubate at 37°C for 12-16 hours.

Each experimental group will have four sets of samples (AC, AD, BC, BD), with each set plated on 1-2 LB plates and incubated overnight at 37°C.

# 5. Growth curve,qPCR and Western Blot

## 5.1 Determination of growth curve

Take nine 50 mL Erlenmeyer flasks and divide them into three groups:
Group 1: Engineered bacteria + LB + antibiotics
Group 2: Wild-type bacteria + LB
Group 3: Empty vector bacteria + LB + antibiotics
Ensure the initial OD600 of each group is 0.2 during inoculation. Cultivate in LB medium for 24 hours. Samples are collected every 4 hours for growth curve analysis.

## 5.2 Inoculation

Distribute the fermentation seed culture prepared on the LB into the remaining three 250 ml sterilized culture bottles, adding at least 5 ml of seed culture to each (using sterile Pasteur pipettes). Incubate at 37°C, 220 rpm for 5-10 hours.

## 5.3 Induction

Add 125 μl of IPTG and L-arabinose stock solution to each bottle (the stock is 1 M, with a final concentration of 1 mM). Incubate overnight at 22°C, 200 rpm for induction.

## 5.4 Total RNA extraction

Materials
Lysozyme (prepare fresh):
Gram-positive: 400 µg/mL Gram-negative: 3 mg/mL
Buffer Rlysis-B (RNase-free)
DEPC-treated ddH₂O (RNase-free water)
Chloroform (molecular biology grade)
Absolute ethanol and 75% ethanol (made with DEPC-H₂O)
RNase-free 1.5 mL tubes, ice, microcentrifuge (4 °C)
Procedure:

1. Harvest cells

 Collect 1 mL late-log culture into a 1.5 mL RNase-free tube. Centrifuge 8,000 rpm, 4 °C, 1 min and discard the supernatant. Add 100 µL lysozyme solution, vortex briefly to resuspend, and incubate 5-10 min at room temperature.

 Lysozyme concentration: 400 µg/mL for Gram-positive bacteria; 3 mg/mL for Gram-negative bacteria.

2. Chemical lysis

 Immediately add 900 µL Buffer Rlysis-B, mix thoroughly (quick vigorous inversion or brief vortex), and incubate on ice for 3 min.

3. Phase separation

 Add 200 µL chloroform, cap tightly, shake vigorously for \~30 s (or invert 15x), incubate 3 min at room temperature, then centrifuge 12,000 rpm, 4 °C, 5 min. Carefully transfer the upper aqueous phase to a new RNase-free tube.

4. Ethanol addition / precipitation

 To the collected aqueous phase, add 1.3 volumes of absolute ethanol, mix gently, incubate 5 min at room temperature, then centrifuge 12,000 rpm, 4 °C, 5 min. Carefully decant the supernatant.

5. Pellet wash

 Wash the pellet with 500 µL of 75% ethanol (prepare with DEPC-H₂O). Centrifuge 12,000 rpm, 4 °C, 3 min and discard the supernatant.

6. Repeat the wash once

 Perform one additional 75% ethanol wash under the same conditions.

7. Dry and dissolve RNA

 Air-dry the pellet at room temperature for \~10 min (do not overdry). Remove any remaining droplets with a pipette tip. Dissolve the RNA in 30-50 µL DEPC-H₂O. Use immediately or store at -80 °C.

## 5.5 cDNA acquisition

Reagents
Total RNA or poly(A)+ RNA (or target RNA)
Oligo(dT)15-20 (0.5 µg/µL) or Random hexamer (0.2 µg/µL) or Sequence-specific primer (20 pmol/µL)
RNase-free ddH₂O
5x Reaction Buffer
RNase Inhibitor (40 U/µL)
dNTP Mix (10 mM each)
AMV Reverse Transcriptase (10 U/µL)

1. Initial mix on ice (bring to 11 µL)

![](https://static.igem.wiki/teams/5938/experiments/fig5-5-1-1.webp)

Mix gently, quick-spin 3-5 s.
Heat 65 °C for 5 min, then ice 30 s, quick-spin again (3-5 s).

2. Add the following on ice (final volume 20 µL)

![](https://static.igem.wiki/teams/5938/experiments/fig5-5-1-2.webp)

Mix gently, quick-spin 3-5 s.

3. Reverse-transcription program
   cDNA synthesis: 42 °C, 30-60 min
   Enzyme inactivation: 95 °C, 5 min (denature RT), then hold on ice

## 5.6 qPCR

10 μL mix, 0.4 μL each of forward and reverse primers, 7.2 μL of DEPC water, and then 2 μL of template were added to make a 20 μL system. The qPCR conditions were a constant temperature section of 95°C for 1 min, a cycle section of 95°C for 10 s, and a cycle of 60°C for 30 s. Fluorescence was read and the number of cycles was 40.

## 5.7 Western Blot

1. Materials

Cell lysis buffer: RIPA or B-PER (+ protease inhibitor cocktail; phosphatase inhibitors if needed)
BCA protein assay kit
4x Laemmli Sample Buffer (final 1x: 62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.002% bromophenol blue; add 100 mM DTT or 5% β-ME as reducer)
SDS-PAGE gels (10-12% resolving + 4-5% stacking)
1x Running Buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS)
PVDF membrane (0.45 µm for >20 kDa; 0.2 µm for small proteins) or NC membrane
PVDF requires 1 min activation in 100% methanol
Transfer Buffer (25 mM Tris, 192 mM Glycine, 20% methanol; add 0.03-0.05% SDS for large proteins)
TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20)
Blocking solution: 5% non-fat milk/TBST (general) or 5% BSA/TBST (phospho-antibodies)
Primary antibody (target-specific or anti-tag His/Flag/Strep) and HRP-conjugated secondary antibody
ECL substrate

2. Sample preparation

* Lysis

 Harvest 1-5 mL induced *E. coli*, wash once with PBS. Add 200-500 µL lysis buffer (+ inhibitors), lyse on ice (sonication or gentle mixing). Spin 12,000 xg, 4 °C, 10 min; collect supernatant.

* Quantify & denature

 Measure protein by BCA. Add 4x sample buffer (to 1x), heat 95 °C, 5 min (or 70 °C, 10 min for aggregation-prone proteins).

* Loading amount

 Load 10 µg total protein per lane.

>
3. SDS-PAGE

Assemble gel, load samples and ladder. Run at 120-160 V until the dye front reaches the bottom (~45-70 min, gel-dependent).

4. Transfer

Activate PVDF in methanol (1 min) → rinse in water → equilibrate in transfer buffer 5-10 min.
Build stack: cathode - sponge - filter - gel - membrane - filter - sponge - anode; remove bubbles.
Wet transfer: 100 V, 60-90 min, 4 °C (or 300-350 mA, 60 min).
Semi-dry: 18-25 V, 30-45 min (instrument-specific).

5. Blocking & antibody incubation

* Block: 5% milk/TBST, RT 1 h.
* Primary antibody: dilute per datasheet (1:2000).

4 °C overnight.

* Wash: TBST 3 x 10 min.
* Secondary antibody : 1:5,000-1:10,000, RT 45-60 min.
* Wash again: TBST 3 x 5-10 min.

6. Detection & imaging

Cover membrane with ECL substrate, incubate 3 min.
Image on a chemiluminescence system.

# 6. Fermentation

## 6.1 Inoculation

Distribute the fermentation seed culture prepared on the TB into the remaining three 250 ml sterilized culture bottles, adding at least 5 ml of seed culture to each (using sterile Pasteur pipettes). Incubate at 37°C, 220 rpm for 5-10 hours.

## 6.2 Induction

Add 125 μl of IPTG and L-arabinose stock solution to each bottle (the stock is 1 M, with a final concentration of 1 mM). Incubate 3 days at 22°C, 200 rpm for induction.

# 7. Response surface analysis

Ferment according to the conditions in the table below. The operation is the same as step 6.

| Std | Run | A:Temperature | B:IPTG | C:l-Arabinose |
| --- | --- | --- | --- | --- |
| 12 | 1 | 28 | 0.9 | 0.2 |
| 4 | 2 | 36 | 0.9 | 0.11 |
| 5 | 3 | 20 | 0.5 | 0.02 |
| 15 | 4 | 28 | 0.5 | 0.11 |
| 3 | 5 | 20 | 0.9 | 0.11 |
| 10 | 6 | 28 | 0.9 | 0.02 |
| 13 | 7 | 28 | 0.5 | 0.11 |
| 9 | 8 | 28 | 0.1 | 0.02 |
| 11 | 9 | 28 | 0.1 | 0.2 |
| 8 | 10 | 36 | 0.5 | 0.2 |
| 6 | 11 | 36 | 0.5 | 0.02 |
| 2 | 12 | 36 | 0.1 | 0.11 |
| 14 | 13 | 28 | 0.5 | 0.11 |
| 1 | 14 | 20 | 0.1 | 0.11 |
| 7 | 15 | 20 | 0.5 | 0.2 |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block extra\_css %}
{% endblock %}
{% block page\_content %}

# 1. Round 1 DBTL: Chassis and Pathway Construction

In the first engineering cycle, our goal was to build a metabolic pathway in *E. coli* capable of synthesizing citronellal. We chose to reconstruct the three-enzyme cascade “geranyl diphosphate → geraniol → citronellal” in the *E. coli* BL21(DE3) chassis, involving three heterologous enzymes: geranyl diphosphate synthase (GPS), geraniol synthase (CsTPS1), and geraniol dehydrogenase (GeDH). To enable flexible and efficient expression, we adopted a multi-plasmid co-expression system: GPS was cloned into pET28a (T7 promoter, IPTG inducible, kanamycin resistance), CsTPS1 into pET21a (T7 promoter, IPTG inducible, ampicillin resistance), and GeDH into pBAD33 (araBAD promoter, L-arabinose inducible, chloramphenicol resistance) (Fig. 1).

![](https://static.igem.wiki/teams/5938/results/results-fig-1.webp)

Fig.1 Recombinant plasmid designs for pET28a-GPS, pET21a-CsTPS1 and pBAD23-GeDH

This plasmid combination ensured compatibility and allowed independent regulation of upstream and terminal enzymes via IPTG and L-ara dual induction, providing convenience for subsequent flux balancing and condition optimization. During construction, the target genes were codon-optimized for *E. coli* and synthesized as fragments (Fig. 2).

![](https://static.igem.wiki/teams/5938/results/results-fig-2.webp)

Fig.2 M: Marker; 1:GeDH; 2:CsTPS1; 3:GPS; 4: pET28a; 5: pET21a; 6: pBAD23

Each fragment was cloned into the corresponding linearized vector by Gibson Assembly, and a triple-plasmid engineered strain was obtained after two rounds of transformation (Fig. 3).

![](https://static.igem.wiki/teams/5938/results/results-fig-3.webp)

Fig.3 Second transformation results

The recombinant colonies were screened and verified using colony PCR. Results showed that GPS, CsTPS1, and GeDH PCR products matched the expected sizes without nonspecific amplification (Fig. 4), indicating correct gene insertion.

![](https://static.igem.wiki/teams/5938/results/results-fig-4.webp)

Fig.4 M: Marker; 1,2:GeDH; 3,4:CsTPS1; 5,6:GPS

Sequencing further confirmed correct sequences, reading frames, and orientations, proving successful construction of the three-enzyme cascade pathway.
This meant we had obtained a strain “theoretically capable of producing citronellal,” laying the foundation for the next round of experiments.

# 2. Round 2 DBTL: Verification of the Performance of Engineered Strains

With the engineered strain constructed, the second cycle focused on validating growth performance and confirming proper transcription and translation of the heterologous pathway. We designed three levels of experiments:

1. Measuring growth curves of engineered and control strains to assess effects of the multi-plasmid dual-induction system.
2. Using qPCR to quantify mRNA fold changes of heterologous genes before and after induction, confirming transcriptional activation.
3. Detecting protein expression of target enzymes by SDS-PAGE/Western Blot to confirm translation.Without induction, the engineered strain's growth curve nearly overlapped with wild-type and empty-vector controls, with similar lag phases, exponential growth rates, and final OD₆₀₀ values (Fig. 5). This indicated that carrying three plasmids did not significantly burden host growth.

![](https://static.igem.wiki/teams/5938/results/results-fig-5.webp)

Fig.5 Group 1: Engineered bacteria + LB + antibiotics; Group 2: Wild-type bacteria + LB; Group 3: Empty vector bacteria + LB + antibiotics

After induction, relative qPCR (normalized to 16S rRNA) showed significant mRNA upregulation: GPS ~16.1-fold,
CsTPS1 ~18.0-fold, and GeDH ~12.6-fold (Fig. 6). This confirmed successful transcriptional activation of the entire pathway under IPTG + L-ara co-induction.

![](https://static.igem.wiki/teams/5938/results/results-fig-6.webp)

Fig.6 Relative expression level of GPS, CsTPS1 and GeDH

Notably, GeDH had slightly lower induction than the other enzymes, likely reflecting promoter strength differences or suboptimal induction conditions, suggesting directions for optimization.

At the protein level, Western Blot successfully detected the three enzymes with clear bands at their expected molecular weights: GPS ~46.9 kDa, CsTPS1 ~63.7 kDa, GeDH ~41.6 kDa (Fig. 7). This confirmed translation and expression of all key enzymes.

![](https://static.igem.wiki/teams/5938/results/results-fig-7.webp)

Fig.7 The Western blot results of GPS, CsTPS1, and GeDH.

Together, growth curve, qPCR, and Western Blot results formed a complete evidence chain from “chassis health” to “transcriptional activation” to “protein expression,” confirming that the pathway was active inside cells and setting the stage for product detection.

# 3. Round 3 DBTL: Product Fermentation and Quantification

The third cycle focused on verifying citronellal production and establishing reliable quantification. Engineered strains were cultured in TB medium, induced at mid-log phase (OD₆₀₀≈0.6) with IPTG + L-ara to initiate citronellal biosynthesis.

High-performance liquid chromatography (HPLC) was used as the primary analysis method. A standard curve was prepared using citronellal standards (0.4-1.6 g/L), achieving excellent linearity (R²≈0.999) (Fig. 8).

![](https://static.igem.wiki/teams/5938/results/results-fig-8.webp)

Fig.8 The standard curve of the citronellal sample.

During HPLC analysis, the citronellal peak was detected and quantified at approximately 14 minutes using a UV detector.
Simultaneously, we conducted parallel UV-Vis spectrophotometry (UV-Vis) measurements as cross-validation. Furthermore,
to evaluate the distribution of the product both inside and outside the cells, we separately measured the citronellal
content in the fermentation supernatant and cell lysate to determine whether subsequent analyses could focus solely on
the supernatant. The HPLC analysis successfully detected the citronellal product. The fermentation supernatant sample
exhibited a target peak at a retention time of around 14 minutes, with the calculated citronellal concentration being
approximately 0.89 g/L (Fig.9)

![](https://static.igem.wiki/teams/5938/results/results-fig-9.webp)

Fig.9: HPLC of TB fermentation supernatant

Correspondingly, citronellal was also detected in the cell lysate sample,
with a concentration of approximately 0.91 g/L (Fig.10)

![](https://static.igem.wiki/teams/5938/results/results-fig-10.webp)

Fig.10: HPLC of cell lysate

The product concentrations of the two samples were very close, and statistical significance
testing confirmed that the difference was not significant (Fig.11). This indicates that the
distribution of citral between the culture supernatant and intracellular fraction is roughly
equivalent. Therefore, the workflow can be simplified by directly measuring the fermentation
supernatant to represent the total yield.

![](https://static.igem.wiki/teams/5938/results/results-fig-11.webp)

Fig.11 Significance Analysis of Concentration Differences between Cell Lysate and Fermentation Supernatant.

In terms of quantification accuracy, concentration results obtained from UV-Vis parallel tests were
consistent with those from HPLC, further enhancing the reliability of the data. Through this round of
fermentation experiments, we clearly demonstrated that the engineered strain is indeed capable of synthesizing
citral, reaching nearly gram-per-liter levels under shake-flask conditions. This not only marks a key milestone
in product generation for the project but also provides a solid quantitative foundation for subsequent optimization.

The third round of results provided three important insights and directions for improvement:
1. The engineered strain successfully “lit up” the synthetic pathway at the product level, achieving citral titers
close to g/L in shake flasks, thereby demonstrating the feasibility of biomanufacturing. 2. No significant difference was
observed in the intra- and extracellular distribution of citral, so future detection can be simplified by analyzing only the
supernatant, improving throughput for strain screening. 3. The consistency between HPLC quantification and UV-Vis detection
established a robust “dual-channel” quantification system, offering a reliable benchmark for process optimization and
performance evaluation.

Based on these findings, we decided to carry out more in-depth engineering optimization in the next round to further improve
production: on one hand, by investigating the effects of induction temperature and inducer concentration on yield in fermentation
process optimization; and on the other, by applying enzyme engineering strategies to modify the rate-limiting enzyme, with the aim
of further enhancing citral synthesis efficiency.

# 4. Round 4 DBTL: Process Optimization and Enzyme Engineering

In the final engineering cycle, our focus shifted from simply achieving production to attaining high production of citral.
To this end, we adopted a dual strategy: process optimization and rational enzyme engineering.

For process optimization, Response Surface Methodology (RSM) was introduced to systematically optimize induction conditions.
Specifically, a Box-Behnken design was used to examine the combined effects of three factors—induction temperature (A), IPTG
concentration (B), and L-ara concentration (C)—each at three levels, on citral yield. A total of 15 shake-flask experiments were
performed to fit a quadratic polynomial model, enabling evaluation of main effects, interactions, and quadratic effects.

In parallel, in silico enzyme engineering of the rate-limiting enzyme GeDH was conducted. First, the protein structure was predicted,
and Funclib was used to identify potential beneficial mutation sites. Next, Rosetta-based molecular docking was applied to calculate
binding conformations, followed by GROMACS molecular dynamics simulations to assess the stability of mutant enzyme-substrate complexes.
Through multiple rounds of scoring, four key mutation sites—Q60R, L125P, F147Y, and A300D—were identified and combined into a quadruple mutant,
expected to enhance the terminal oxidation step from geraniol to citral.Under the optimized RSM conditions, citral yield reached 1.01 g/L
(Fig.12), representing an improvement over the previous round.

![](https://static.igem.wiki/teams/5938/results/results-fig-12.webp)

Fig.12 Optimal fermentation conditions predicted by response surface HPLC

Meanwhile, the GeDH quadruple mutant strain achieved an even higher yield under the same conditions, with HPLC quantification showing 1.36
g/L—approximately a 53% increase compared to the unmodified strain (Fig.13). This significant improvement demonstrates the practical effectiveness
of rationally designed mutations in enhancing flux through the terminal step.

![](https://static.igem.wiki/teams/5938/results/results-fig-13.webp)

Fig.13 HPLC fermentation under optimal conditions for the mutant strain

By combining process parameter optimization with enzyme engineering, we successfully elevated citral titers to
a new level. The fourth round experiments deepened our understanding of the engineered system and provided clear
guidance for future work:1. Induction temperature is a critical lever for yield improvement—low-temperature induction
markedly enhanced citral synthesis efficiency and should be adopted as the standard condition.2. Dual-inducer
optimization is essential—IPTG exhibited an optimal effective concentration, beyond which no further yield increase
was observed. Optimal results required coordinated tuning with L-ara levels.3. Enzyme engineering is effective—rational
mutations of the rate-limiting enzyme significantly boosted pathway flux, and when combined with process optimization,
the gains exceeded those of either strategy alone.

Accordingly, we established 20 °C, IPTG 0.46 mM, and L-ara 0.20% as the current optimal induction conditions, and confirmed
the use of “direct HPLC quantification of the fermentation supernatant, with UV-Vis as auxiliary” as our strategy for yield
evaluation. Moving forward, we plan to extend the successful GeDH engineering approach to upstream enzymes, applying saturation
or combinatorial mutagenesis at key sites. For scale-up studies, additional factors such as dissolved oxygen, pH, and induction
timing will be incorporated into the next round of RSM optimization, in order to further enhance volumetric productivity and ensure
batch-to-batch consistency.

Through iterative optimization in each cycle, we aim to ultimately achieve efficient and stable citral production.

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