iGEM 2024 — Team shanghai-biox

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% extends "layout.html" %}
{% block title %}Project Safety{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
{% block page\_content %}

![Project Banner](https://static.igem.wiki/teams/5207/banner/enginering.svg)

### Index

* [Overview](#overview)
* [Design](#design)
* [Build](#build)
* [Test: Product Detection](#product-detection)
* [Learn](#learn)
* [Reference](#reference)

## Overview

In order to acheive the synthesis of the precursors of key bioactive compounds from Salvia plants such as S. miltiorrhiza, we utilized yeast as the chassis organism and employed the GoldenGate assembly method to construct two composite parts, TY9 and TY10. We also conducted tests on vector construction and fermentation processes. The results indicate that we have successfully synthesized tanshinones derivatives and carnosic acid, which are expected to be applied in the treatment of myocarditis.

## Design

To realize the production of tanshinones precursor and carnosic acid, the active ingredients of Salvia plants, on the yeast platform, we planned to integrate various key enzyme genes, SmGGPPS, SmCPS, SmKSL, SmCPR, CYP76AH1, CYP76AH3, CYP76AH22, and CYP76AK6, respectively, into pYTK096 vector, and to select the corresponding genes for transcription and termination to control the expression of these enzymes in Saccharomyces cerevisiae. Promoters and terminators for transcription are used for controlling the expression of these enzyme genes. Detailed design please to our design page.

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s1.jpg)

Figure 1 Gene circuit design of tanshinones (TY9) and carnosic acid (TY10)
production

As shown in the following figure: we constructed the plasmid vectors of TY9 and TY10 by three steps

1. **Level 0:** Cloning the sequences containing the target genes by PCR reaction, and inserting them into vector pYTK001 by BsmBI respectively,
2. **Level 1:** Combining the modules of each Level 0, constructing them into vector pYTK095 after ligating them by BsaI zymography,
3. **Level 2:** Combination of individual Level 1 vectors, constructed into vector pYTK096 after ligation by BsmBI digestion.

(Detailed design can be found in our [Design page](https://2024.igem.wiki/shanghai-biox/design))

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s2.jpg)

Figure 2 Flowchart for Level-0 to Level-2 assembly

## Build

### Level 0 Plasmid Construction

Firstly, we designed primer **F: GCATCGTCTCATCGGTCTCAT(ATG) + CDS-5'** and primer
**R: ATGCCGTCTCAGGTCTCAGGAT + CDS-3'**, which contain the BsmBI cleavage site, and cloned
the sequences containing CDS of the target genes by PCR reaction. The target genes are
*SmGGPPS*, *SmCPS*, *SmKSL*, *SmCPR*, *CYP76AH1*,
*CYP76AH3*, *CYP76AH22*, *CYP76AK6*, which were obtained from the template DNA.

CDS sequences of *Salvia* was amplified by PCR and verified (as shown in Figure
3):

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s3.jpg)

Figure 3 Validation plot of target gene gel electrophoresis

According to Figure 3, we can see that the target genes *SmGGPPS*, *SmCPS*,
*SmKSL*, *SmCPR*, *CYP76AH1*, *CYP76AH3*, *CYP76AH22*, and *CYP76AK6*
have obvious DNA bands at the corresponding positions as expected, and the construct validation is
successful.

Then they were inserted into the vector **pYTK001** (Figure 4) respectively, which
contains the CDS element of the key enzyme gene of the diterpene pathway, and this step is the
construction of Level 0. The construction system is as follows:

* 1) pYTK001-PTDH3 (or PCCW12, PPGK1, PTEF1, PTEF2);
* 2) pYTK001-SmGGPPS (or SmCPS1, SmKSL, SmCPR, CYP76AH1, CYP76AH3, CYP76AH22, CYP76AK6);
* 3) pYTK001-TENO1 (or TSSA1, TADH1, TTDH1, TENO2).

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s4.jpg)

Figure 4 Snapgene diagrams of pYTK001

The monoclonal strains that did not show fluorescence were transformed in *E. coli*
DH10B. We selected under UV light for colony PCR amplification and validation as shown in Figures 5-6 below:

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s5.jpg)

Figure 5 Fluorescent labeling screening of pYTK001

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s6.jpg)

Figure 6 Validation plot of pYTK001 gel electrophoresis

### Level 1 Plasmid Construction

According to Figure 6 electrophoresis diagram, we can see that Level 0 plasmids all have obvious DNA bands at the corresponding positions, which is consistent with our theory, and the construction is verified successfully. Next, we combined each Level 0 junction module, promoter, CDS, terminator, and ending module, and constructed them into vector pYTK095 (Figure 7) through BsaI digestion and ligation, and this step was the construction of Level 1. The construction system is as follows:

* pYTK095-PTDH3-SmGGPPS-TENO1
* pYTK095-PCCW12-SmCPS1-TSSA1
* pYTK095-PPGK1-SmKSL-TADH1
* pYTK095-PTEF2-SmCPR-TTDH1
* pYTK095-PTEF1-CYP76AH1/AH22-TENO2
* pYTK095-PTEF2-CYP76AH3/AK6-TTDH1

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s7.jpg)

Figure 7 Snapgene diagrams of pYTK095

After transformation in *E. coli* DH10B, we selected the single bacterial colony (pYTK095) that does not fluoresce under UV light from the petri dishes. It was used for colony PCR amplification and validation as shown in Figure 8-9 below:

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s8.jpg)

Figure 8 Fluorescent labeling screening of pYTK095

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s9.jpg)

Figure 9 Validation plot of pYTK095 gel electrophoresis

### Level 2 Plasmid Construction

According to Figure 9, we can see that Level 1 plasmids all have obvious DNA bands at the corresponding positions, which is consistent with our theory, and the construction verification is successful. Then, we combined the Level 1 vectors and constructed them into pYTK096 vector (Figure 10) by BsmBI digestion and ligation, and successfully constructed the Level 2 vector. The construction system is as follows:

* TY9: pYTK096- PTDH3-SmGGPPS-TENO1: PCCW12-SmCPS1-TSSA1: PPGK1-SmKSL-TADH1: PTEF2-SmCPR-TTDH1: PTEF1-CYP76AH1-TENO2: PTEF2-CYP76AH3-TTDH1
* TY10: pYTK096-PTDH3-SmGGPPS-TENO1:PCCW12-SmCPS1-TSSA1: PPGK1-SmKSL-TADH1:PTEF2-SmCPR-TTDH1:PTEF1-CYP76AH22-TENO2:PTEF2-CYP76AK6-TTDH1

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s10.jpg)

Figure 10 Snapgene diagrams of pYTK096

Similarly, after transformation in *E. coli* DH10B identified positive monoclones by resistance, fluorescent labeling screening, and colony PCR, the results are shown in Figures 11-12 below:

|  |  |
| --- | --- |
| ![Image 1](https://static.igem.wiki/teams/5207/engineering/s11.jpg) | ![Image 2](https://static.igem.wiki/teams/5207/engineering/s12.jpg) |

Figure 11 Fluorescent labeling screening of pYTK095

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s13.jpg)

Figure 12 Validation plot of pYTK096 gel electrophoresis

According to Figure 11, we can see that the one that does not show green fluorescence is the recombinant plasmid we want, which corresponds to the colony PCR agar gel image, and the recombinant plasmid is successfully constructed. Next, we extracted the corresponding plasmid DNA after amplification and culture, and used it for transformation in yeast cells.

The constructed vector was linearized by NotI endonuclease and then transferred into BY4742 yeast receptor cells, which were screened by -ura-deficient medium, and the yeast genomic DNA was extracted for PCR identification to determine the positive single clones. The validation results were as follows:

1) URA-deficient medium screening results (URA-deficient SD):

|  |  |
| --- | --- |
| ![Image 1](https://static.igem.wiki/teams/5207/engineering/s14.jpg) | ![Image 2](https://static.igem.wiki/teams/5207/engineering/s15.jpg) |

Figure 13 Graph of the results of the screening of URA-deficient media
(left TY9, right TY10)

2）Insert fragment PCR assay results:

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s16.jpg)

Figure 14 Gel validation of TY9 and TY10 recombinant plasmids in yeast

The successful monoclonal plasmid was expanded in culture, shaken small overnight and preserved, and the strain was further fermented and cultured using liquid YPDA medium for 5 days and then centrifuged to collect the organisms.

|  |  |
| --- | --- |
| ![Image 1](https://static.igem.wiki/teams/5207/engineering/s17.jpg) | ![Image 2](https://static.igem.wiki/teams/5207/engineering/s18.jpg) |

Figure 15 Overnight culture and scale-up of yeast small shake
fermentation

|  |  |
| --- | --- |
| ![Image 1](https://static.igem.wiki/teams/5207/engineering/s19.jpg) | ![Image 2](https://static.igem.wiki/teams/5207/engineering/s20.jpg) |

Figure16 Schematic diagram of the fermented bacterial liquid

## Test: Product Detection

Saccharomyces cerevisiae cells were extracted with methanol by Ultrasonic Cell Disruption for 1 h. After centrifugation at low temperature, the supernatant extract was filtered with a filter membrane, and the filtered samples were transferred to liquid phase vials with lined tubes for Q-Exactive analysis, and the results were as follows:

TY9: as shown in Figure 17, the product of TY9 is mainly composed of two compounds 1,2 which appeared near 6 and 9 minutes, with molecular weights of 317 and 299 under positive mode, respectively. Through literature analysis and discussed with our instructor, we confirmed that the compound MW of 316 is 11-hydroxy-sugiol, and MW 298 has not been found to point to any specific substance at this point in time.

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s21.jpg)

Figure17 Chromatogram and mass spectrogram of S. c BY4742/TY9 extract

TY10: as can be seen in Figure 18, where Figure 18-A is the result of the TY10 mass spectrometry test, showing that it contains two main compounds 3,4, Figure 18-B is the chromatogram of the carnosic acid (CA) standard, and Figure 18-C is the chromatogram of the carnosol (CV) standard.

![Synthetic Biology Image](https://static.igem.wiki/teams/5207/engineering/s22.jpg)

Figure18 Chromatogram of S. c BY4742/TY10 extract (A), carnosic acid (CA) standard (B), and carnosol (CV) standard (C); (D) CA and (E) CV standard curves

As can be seen in Figure 17-18(A-C), the 11-hydroxy-sugiol, CV and CA confirmed the correct synthesis of the products by the mass-to-charge ratios and intensities of the absorption peaks where they are located and the molecular masses compared to the standards, and the 11-hydroxy-sugiol is more predominant in the yeast cell fermentation broth extracts. In Figure 18(D-E), we used CV and CA standards and their corresponding peak areas to derive the corresponding equations:

Equation for CA: **y=2E+0.6x+1356.6 (R2=0.9997)**
Equation for CV: **1E+0.6x+1252.5 (R2=0.9993)**

The yield of CA in the test was calculated to be 0.268 mg/ml, and similarly, the yield of CV in the test can be obtained to be 0.857 mg/ml. Further, the content of the extracted CA in 3 L of fermentation broth in the experiment was obtained to be 0.178 mg/L, and the content of CV was obtained to be 0.571 mg/L.

## Learn

In summary, we successfully synthesized key bioactive compounds, including tanshinone derivatives and carnosic acid, using *Saccharomyces cerevisiae* as a platform. By utilizing the GoldenGate assembly method, we constructed two composite parts, TY9 and TY10, integrating multiple key enzyme genes to achieve the synthesis. Through thorough validation via plasmid construction, PCR amplification, and liquid chromatography-mass spectrometry (LC-MS), we confirmed the production of 11-hydroxy-sugiol, carnosic acid (CA), and carnosol (CV). The yields of CA and CV were calculated, showing promising results for future application in myocarditis treatment.

However, there are areas for improvement:

* **Further Analysis of Unknown Compounds and Metabolic Pathway Investigation:** The unidentified compound with a molecular weight of 298 is likely a precursor in the tanshinone biosynthesis pathway. Further structural analysis and metabolic pathway mapping are required to confirm its role and complete the synthesis of tanshinone derivatives on the yeast platform. This investigation will focus on identifying all intermediates and optimizing the entire metabolic pathway for efficient production of tanshinones.
* **Validation of Anti-inflammatory Efficacy:** While we have successfully synthesized tanshinone derivatives and carnosic acid, the next crucial step is to experimentally validate their anti-inflammatory properties. This includes conducting in vitro and in vivo assays to confirm the efficacy of these compounds in treating myocarditis and other inflammatory conditions, ensuring their potential for therapeutic use is realized.Optimizing our detection and characterization methods would help address this issue. Additionally, increasing the overall yield of CA and CV is necessary for large-scale application, and refining the fermentation process could help improve these outcomes.
* **Boosting Production:** Boost the production of these bioactive compounds by optimizing the fermentation parameters, increase enzyme expression levels, and explore alternative engineering strategies.

## References

1. Novarina D, Koutsoumpa A, Milias-Argeitis A. A user-friendly and streamlined protocol for CRISPR/Cas9 genome editing in budding yeast[J]. STAR protocols, 2022, 3(2): 101358.
2. Guo et al., 2016, New Phytologist，210: 525–534.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

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Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project and what you learned from them. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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