iGEM 2025 — Team szsais-baoan

Notebook

Overview

Since the launch of relevant practical research in March, the team has clearly identified the efficient
biosynthesis of salidroside as the core research direction. On this basis, we have systematically
reviewed cutting-edge literature in the field and conducted in-depth studies on theoretical systems
related to the optimization of biosynthesis efficiency. From June 20th to August 14th, through nearly
two months of continuous experimental exploration and multiple rounds of expert discussions, we finally
succeeded in achieving the efficient biosynthesis of salidroside in the laboratory environment. This
landmark achievement has laid a solid foundation for the team's subsequent technological breakthroughs
in the field of synthetic biology and further strengthened our confidence in exploration.

**Lab rules:**

1. Wear gloves and lab coat before interacting with any lab materials (Wear goggles if handling harzardous objects).

2. Individuals with long hair should put back their hair.

3. Do not drink or eat anything in the lab.

4. Do not run, or fight (horse play) in the lab, and especially when an individual is holding lab material.

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Engineering

![](https://static.igem.wiki/teams/5954/logo/menu.webp)

# Menu

* [##### ⚪ Overview](#one)
* [##### ⚪ Cycle 1: Design and construct a biosensor responsive to salidroside](#two)
* [##### ⚪ Cycle 2: Construction of the salidroside synthesis pathway in *Escherichia coli* chassis strains](#three)
* [##### ⚪ Cycle 3: Enhance the endogenous synthesis pathway in *Escherichia coli*](#four)

- Overview -

We have consistently adhered to the "Design-Build-Test-Learn" cyclic paradigm to advance this project. This year's project can be broadly divided into three cyclic phases: in the first cycle, we designed and constructed a biosensor responsive to salidroside; in the second cycle, we established the salidroside synthesis pathway in the *Escherichia coli* chassis strain; and finally, in the third cycle, we optimized the salidroside-producing performance of the engineered strain by overexpressing the endogenous genes.

![engineering1](https://static.igem.wiki/teams/5954/engineering/engineering1.webp)

Figure 1: Design Framework for Efficient Synthesis of Salidroside in *Escherichia coli*

Cycle 1:

- Design and construct a biosensor responsive to salidroside -

· Design

This project developed a synthetic biosensor with the transcription factor mutant HucRSD to detect salidroside. It has three DNA fragments working together for sensitive, visual detection.

HucRSD (four mutations) in the sensing module specifically recognizes salidroside: binding HucO to block transcription without substrate, forming a complex to activate expression when substrate is present.

The reporting module links GFP (fluorescent quantification) and LacZ (enzyme checks). Controlled by strong promoter P23119 with RBS and terminators, it’s efficient and sensitive.

![engineering2](https://static.igem.wiki/teams/5954/engineering/engineering2.webp)

Figure 2: A biosensor based on the transcription factor mutant HucRSD

· Build

We constructed all target plasmids by Gibson Assembly, attaching Lacz and HucR on Vector. HucO is connected with HucR, acting as a repressor until it combines with salidroside and falls off.

### Results for pRB1k-pHuc-eGFP-LacZ:

![engineering3](https://static.igem.wiki/teams/5954/engineering/engineering3.webp)

Figure 3: pRB1k-pHuc-eGFP-LacZ Plasmid Map

![engineering4](https://static.igem.wiki/teams/5954/engineering/engineering4.webp)

Figure 4: pRB1k-pHuc-eGFP-LacZ Plasmid Construction Process

· Test

Measurements using a microplate reader revealed a linear relationship between salidroside concentration and biosensor absorbance. Absorbance increased proportionally with rising salidroside concentration, confirming the accuracy of our results. A sixfold difference in absorbance was observed between samples containing 0.0 g/L and 2.0 g/L salidroside. Testing with salidroside standards demonstrated that our plasmid specifically responds to salidroside.

![engineering5](https://static.igem.wiki/teams/5954/engineering/engineering5.webp)

Figure 5: Bio-sensor Effect Test

Subsequently, the salidroside produced by the salidroside-synthesizing bacterial culture was divided into two batches. One batch was subjected to HPLC analysis, while the other was added to the biosensor strain for fermentation, followed by absorbance measurement using a microplate reader.

· Learn

In conclusion,we've successfully built a system that can detect the presence and concentration of salidroside. With the mutant HucRSD, we've achieved a regulatory response to salidroside. When salidroside is added, the fluorescent proteins GFP and LacZ are turned on, and they give quantifiable results, showing different shades of yellow to indicate the concentration differences.

Cycle 2:

- Construction of the salidroside synthesis pathway in *Escherichia coli* chassis strains -

· Design

Through an extensive literature review, we found that *Escherichia coli* is incapable of autonomously synthesizing salidroside. Therefore, we set out to construct its de novo synthesis pathway, with the biosynthesis of tyrosol as the core intermediate step. First, two heterologous genes, Kivd from *Lactococcus lactis* and PAR from *Rosa damascena*, were introduced to form an enzyme module, enabling the efficient biosynthesis of tyrosol using glucose. Salidroside is produced through the glycosylation of tyrosol catalyzed by UGTs (glycosyltransferases), and this reaction determines the yield and regioselectivity of the product. Hence, screening for highly efficient UGTs is crucial for enhancing the catalytic efficiency of the conversion from tyrosol to salidroside, thereby increasing the yield of salidroside.

![engineering7](https://static.igem.wiki/teams/5954/engineering/engineering6.webp)

Figure 6: Constructing the Salidroside Synthesis Pathway

· Build

We amplified the target genes KivD and PAR by PCR; digested the vector pY97a, and constructed the recombinant plasmid pY97a-P23107-KivD-PAR using Gibson assembly technology.

### Results for pY97a-P23107-KivD-PAR:

![engineering7](https://static.igem.wiki/teams/5954/engineering/engineering7.webp)

Figure 7: pY97a-P23107-KivD-PAR Plasmid Construction Process

We found 33 different UGT-protein series from different species on the NCBI website, and we analysed their similarity, we drew a phylogenetic tree, then we choosed UGT85A1 and UGT85A1A21G which both from *Arabidopsis thalinana*, *UGT72B14* which from *Rhodiola sachalinensis*, and RrUGT33 from *Rhodiola rosea L*. We let the company synthesize the pY97a-P23119-UGTs-P23107-KivD-PAR plasmids, each containing one of the four genes.

![engineering8](https://static.igem.wiki/teams/5954/engineering/engineering8.webp)

Figure 8: Screening glycosyltransferases from different sources

· Test

The successfully constructed recombinant plasmids were transformed into competent BW25113 cells for induced expression. After LB activation and ZY-induced expression for 16 hours, bacterial cultures with an OD600 of 3 were collected in centrifuge tubes and centrifuged. Then, 200 µL of M9 fermentation medium was added for fermentation, and the cultures were incubated in a shaker for 16 hours. The cultures were centrifuged again, and the supernatant was collected using a 1 mL syringe fitted with a filter membrane. The filtered supernatant was transferred to liquid-phase vials and sent to a company for testing.

After we received the test results from the HPLC (high performance liquid chromatography), we constructed a graph to present the data. The graph is plotted in a way where the salidroside productivity(mg/L) is aligned on the Y-axis against the type of the UGT sample, which is shown on the X-axis.

The four types of UGT samples were each tested for three trials in order to guarantee the accuracy of the resulting data. The error and dispersion of the data is indicated on the graph by the error bar and dots located on top of the columns.

![engineering9](https://static.igem.wiki/teams/5954/engineering/engineering9.webp)

Figure 9: Detecting Glycosyltransferases from Different Sources Using HPLC

· Learn

By inserting the exognous gene KivD, PAR,and UGT to the *E.coli*,we find a effective way to produce biosynthesis salidroside. Indeed, the test results demonstrate that all the exognous genes we select have contribute to the synthesis of salidroside. What's more, we discovered that UGT72B14 has the highest productivity of biosynthesis salidroside. Average amount of UGT85A1 was 50.95mg/L, and average amount of UGT72B14 was 72.96mg/L, which means that the productivity of UGT72B14 increased 43% more than productivity of UGT85A1. In conclusion, we decide to use UGT72B14 as our enzyme to catalyze the UDP-Glucose and the tyrosol produce salidroside.

Cycle 3:

- Enhance the endogenous synthesis pathway in *Escherichia coli* -

· Design

Our goal is to enhance the biosynthetic pathways of tyrosol and uridine diphosphate glucose (UDPG), both of which are precursors required for salidroside production. To improve the efficiency of microbial salidroside production by optimizing the biosynthesis of these two primary precursors, we overexpress the endogenous *Escherichia coli* genes GalU and Pgm to increase intracellular UDPG levels. Meanwhile, to boost tyrosol production, we introduce feedback-resistant mutant genes AroGfbr and TyrAfbr. These mutants can maintain metabolic flux under high-load production conditions and avoid inhibition by downstream metabolites.

![engineering10](https://static.igem.wiki/teams/5954/engineering/engineering10-new.webp)

Figure 10: Overexpression of endogenous key genes in the salidroside synthesis pathway

· Build

We amplified the target genes GalU, Pgm, AroGfbr, and TyrAfbr by PCR; digested the vectors pLB1s and pSB1c; and constructed the recombinant plasmids pLB1s-GalU-Pgm and pSB1c-AroGfbr-TyrAfbr using Gibson assembly technology.

### Results for pLB1s-GalU-Pgm:

![engineering11](https://static.igem.wiki/teams/5954/engineering/engineering11.webp)

Figure 11: pLB1s-GalU-Pgm Plasmid Map

![engineering12](https://static.igem.wiki/teams/5954/engineering/engineering14.webp)

Figure 12: pLB1s-GalU-Pgm Plasmid Construction Process

### Results for pSB1c-AroGfbr-TyrAfbr:

![engineering13](https://static.igem.wiki/teams/5954/engineering/engineering13.webp)

Figure 13: pSB1c-AroGfbr-TyrAfbr Plasmid Map

![engineering14](https://static.igem.wiki/teams/5954/engineering/engineering12.webp)

Figure 14: pSB1c-AroGfbr-TyrAfbr Plasmid Construction Process

· Test

The successfully constructed recombinant plasmids were transformed into competent BW25113 cells for induced expression. After LB activation and ZY-induced expression for 16 hours, bacterial cultures with an OD600 of 3 were collected in centrifuge tubes and centrifuged. Then, 200 µL of M9 fermentation medium was added for fermentation, and the cultures were incubated in a shaker for 16 hours. The cultures were centrifuged again, and the supernatant was collected using a 1 mL syringe fitted with a filter membrane. The filtered supernatant was transferred to liquid-phase vials and sent to a company for testing.

In order to evaluate the effect of our biosynthesis pathway enhancments, we submitted four different samples for testing the salidroside yield (mg/L) using HPLC technique, including:

●No enhancement, use as control data (UGT72B14)

●Only the shikimate biosynthesis pathway enhancement plasmid (UGT72B14-AroGfbr-TyrAfbr)

●Only the UGP-glucose biosynthesis pathway enhancement plasmid (UGT72B14-GalU-Pgm)

●Both enhancement plasmids (UGT72B14-AroGfbr-TyrAfbr-GalU-Pgm).

![engineering15](https://static.igem.wiki/teams/5954/engineering/engineering15-new.webp)

Figure 15: Results of HPLC detection of overexpressed key genes

We performed a side-by-side comparison of the detection results from the biosensor fermentation and the HPLC analysis. The consistent trend between the two methods confirms that the trend we detected using the biosensor, although approximate, is accurate.

![engineering16](https://static.igem.wiki/teams/5954/engineering/engineering16.webp)

Figure 16: Comparison of HPLC measurement results with biosensor measurement results

· Learn

According to HPLC results, comparing our results to the control data bar, we are able to visualize that the enhancement of shikimate and UDP-glucose biosynthesis pathway lead to the increase in yield of salidroside. When both shikimate and UDP-glucose biosynthesis pathway are enhanced, yield of salidroside has multiplied about 2.86 times, achieving 208.32mg/L.

· Conclusion

Following a rigorous "Design-Build-Test-Learn" methodology, our project successfully engineered an *E. coli* strain for the efficient biosynthesis of salidroside. Key engineering successes include the construction of a novel, low-cost biosensor, and the establishment of a functional de novo synthesis pathway. Through systematic optimization, we achieved a final salidroside yield of 208.32 mg/L. This work not only provides a viable platform for sustainable salidroside production but also the utility of our custom-built biosensor for accelerating future metabolic engineering projects.

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

## What Should this Page Contain?

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Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

* Clearly and objectively describe the results of your experiments and research.
* Use tables, graphs, and images to visualize your data.
* Discuss the implications of your findings in the context of your project goals.
* Highlight any trends, patterns, or significant observations.
* Include an analysis summary section to synthesize your findings and discuss their overall significance.
* Outline your future plans for the project, including potential applications, further research, and development.
* Explain what went wrong and what you learned from these experiences.
* Discuss potential improvements or alternative approaches.
* Emphasize the importance of scientific honesty and transparency.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 EPFL](https://2024.igem.wiki/epfl/results)
* [2024 UToronto](https://2024.igem.wiki/utoronto/results)
* [2024 Marburg](https://2024.igem.wiki/marburg/results)
* [2024 Sydney-Australia](https://2024.igem.wiki/sydney-australia/results)
* [2024 Toulouse-INSA-UPS](https://2024.igem.wiki/toulouse-insa-ups/results)
* [2024 BNDS-China](https://2024.igem.wiki/bnds-china/results)
* [2024 Vilnius-Lithuania](https://2024.igem.wiki/vilnius-lithuania/results)
* [2024 XMU-China](https://2024.igem.wiki/xmu-china/results)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/results)

{% endblock %}