iGEM 2025 — Team fudan

---
title: Notebook
authors:
  - name: Yuxin Duan
    url: /fudan/team/#Yuxin
    avatar: https://static.igem.wiki/teams/5643/pageimage/team/dyx-a.webp
layout: igem
heroImage: https://static.igem.wiki/teams/5643/header/notebook.webp
description: On this page, we document our project's journey, tracking the key developments and significant progress we have made from inception to freeze.
---

## January

From January 6th to 15th, we held the ten-day [winter training](https://2018.igem.org/Team:Fudan/Heritage#section2) on synthetic biology and the iGEM competition.

The training was structured into three components:

1. Sharing experiences from [Fudan iGEM 2024](https://2024.igem.wiki/fudan/team) members to prepare new members with essential skills and knowledge while allowing them to select their own focuses based on interest.
2. Ice-breaking among new members to build effective communication.
3. Through brainstorming and three rounds of idea competitions, we conceptualized potential project proposals and ultimately defined the research direction for iGEM 2025, with further refinements in subsequent phases.

<div style="text-align: center;" id="fig1">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/january-trainingschedule.webp" style="width:100%; max-width:100%;max-width:100%">
    <div>
        <span style="color:gray">Figure 1. The schedule of the winter training</span>
        <br><br>
    </div>
</div>

Through these training programs, we ensured seamless transfer of knowledge and experience between alumni and new members, maintained team continuity, and fostered team growth. We also actively sought valuable feedback from faculty, inviting their suggestions on our brainstorming initiatives. Ultimately, inspired by the insights of alumnus [Kai Tong](https://www.scopus.com/authid/detail.uri?authorId=57224397364) and under the mentorship of Professor [Ratcliff](https://ratclifflab.biosci.gatech.edu/), a leading figure in experimental evolution, we selected multicellular yeast as our chassis organism and developed the initial concept for our subsequent project, [*DR.sTraTeGY*](/design/) (Drug Resistance mutation Tracking Technology based on Grape Yeast).

<div style="text-align: center;" id="fig2">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/january-groupphoto.webp" style="width:80%">
    <div>
        <span style="color:gray">Figure 2. All new &amp; old members who participated in the 2025 winter training</span>
        <br><br>
    </div>
</div>

<div style="text-align: center;" id="fig3">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/january-training.webp" style="width:80%">
    <div>
        <span style="color:gray">Figure 3. Our multicellular yeast brainstorming project</span>
        <br><br>
    </div>
</div>

## February

During the Spring Festival, we organized into groups based on individual skills and interests to conduct modular research. We thoroughly studied previous iGEM projects and compiled our findings by the end of February, which significantly enhanced our understanding of the iGEM [Judging Handbook](https://competition.igem.org/judging/handbook). Concurrently, we clarified each team member's tasks and roles to advance the project through further literature investigation.

<div style="text-align: center;" id="fig4">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/notebook-fig4.avif" style="width:100%;max-width:100%">
    <div>
        <span style="color:gray">Figure 4. Our research work conducted during the winter break</span>
        <br><br>
    </div>
</div>

## March

We held regular meetings to refine the design of both dry and wet-lab experiments based on our winter break preparations. These collaborative sessions helped clarify team responsibilities and establish workflows for various experimental tasks. During the meetings, each team member reported on the progress of their assigned tasks. We developed [fluorescent molecular clock](/design/#timer-tracking-cell-lineage) as a real-time tracker to monitor the evolutionary history of multicellular yeast and uploaded clothing designs featuring our project logo to the Animal Crossing platform.

Additionally, we corresponded with Professor Ratcliff via email and received valuable suggestions for organizing education initiatives. Based on practical considerations, we identified [butterfly children (patients with Epidermolysis Bullosa)](/inclusivity/#breaking-the-silence-the-first-glimpse-of-visibility)as the target group for our inclusivity efforts. We subsequently hosted [Garden Party](/education/#activity-4-garden-party), using interactive games to popularize synthetic biology knowledge in an engaging format.

<div style="text-align: center;" id="fig5">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/notebook-fig1.avif" style="width:100%;max-width:100%">
    <div>
        <span style="color:gray">Figure 5. Our project progress and some photos from public events</span>
        <br><br>
    </div>
</div>

## April

On April 15th, we conducted an interview with Professor Huang to advance our investigation into fungal drug resistance mechanisms. Our discussion focused on the primary evolutionary pathways of fungal resistance and explored the application of multicellular systems in resistance monitoring.

This discussion clarified the crucial role of chromosomal aneuploidization in the development of drug resistance, providing key theoretical support for constructing our multicellular yeast monitoring platform.

<div style="text-align: center;" id="fig6">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/notebook-fig2.avif" style="width:80%">
    <div>
        <span style="color:gray">Figure 6. Our discussion with Professor Huang</span>
        <br><br>
    </div>
</div>

## May

On May 11th, our team secured the second prize at the Shanghai Stock Exchange Cup Haiju Yingcai Innovation and Entrepreneurship Competition.

On May 13th, we attended the lecture "The Art of Interviewing: From Research Questions to Profound Dialogue" and subsequently integrated enhanced interviewing techniques into our [Human Practices](/human-practices/) initiatives. Concurrently, through an interview with Professor Zhu from Huashan Hospital, we received valuable guidance to transition our research focus from "strain screening" to "drug screening," while validating the correlation between our multicellular yeast system and clinical strains to ensure its practical applicability.

On May 16th, we collaborated with [CJUH-JLU-China](/education/#academic-depth-exchanges-with-jilin-university) to refine our educational outreach strategies, implementing personalized science communication approaches tailored to the audience's age and educational background.

On May 17th, we connected with middle school students in Henan Province through letter exchanges, effectively promoting iGEM and our project's vision.

<div style="text-align: center;" id="fig7">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/notebook-fig3.avif" style="width:100%;max-width:100%">
    <div>
        <span style="color:gray">Figure 7. Photos from our business roadshow and interview with experts</span>
        <br><br>
    </div>
</div>

## June

On June 27th - 28th, we attended The Chinese Biopharmaceutical Association (CBA-China) Annual Conference 2025 and engaged in project discussions with teams including [Nanjing-China](https://2025.igem.wiki/nanjing-china/), [ZJU-China](https://2025.igem.wiki/zju-china/), [ShanghaiTech-China](https://2025.igem.wiki/shanghaitech-china/), and [SJTU-BioX-Shanghai](https://2025.igem.wiki/sjtu-biox-shanghai/).

On June 30th, we partnered with [CSU-China](https://2025.igem.wiki/csu-china/) to co-organize an online lecture focusing on synthetic biology as a cutting-edge interdisciplinary field, introducing high school students to career development paths and prospects in life sciences.

We also jointly produced the children's [picture book "My Microbe Friends"](/education.html#my-microb-friends%E2%80%94%E2%80%94picturebook) with [AFMU-China](https://2025.igem.wiki/afmu-china).

<div style="text-align: center;" id="fig8">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/june-summary.webp" style="width:100%;max-width:100%">
    <div>
        <span style="color:gray">Figure 8. Our members attending conferences and organizing lectures</span>
        <br><br>
    </div>
</div>

## July

On July 7th, we named our multicellular yeast "[Grape Yeast](/design/)" based on its morphological characteristics and officially designated the project as "[*DR.sTraTeGY*](/description/)". During the summer vacation, we conducted a series of wet-lab experiments including yeast culturing, competent cell preparation, plasmid extraction, and [NotI](https://www.neb.com/en/products/r3189-noti-hf) enzyme digestion followed by yeast transformation.

Furthermore, we collaborated with [USTC](https://2025.igem.wiki/ustc/), [Nanjing-China](https://2025.igem.wiki/nanjing-china/), [SJTU-BioX-Shanghai](https://2025.igem.wiki/sjtu-biox-shanghai/), and [ZJU-China](https://2025.igem.wiki/zju-china/) to organize educational initiatives, producing instructional videos on microbial painting. Throughout mid-to-late July, we conducted multiple [microbial science outreach activities](/education.html#interactive-science-workshop) in various communities, successfully inspiring children's interest in scientific exploration.

<div style="text-align: center;" id="fig9">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/july-wetlabandhp.webp" style="width:80%">
    <div>
        <span style="color:gray">Figure 9. Our wet-lab and Human Practice in communities and schools</span>
        <br><br>
    </div>
</div>

## August

From August 6th to 8th, we participated in the Conference of China iGEM Community ([CCiC](https://mp.weixin.qq.com/s/Khat--iWCXM0JxiwPnq1Ng)), where we engaged with over 100 teams in focused discussions on Human Practices and exchanged valuable project insights, witnessing firsthand the dynamic progress of synthetic biology in China.

During this period, our conversation with Ms. Su revealed that Human Practices should fundamentally employ "oneself as methodology" to design reflexive activities through examining our relationship to the project theme. This approach ensures profound integration of engagement with research objectives while maintaining methodological rigor to reduce bias.

Meanwhile, we collaborated with 33 iGEM teams to compile the ["Debunking Synthetic Biology Myths" handbook](https://static.igem.wiki/teams/5643/pageimage/collaboration/smashing-synthetic-biology-rumours-science-brochure-compressed.pdf). This guide clarifies common misconceptions and aims to help the public identify and avoid misinformation related to synthetic biology.

<div style="text-align: center;" id="fig10">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/august-ccic.webp" style="width:100%;max-width:100%">
    <div>
        <span style="color:gray">Figure 10. Our journey at CCiC</span>
        <br><br>
    </div>
</div>

## September

On September 3rd, we took part in [the Star Forum](https://mp.weixin.qq.com/s/b8w70TE68bhm5pWgQCQuBA) and listened to Professor Jianpeng Ma's presentation on the deep integration of AI and life sciences. He analyzed how algorithms create value in biological fields and explored future trends and potential challenges.

On September 11th, we organized a joint project sharing session with [CSU-China](https://2025.igem.wiki/csu-china/), [SZU-China](https://2025.igem.wiki/szu-china/), [SUSTech-China](https://2025.igem.wiki/cjuh-jlu-china/collaboration), [SCAU-China](https://2025.igem.wiki/scau-china/) and ULink Education Group. Through these exchanges, we introduced synthetic biology concepts and presented our project in detail, while collecting valuable feedback and diverse perspectives.

In late September, we implemented targeted outreach initiatives for communities affected by fungal diseases. We conducted [photovoice workshops](/inclusivity/#_3-1-facilitating-dialogue-photovoice) and [healthcare accessibility mapping](/inclusivity/#sensory-friendly-research-design) for patients, organized [collaborative art exhibitions](/inclusivity/#_3-2-co-creation-in-action-the-art-experience-exhibition) and [handicraft charity sales](/inclusivity/#_1-1-art-pop-up-and-co-creation-charity-with-eb-patients) for Butterfly Children, and delivered elderly-focused seminars exposing "high-tech scams" while educating about legitimate synthetic biology-based drug development.

Over the course of one month, we jointly developed an [educational mini-game](https://gd.games/ruoxi_cao/mint-picture--platformer----bi) with USTC that visualizes gene editing processes through intuitive gameplay mechanics.

Our laboratory team proceeded with wet-lab experiments and analyzed the results using fluorescence microscopy, flow cytometry, and deep sequencing.

<div style="text-align: center;" id="fig11">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/september-summary.webp" style="width:100%;max-width:100%">
    <div>
        <span style="color:gray">Figure 11. Our education workshops and inclusiveness practices</span>
        <br><br>
    </div>
</div>

## October

We finalized the authorship for different web pages and divided the tasks to start writing the pages and summarizing previous work. Meanwhile, all members worked 24x7, analyzing wet-lab results, refining dry-lab work and optimizing the project presentation video until the final deadline.

<div style="text-align: center;" id="fig12">
    <img src="https://static.igem.wiki/teams/5643/pageimage/notebook/october-wiki.webp" style="width:80%">
    <div>
        <span style="color:gray">Figure 12. Wiki page assignments and writing</span>
        <br><br>
    </div>
</div>

And, Paris - we are coming!

---
title: Engineering
authors:
  - name: Zhiqin Wang
    url: /fudan/team/#Zhiqin
    avatar: https://static.igem.wiki/teams/5643/pageimage/team/wzq-a.webp
layout: igem
heroImage: https://static.igem.wiki/teams/5643/header/eng.webp
description: On this page, we detail how our Recorder module was developed through Design–Build–Test–Learn (DBTL) cycles.
---

## DBTL Cycles: Optimization of the Reporter Module

In our [Recorder](/design/#recorder) module, we designed and tested a fluorescent reporter system to monitor changes induced by EMS mutagenesis. This module involved four distinct promoters driving seven of our EMS-optimized fluorescent proteins. In completing this module, we encountered and flexibly resolved several key challenges through the Design-Build-Test-Learn (DBTL) cycle.

For the entire project design, please refer to our [Design](/design/) page; for specific experimental protocols, please check the [Experiments](/experiments/) page.

### Iteration 1: Restriction Site Conflict in Level 0 Construction

#### Design

Based on literature, we selected four promoters, pSTM1 (BBa_25FQBGJW), pRNR2 (BBa_K3748013), pOST1 (BBa_259JX52V), and pTDH3 (BBa_K3190001) with distinct mutation patterns[^1].

- **pSTM1** : Tends to increase expression
- **pRNR2**: Tends to decrease expression
- **pOST1**: Remains stable
- **pTDH3**: A strong constitutive benchmark promoter

We designed primers to amplify their genomic sequences for integration into the pYTK001 in Yeast Toolkit (YTK, Lee et al., 2015)[^2].

#### Build

We used the standard YTK process: Level 0 parts were amplified with flanking [BsmBI](https://www.neb.com/en/products/r0739-bsmbi-v2) and [BsaI](https://www.neb.com/en/products/r3733-bsai-hf-v2) sites and cloned into the pYTK001 entry vector via [BsmBI](https://www.neb.com/en/products/r0739-bsmbi-v2) [Golden Gate assembly](/experiments/#golden-gate-assembly).

#### Test

We successfully built the Level 0 plasmids for three promoters (pSTM1, pOST1, and pTDH3).

However, the pRNR2 promoter sequence contained internal [BsaI](https://www.neb.com/en/products/r3733-bsai-hf-v2) sites. During assembly, that site was cleaved, leading to fragmented DNA and extremely low efficiency in subsequent Level 1 assembly.

#### Learn

Internal [Type II](https://www.neb.com/en/tools-and-resources/feature-articles/everything-you-ever-wanted-to-know-about-type-ii-restriction-enzymes)S restriction sites (especially [BsmBI](https://www.neb.com/en/products/r0739-bsmbi-v2) and [BsaI](https://www.neb.com/en/products/r3733-bsai-hf-v2)) in a part drastically reduce Golden Gate efficiency by cleaving the fragment and making it unavailable for ligation. For pRNR2, the standard protocol was unreliable due to this unavoidable molecular interference.

### Iteration 2: Optimized Assembly and Initial Functional Failure

#### Design

To resolve the pRNR2 issue without mutating the promoter sequence:

1. Sequence Check: We confirmed that the 4-bp overhangs generated by the internal [BsaI](https://www.neb.com/en/products/r3733-bsai-hf-v2) sites did not match the YTK's standardized overhangs, meaning fragments would self-ligate rather than misassemble.
2. Protocol Optimization: We adapted a solution from other researchers: adding a final 30-minute T4 DNA Ligase reaction after the thermal cycles to maximize the repair and ligation of all ends[^3].

#### Build

We executed the Golden Gate assemby as normal, followed by a dedicated 30-minute incubation with additional T4 DNA Ligase at 16∘C. This extended ligation step was designed to maximize the re-ligation of the temporarily fragmented pRNR2 sequence, leveraging the non-cognate nature of its internal overhangs.

#### Test

Final plasmids were verified by [NotI](https://www.neb.com/en/products/r3189-noti-hf) digestion and [agarose gel electrophoresis](/experiments/#other-experimental-methods). Successfully transformed yeast colonies were confirmed via colony PCR for integration. Functionally, promoter activity was observed using a microscope, and quantified by flow cytometry.

However, a new problem emerged: our near-infrared fluorescent proteins EMSfp642 (BBa_25GARG3E) and EMSfp643 (BBa_2599SI53) exhibited very weak fluorescence and a low signal-to-noise ratio, making them ineffective as reporters.

#### Learn

The modified Golden Gate protocol was successful in overcoming the restriction site conflict. We successfully assembled the pRNR2-driven composite parts for yeast expression.

**New Problem:** Near-infrared fluorescent proteins require the chromophore Biliverdin (BV) for fluorescence. Although S. cerevisiae harbors the HMX1 gene (a heme oxygenase homolog), the BV produced by endogenous Hmx1 was insufficient to saturate iRFP and smURFP, resulting in poor signal. Functional success required external modification.

### Iteration 3: Chromophore Supplementation for Functional Enhancement

#### Design

To solve the low near-infrared fluorescence, we modified the protocol by adding an exogenous chromophore. Literature indicated that phycocyanobilin (PCB) treatment could increase infrared fluorescent proteins fluorescence tenfold.[^4] We added a PCB incubation step into our functional assay.

#### Build

We implemented the new standard operating procedure: yeast cultures were incubated with PCB for 1 hour prior to fluorescence measurement (see details on our [Experiments](/experiments/) page).

#### Test

[Quantitively](https://gitlab.igem.org/2025/fudan/-/tree/main/measurement/cytoflex?ref_type=heads) analysis via flow cytometry (our raw data at DOI: 10.5281/zenodo.17293146 ) showed a significant increase in fluorescence intensity for both EMSfp642 (Log10_Fold Change = 1.85) and EMSfp643 (Log10_Fold Change = 2.36), achieving an acceptable signal-to-noise ratio.

<div style="text-align: center;" id="fig1">
    <img src="https://static.igem.wiki/teams/5643/pageimage/engineering/pcb-analysis-grouped-by-sample-final-colors-capsize-r660-a-fixedtitle.webp" style="width:50%">
    <div>
        <span style="color:gray">Figure 1. Fluorescent Inteansity Change after PCB treatment</span>
        <br><br>
    </div>
</div>

#### Learn

The functional output of the near-infrared reporters is decoupled from the successful DNA construction. Achieving sufficient fluorescence for EMSfp642 and EMSfp643 in yeast requires the supplementation of the chromophore precursor PCB, as the native BV pathway is rate-limiting. This experience mandates that all future experiments using BV-dependent fluorescent proteins in *S. cerevisiae* must include the PCB incubation step to ensure reliable and quantifiable results.

## Other DBTL Examples

These DBTL cycles are just one example of our project engineering processes. Other examples are available on our [Software Tool](/software/#development-process-dbtl-cycle), [Model](/model/#improvement-log), [Education](/education/theoretical-foundation-constructionism), [Inclusivity](/inclusivity/#) and [Entrepreneurship](/entrepreneurship/#our-action) pages.

## Reference

[^1]: Hodgins-Davis, A., Duveau, F., Walker, E. A., & Wittkopp, P. J. (2019). Empirical measures of mutational effects define neutral models of regulatory evolution in *Saccharomyces cerevisiae*. *Proceedings of the National Academy of Sciences of the United States of America*, *116*(42), 21085–21093. DOI: 10.1073/pnas.1902823116

[^2]: Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. *ACS synthetic biology*, *4*(9), 975–986. DOI: 10.1021/sb500366v

[^3]: Estelle Lab. (2025). Golden Gate Assembling protocol (Unpublished). University of California, San Diego. Retrieved from https://labs.biology.ucsd.edu/estelle/Moss_files/MossGG_Assembling.pdf

[^4]: Sakai, K., Kondo, Y., Fujioka, H., Kamiya, M., Aoki, K., & Goto, Y. (2021). Near-infrared imaging in fission yeast using a genetically encoded phycocyanobilin biosynthesis system. *Journal of cell science*, *134*(24), jcs259315. DOI: 10.1242/jcs.259315

---
title: Experiments
authors:
  - name: Yixuan Lu
    url: /fudan/team/#Yixuan
    avatar: https://static.igem.wiki/teams/5643/pageimage/team/lyx-a.webp
layout: igem
heroImage: https://static.igem.wiki/teams/5643/header/exp.webp
description: On this page, we describe detailed protocols for Golden Gate assembly, yeast transformation and genome integration validation, as well as yeast imaging and flow cytometry analysis.
---

## Golden Gate Assembly

As described in Shaw et al. (2023)[^1], briefly:

### Assembly Reaction

1. Normalize the concentration of all DNA parts prior to assembly. Use a [Nanodrop](https://www.thermofisher.com/order/catalog/product/hk/en/ND-2000) for quantification.

> Accurate DNA quantification and normalization are crucial for high assembly efficiency.

- **Inserts:** Dilute to a final concentration of 50 fmol/&mu;L.
- **Plasmid Backbones:** Dilute to a final concentration of 25 fmol/&mu;L.

2. Prepare the Golden Gate reaction mixtures in PCR tube:

> Assemble reactions on ice to prevent premature enzyme activity, and put the enzymes back immediately after use!

> For multiple reactions, create a master mix. The total number of reactions can be overcounted by 0.2 allowing for the inaccuracy of pipettes.

   |       |                      Reagent                       |        Volume (&mu;L)         |   Final Concentration/Amount    |
   | :---: | :------------------------------------------------: | :---------------------------: | :-----------------------------: |
   | **1** |      Each DNA Insert/Plasmid (50 fmol/&mu;L)       |              0.5              |             25 fmol             |
   | **2** |          Plasmid Backbone (25 fmol/&mu;L)          |              0.5              |            12.5 fmol            |
   | **3** |              10x T4 DNA Ligase Buffer              |               1               |               1x                |
   | **4** |            T4 DNA Ligase (400 U/&mu;L)             |              0.5              |              200 U              |
   | **5** | Restriction Enzyme ([BsaI](https://www.neb.com/en/products/r3733-bsai-hf-v2) or [BsmBI](https://www.neb.com/en/products/r0739-bsmbi-v2) or [BpiI](https://www.thermofisher.com/order/catalog/product/FD1014)) | 0.5 | 10 U ([BsaI](https://www.neb.com/en/products/r3733-bsai-hf-v2)) or 5 U ([BsmBI](https://www.neb.com/en/products/r0739-bsmbi-v2)/[BpiI](https://www.thermofisher.com/order/catalog/product/FD1014)) |
   | **6** |                Nuclease-Free Water                 | to a final volume of 10 &mu;L |                -                |
   |       |                     **Total**                      |            **10**             |                                 |

> Do not confuse [BpiI](https://www.thermofisher.com/order/catalog/product/FD1014) (for marker assembly with high efficiency) with [BsaI](https://www.neb.com/en/products/r3733-bsai-hf-v2) or [BsmBI](https://www.neb.com/en/products/r0739-bsmbi-v2), which is an isoschizomer of BbsI.

3. Place the reaction tubes in a thermocycler and run the following program:

   - For level 1 assembly:
     a. **Cycling (25 cycles):**

     - **Digestion:** For reactions with more than 6 inserts, increase digestion time to 5 min.
       - For [BsaI](https://www.neb.com/en/products/r3733-bsai-hf-v2): 37°C for 2 min.
       - For [BsmBI](https://www.neb.com/en/products/r0739-bsmbi-v2): 42°C for 2 min.
     - **Ligation:** 16°C for 5 min.

     b. **Final Digestion:** 55°C for 10 min.
     c. **Heat Inactivation:** 80°C for 10 min.
     d. **Hold:** 16°C

   - For marker assembly:
     a. **Cycling (10 cycles):**

     - **Digestion:** 37°C for 2 min.
     - **Ligation:** 16°C for 5 min.

     b. **Final Digestion:** 55°C for 10 min.
     c. **Heat Inactivation:** 80°C for 10 min.
     d. **Hold:** 16°C

> For inserts containing internal [BsmBI](https://www.neb.com/en/products/r0739-bsmbi-v2) and [BsaI](https://www.neb.com/en/products/r3733-bsai-hf-v2) sites, we added a dedicated 30-minute incubation with additional T4 DNA Ligase at 16°C after the thermal cycles to maximize the repair and ligation of all ends.

### Bacteria Transformation and Plating

1.  Thaw [Turbo](https://www.neb.com/en/products/c2984-neb-turbo-competent-e-coli-high-efficiency) competent *E. coli* on ice, within 5 min aliquot 30 &mu;L for each assembly reaction.
2.  Add 2 &mu;L Golden Gate reaction mixture to the cells. Gently mix.
3.  Incubate on ice for 25 min.
4.  Heat shock the bacteria at 42°C for 45 s.
5.  Immediately return the tube to ice for another 2 min.
6.  Add 500 &mu;L of SOC medium (pre-warmed to 37°C).
7.  Incubate at 37°C with rotating for 1 h.
8.  Plate 100-200 &mu;L of the culture onto an LB agar plate containing the appropriate antibiotic.
9.  Incubate plates at 37°C for 8-12 hours before picking the colony.

> Ensure the antibiotic on the plate matches the resistance marker of the destination plasmid backbone (kanamycin or ampicillin), not chloramphenicol.

### Colony Screening

For level 0 and level 1 Golden Gate assembly:

- Screen bacterial colonies for the loss of the fluorescent dropout cassette (level 0, loss GFP; level 1, loss RFP) using a blue light transilluminator.
- Pick 2-4 non-fluorescent (correctly assembled) colonies for each construct.

For marker assembly:
- Pick a random colony from the plate.

Then,

1.  Inoculate colonies into liquid LB medium with the appropriate antibiotic and grow for 6-10 hours.
2.  Perform a [plasmid miniprep](#other-experimental-methods) to isolate the assembled plasmid DNA.
3.  Validate the plasmid structure using [NotI](https://www.neb.com/en/products/r3189-noti-hf) and [agarose gel electrophoresis](#other-experimental-methods).
4.  Analyze the fragment sizes to confirm the correct assembly.

### Expanded Golden Gate

Inspired by Sorida et al. (2023)[^2], briefly:

1. Prepare the digestion mixtures in PCR tube:

   |       |           Reagent           |    Volume (&mu;L)    |
   | :---: | :-------------------------: | :------------------: |
   | **1** | Restriction Enzyme 1 (XhoI) |         0.2          |
   | **2** | Restriction Enzyme 2 (XbaI) |         0.2          |
   | **3** |  10x DNA Ligase Buffer      |          1           |
   | **4** |           Vector            |      500 ng       |
   | **5** |     Nuclease-Free Water     | Bring up to 10 &mu;L |
   |       |          **Total**          |        **10**        |

2. Incubate the reaction at 37°C for 4 h, and then gel extract the large DNA fragment (&gt; 1.77 kb).

3. Take 1 &mu;L digested vector, add 0.25 &mu;L each of new 5' and 3' HA, 0.25 &mu;L [BsmBI](https://www.neb.com/en/products/r0739-bsmbi-v2), and 0.25 &mu;L T4 DNA Ligase to a total reaction volume of 2 &mu;L, and then conduct 42°C - 16°C 2-min cycling of Golden Gate assembly for 25 cycles. Preform [bateria transformation](https://2023.igem.wiki/fudan/experiments/#plasmid-transformation) as described above.

4. Once correct bacteria clones are obtained, use [BpiI](https://www.thermofisher.com/order/catalog/product/FD1014) to [introduce the selection marker into the vector](#golden-gate-assembly).

## Site-Directed Mutagenesis PCR

As described in Liu et al. (2008)[^3], briefly:

### Primer Design

Primers were designed as in Liu et al. (2008) Figure 2[^3]. The non-overlapping sequences are larger (significantly larger than suggested in [^4])than the complementary sequences to make the melting temperature of non-overlapping sequences (Tm no, 47.5-60.2) 5 to 10°C higher than the melting temperature of primer-primer complementary sequences (41.1-50.3 Tm pp < Tm no).

### PCR Reaction Setup

1. Prepare the PCR master mix in a 0.2-mL microcentrifuge tube on ice:

   |       |             Reagent              | Final Concentration/Amount |
   | :---: | :------------------------------: | :------------------------: |
   | **1** | Template DNA / Bacteria Solution |      1-5 ng / 1 &mu;L      |
   | **2** |             F-primer             |          1 &mu;M           |
   | **3** |             R-primer             |          1 &mu;M           |
   | **4** |               dNTP               |         0.5 &mu;M          |
   | **5** |              Phanta              |         0.2 &mu;L          |
   | **6** |        10x Phanta Buffer         |         12.5 &mu;L         |
   | **7** |        ddH<sub>2</sub>O         |    Bring up to 25 &mu;L    |
   |       |            **Total**             |           **25**           |

2. Place the reaction tubes in a thermocycler and run the following program:

   a. **Initial Denaturation:**
   - For template DNA: 95°C for 30 s.
   - For bacteria solution:  95°C for 3 min.

   b. **Amplification (12 cycles):**

   -   **Denaturation:** 95°C for 15 s.
   -   **Annealing:** T<sub>m no</sub> - 5°C for 15 s.
   -   **Extension:** 72°C for appropriate time (according to the length of the template constructs, for Phanta DNA polymerase is 30-60 s/kb)

   c. **Final Annealing:** T<sub>m pp</sub> - 5°C for 15 s.
   d. **Final Extension:** 72°C for 10 min.

3. While verifying 10&mu;L PCR products by [agarose gel electrophoresis](https://2022.igem.wiki/fudan/experiments#agarose-gel-electrophoresis); treat the remaining 15&mu;L PCR products with 0.5 &mu;L [DpnI](https://www.neb.com/en/products/r0176-dpni) at 37°C for > 4 h. Once verified and DpnI digestion completed, the PCR products were [transformed](#other-experimental-methods) into *E. coli*.

## Yeast Transformation

As described in Shaw et al. (2023)[^1]. Briefly:

### One-day Competent Yeast Preparation

1. Pick colony from plate into 3 mL growth medium for overnight growth at 30°C, 250 rpm.
2. Do a 1:20 dilution of saturated overnight culture into fresh growth medium (YPD) and incubate at 30°C, 250 rpm.

   1 mL culture = 1 transformation

3. Grow up yeast to OD600 ~0.7 (about 4 h).
4. Pellet cells at 3000 *g* for 3 min, room temperature; use large centrifuge if needed.
5. Wash with 0.5x volume 0.1 M LiOAc and pellet cells at 3000 *g* for 3 min in large centrifuge, room temperature.
6. Resuspend cells in 0.1 M LiOAc, 100 &mu;L per transformation
7. Aliquot 100 &mu;L of cells into individual 1.5-mL tubes and pellet at 8000 rpm for 1 min at room temperature on small tabletop centrifuge and remove supernatant.

### DNA Digestion

This step linearizes plasmid DNA prior to yeast transformation to increase integration efficiency.

> This step can be done during the period when the yeast grows to OD600 ～0.7.

1. Set up the digestion reaction in a sterile microcentrifuge tube. For a 20 &mu;L reaction, combine the following:

   |       |       Reagent       |        Volume (&mu;L)         |
   | :---: | :-----------------: | :---------------------------: |
   | **1** |       [NotI](https://www.neb.com/en/products/r3189-noti-hf)       |              0.1              |
   | **2** | 10x CutSmart Buffer |               2               |
   | **3** |  ddH<sub>2</sub>O  |     Bring up to 20 &mu;L      |
   | **4** |         DNA         | Variable Amount (600-1000 ng) |
   |       |      **Total**      |            **20**             |

2. Incubate the reaction for at least 4 h at 37°C. Run 4 &mu;L digestion products if needed by [agarose gel electrophoresis](https://2022.igem.wiki/fudan/experiments#agarose-gel-electrophoresis).

### PEG/Lithium Acetate Preparation

1. Ahead of [heat shock](#heat-shock), prepare the following reagents:

   - Fresh prepared PEG-LiOAc mixture

     |       | Filter Steriled Reagent | Volume per transformation (&mu;L) |
     | :---: | :----------------: | :-------------------------------: |
     | **1** | PEG-4000 (50% w/v) |                260                |
     | **2** |   LiOAc (1.0 M)    |                36                 |
     |       |     **Total**      |              **296**              |

   - DNA cocktail

     |       |        Reagent         | Volume per transformation (&mu;L) |
     | :---: | :--------------------: | :-------------------------------: |
     | **1** | Boiled [ssDNA](https://www.pombevolution.eu/labwiki/index.php/SsDNA) (2 mg/mL) |                50                 |
     | **2** | DNA + ddH<sub>2</sub>O |                14                 |
     |       |       **Total**        |              **64**               |

### Heat Shock

1. Resuspend [cell pellet](#one-day-competent-yeast-preparation) with 64 &mu;L of DNA cocktail.
2. Mix cell-DNA suspension with 296 &mu;L of PEG-LiOAc mixture and incubate at room temperature for 30 min.
3. Transfer tubes to 42°C water bath for 15 min, or longer if needed (up to 45 min).
4. Centrifuge 8000 rpm on small bench top centrifuge for 30 s and remove supernatant.
5. Depending on the selection marker:

> Be careful not to mix up the selection markers!

   - Auxotrophic selection marker: Add 0.5 mL H<sub>2</sub>O and leave for 10 mins at room temperature to resuspend.
   - Antibiotic selection marker: Recover in 0.5 mL YPD for 2-3 h at 30°C rotating.

6. Plate desired volume on plates.
7. Incubate plates for 2-3 days upside down at 30°C.

### Yeast Frozen Competent Cell Preparation

For the convenience of the transformation experiments, yeast competent cells can be prepared ahead and stored for a long time.

1. Pick colony from plate into 5 mL growth medium for overnight growth at 30°C, 250 rpm.
2. Do a 1:100 dilution of saturated overnight culture into fresh growth medium and incubate at 30°C, 250 rpm.

   2 mL culture = 1 competent cell aliquot = 1 transformation

3. Grow up yeast to OD600 ~0.75.
4. Pellet at 4000 *g* for 5 min in large centrifuge.
5. Wash with 0.5x volume H<sub>2</sub>O.
6. Pellet at 4000 *g* for 5 min in large centrifuge.
7. Resuspend in 0.025x volume frozen competent cell solution (filter sterilize):

   |       |      Reagent      | Volume (mL) |
   | :---: | :---------------: | :---------: |
   | **1** |   Glycerol (5%)   |     2.5     |
   | **2** |    DMSO (10%)     |      5      |
   | **3** | ddH<sub>2</sub>O |    42.5     |
   |       |     **Total**     |   **50**    |

8. Aliquot 50 &mu;L of cell suspension into 1.5 mL tubes and put into -80°C freezer for long term storage.

On the day of transformation:

1. Thaw frozen competent cells on ice, ~5 min.
2. Centrifuge cells at 8000 rpm on small bench top centrifuge for 30 s and remove supernatant.
3. Conduct [heat shock](#heat-shock) for digested DNA.

## Yeast Colony PCR

As described in Shaw et al. (2023)[^1], briefly:

1. Pick a single colony from solid medium and resuspend it in 50 &mu;L of sterile water; or use 1 &mu;L of liquid culture.
2. Combine the following components in a PCR tube:

   |       |               Reagent                | Volume (&mu;L) |
   | :---: | :----------------------------------: | :---------: |
   | **1** |           Yeast Suspension           |      1      |
   | **2** |        Forward Primer (10 &mu;M)        |     0.5     |
   | **3** |        Reverse Primer (10 &mu;M)        |     0.5     |
   | **4** |           ddH<sub>2</sub>O           |      3      |
   | **5** | 2x [Phire](https://www.thermofisher.com/order/catalog/product/F160S) Plant Direct PCR Master Mix |      5      |
   |       |              **Total**               |   **10**    |

3. Put tubes into a thermocycler and run the following program:

   a. **Initial Denaturation:** 98°C for 5 min.
   b. **Cycling (30 cycles):**

   -   **Denaturation:** 98°C for 5 s.
   -   **Annealing:** 57°C for 5 s.
   -   **Extension:** 72°C for 30 s.

   c. **Final Extension:** 72°C for 30 s.

### Quick Yeast Genome DNA Purification

If the routine genotyping above fails, the following protocol was used to quickly extract good quality genome for PCR fragments < 3.5kb with DNA polyermase[^5].

1. Pick one yeast colony from the plate into 100 &mu;L of water or spin down 100 &mu;L of saturated liquid yeast culture in 1.5-mL tube.
2. Resuspend cells in 100 &mu;L of lysis buffer (200 mM LiOAc, 1% SDS)
3. Incubate for 5 min at 70°C.
4. Add 300 &mu;L of 96-100% ethanol and vortex well.
5. Pellet DNA and cell debris at 15000 *g* for 3 min and remove ethanol.
6. Wash pellet with 200 &mu;L ethanol, spin down and remove as much ethanol as possible.
7. Dry pellet with lid open for 5-10 min.
8. Dissolve pellet in 100 &mu;L of H<sub>2</sub>O or TE and spin down cell debris for 15 s at 15000 *g*.
9. Transfer supernatant to fresh tube.
10. Use 1 &mu;L of supernatant for PCR.

## Grape Yeast Settling Selection

As described in Tong et al. (2025)[^6], briefly:

1. Grow the diploid and tetraploid yeast with *ACE2* deletion in 3 mL of YPD media with and without G418 at 30°C with shaking at 250 rpm for 24 h.
2. Transfer 1 mL of the culture into a 1.5-mL microcentrifuge tube.
3. Let clusters settle on the bench for 3 min.
4. Transfer the bottom 50 &mu;L into 3 mL of fresh YPD media for the next day of growth.
5. Archive a glycerol (20%) stock for each population every 7 days.

### Sedimentation Rate Measurement

1. Grow the diploid and tetraploid yeast with *ACE2* deletion of certain generation until OD600 > 1.
2. After sufficient pipetting, take 1 mL fresh yeast culture to measure its OD600.
3. Record the change of OD600 value with a camera until it declines under 0.5, and then calculate the sedimentation rate.

## Yeast Growth Curves

1. Grow yeast cultures in YPD at 30°C with 220 rpm shaking until OD600 > 1.
2. Normalized OD600 reading usng fresh YPD among cultures.
3. Dilute the cultures at 1:20 into 4 mL of fresh YPD and incubate at 30°C with 220 rpm shaking.
4. Every 1 h, 200&mu;L of the cultures was sampled, and mixed with 800&mu;L of ddH<sub>2</sub>O to achieve a 5-fold dilution.
5. Measure OD600 of the diluted samples and draw the growth curve.

## Flow Cytometry of Unicellular Yeast

Flow cytometry is performed to detect the fluorescence of [TU recorder](/design/#recorder) transformed into BY4741 or BY4742 (unicellular yeast). We did not use chitinase to treat multicellular yeast for this analysis.

1. Pick strains into 3 mL synthetic complete (SC) medium and grow them at 30°C with 250 rpm shaking overnight.
2. Take 2 mL cultures into a 2-mL tube and pellet the cells by spinning at 8,000 *g* for 1 min.

   - For cells whose flourescence from smURFP or miRFP670-2, take 1 mL of fresh yeast culture (OD600 > 1), centrifuge the cells at 8,000 *g* for 1 min and resuspend the pellet in 100 &mu;L SC medium, and then add 10 &mu;L of 1 mM PCB shaking for 1 h in the dark[^7].

> We greatly appreciate Dr. Chuanwei Yang and Dr. Lin Li for providing us with ready to use phycocyanobilin (PCB) 1 mM solution in our initial test, and Dr. Hongtai Liu for sending PCB powder on the same day which is enough for the following year!

3. Wash the pellet with PBS and resuspend the cell in 1 mL PBS.
4. Measure cell fluorescence using CytoFlex LX according to the following settings: FSC 300 V, SSC 350 V, gating for singlets using FSC-A vs FSC-H. Record 4 channels of fluorescence (Y585-A, B525-A, R660-A, V450-A) at the same time.

> For yeast, set FSC-A vs FSC-H with logarithmic axes (Log Axis), and adjust the axis scale as needed so that the cell population is centered in the plot.

> Use a parallelogram gate in the plot to enclose the region where FSC-A and FSC-H are approximately proportional (this region represents single cells).

## EMS Mutagenesis

As described in Hodgins-Davis et al. (2019)[^8], briefly:

1. Wash a 2 mL aliquot of saturated culture (~10<sup>7</sup> cells/mL) twice in 5 mL of H<sub>2</sub>O, and then resuspend in 2 mL of sodium phosphate buffer (0.1 M, pH = 7).
2. Divide the culture into two 1 mL aliquots in 1.5-mL microcentrifuge tubes.
3. Add 10 &mu;L EMS (99%) to one sample (EMS treatment), but not the other (sham treatment), and seal both samples with parafilm.
4. Incubate both samples at 30°C on a rotor for mixing for 70 min.
5. Treat both samples with 1 mL of neutralizing sodium thiosulfate (5% in ddH<sub>2</sub>O).
6. Pellet and resuspend cells in 1 mL of sodium thiosulfate (5%), twice in 1 mL of H<sub>2</sub>O, and finally suspend cells in 1 mL of YPD.
7. 1/8 of either EMS treated or sham samples were seeded into 3 mL YPD, culture at 30°C for 10 h before [flow cytometry](#flow-cytometry-of-single-cell-yeast).

## Grape Yeast Cluster Size Measurement

As described in Tong et al. (2025)[^6], briefly:

### Strain Revival

1. Revive strains from glycerol stocks by growing them on YPD plates at 30°C for 2 days.
2. Inoculate each strain into 5 mL of YPD media. Grow at 30°C with 220 rpm shaking for 3 days, performing daily settling selection before transferring to fresh media.
3. On the last day, after settling selection and transfer, grow the cultures for 24 hours. Sample them at 4 hours (exponential phase) and 24 hours (stationary phase) for measuring cluster size.

### Sample Preparation for Imaging

1. Gently shake each culture by hand. In a 24-well plate, add an appropriate volume of the culture (1–250 &mu;L) to H<sub>2</sub>O containing 10 &mu;L of 16% (w/v) formaldehyde, making a final volume of 510 &mu;L per well.
2. For macroscopic strains, use wide-bore 1-mL tips and sample twice from each culture into two separate wells.
3. Gently shake the 24-well plate to evenly spread out the clusters and allow 5–10 minutes for clusters to settle.

### Image Acquisition and Analysis

Use a microscope to scan the entire well. Take and stitch 5 x 8 bright-field, shading-corrected images with 10% overlap at x4 magnification.

Use [ImageJ](https://imagej.net/ij/) for a semi-automated image analysis:

1. Segment clusters using auto local thresholding and split touching clusters using seed-based watershed.
2. Perform manual correction as needed.
3. Measure the cross-sectional area of each cluster.
4. Remove segmented objects with an area below 40 &mu;m^2.
5. Using R, convert cluster areas to cluster volumes and radiuses, treating clusters as perfect spheres.
6. For each strain, calculate its biomass-weighted mean cluster radius by first calculating its mean cluster volume weighted by cluster volume, and then converting it to a radius.

## Imaging-based Ploidy Measurement

As described in Tong et al. (2025)[^6], briefly:

In every experiment, two control strains with known ploidy, namely, the engineered grande diploid and tetraploid grape yeast, also went through the same procedure of sample preparation and imaging.

### Propidium Iodide (PI) Staining

1. Grow each strain of interest in YPD media to mid-log phase.
2. Transfer 250 &mu;L of the culture into a 1.5-mL microcentrifuge tube.
3. Pellet the clusters by spinning at 5,000 *g* for 1 min and wash them with 1 mL of H<sub>2</sub>O.
4. Fix and permeabilize the cells in 1 mL of 70% ethanol at room temperature for 2 h with end-to-end rotation on a mini-rotator at a speed of 40 rpm.
5. Centrifugat for ethanol removal and wash the pellet with 1 mL of 50 mM sodium citrate twice.
6. Resuspend the clusters in 200 &mu;L of 50 mM sodium citrate containing 0.5 mg/ml RNase A and incubate them in a 37°C heat block for 2 h with gentle inversion every 30 min (as clusters settle over time).
7. After RNA digestion, add 5  &mu;L of 1 mg /mL propidium iodide and incubate the mixture in a 30°C incubator in dark overnight with rotation on a mini-rotator at  at a speed of 10 rpm to keep clusters from settling.
8. Propidium iodide-stained clusters can be stored at 4°C for no longer than 1 week before imaging.

### PI Fluorescent Imaging

1. Crush 5 &mu;L of propidium iodide-stained clusters into a single layer of cells between a microscope slide and a coverslip, seal the coverslip with nail polish.
2. For each sample, take 14-bit images of approximately 5 FOVs at x100 magnification.
3. For each FOV (fields of view), set the focus by manually moving to a *z*-plane in which cells look grey (rather than brighter or darker) in the brightfield channel; do not adjust the focus using red fluoresent channel.
4. Image the flattened clusters in the red fluorescent channel (excitation laser 561 nm in our case) at the focal plane as well as one *z*-plane 0.3 &mu;m above and below the focal plane (three *z*-planes in total) to capture the propidium iodide-stained nuclei, and then imaged in the brightfield channel at the focal plane.

> Set the exposure and gain of the fluorescent channel such that the brightest pixels in the tetraploid control strain are approximately 80% of the maximal allowed pixel value while minimizing photobleaching and noise.

### Image Analysis

Images were quantitatively analyzed by [ImageJ](https://imagej.net/ij/).

1. Perform maximum intensity projection of the three *z*-planes taken in the fluorescent channel and used the resulting image for segmentation and fluorescence quantification.
2. Segment nuclei and filter them to include only single round nuclei.
3. Measure the total propidium iodide fluorescence intensity of each nucleus with background subtraction, in which the background fluorescence is the median propidium iodide fluorescence intensity of the cytoplasm in the cluster that the focal nucleus is in. The resulting nuclear propidium iodide intensity scales linearly with the DNA content.

Continue the analysis in R.

1. For each sample, remove tiny segmented objects with areas two median absolute deviations below median (which are nucleus segmentation artefacts).
2. Manually remove FOVs with outlier distributions of nuclear propidium iodide intensity. The distribution of nuclear propidium iodide intensity in a clonal strain contains two peaks that correspond to G1-phase and G2-phase cells.
3. Estimate the DNA content of each nucleus by dividing its propidium iodide intensity with the propidium iodide intensity of a haploid genome, which was estimated by averaging across the two ploidy control strains, that is, mean (G1 peak intensity of diploid control strain/2, G1 peak intensity of tetraploid control strain/4).
4. Estimate the DNA content of a clonal strain as the DNA content of its G1 peak.

## Chitin staining

We stained cells with [calcofluor white](https://www.sigmaaldrich.com/HK/en/product/sial/18909) as described in Tong et al. (2025)[^6], briefly:

1. Sample 150&mu;L from the cell culture from the ancestor.
2. Remove the supernatant through an iterated process of centrifugation and pipetting medium removal.
3. Build 15&mu;L of 1 mg/mL calcofluor solution into 500&mu;L 1x phosphate-buffered saline solution (PBS) and mixed with the yeast pellet.
4. Incubate the sample in the dark at room temperature for 25 min.
5. Remove the calcofluor media by centrifugation and pipetting.
6. Add 200 &mu;L 1x PBS on top of the pellet. 5&mu;L of this cell suspension was pipetted onto a clean glass slide and covered with a coverslip for microscopy analysis.

## Yeast Live-cell Imaging in Agarose

1. Grow yeast overnight in 3 mL of YPD at 30°C with 220 rpm shaking.
2. Dilute the overnight cultures 1:20 into fresh SC media and incubate at 30°C with 220 rpm shaking until OD600 between 0.7-1.
3. Prepare 0.4% low melting agarose in SC media, and keep at 42°C.
4. Mix appropriate amout of yeast with melted agarose, vortex, and drop a total of ∼500&mu;L onto a glass-bottom dish allow it to solidify. Cover the agarose pad with SC media if needed.
6. Observe cells immediately using a fluorescence microscope equipped with appropriate filters. Maintain stage temperature at 30°C.

## Mating of *a* and &alpha; Yeast Strains

As described in Tong et al. (2025)[^6], briefly:

1. Inoculate *a* and *&alpha;* strains separately into liquid medium and grow overnight at 30°C until cultures reach saturation.
2. Mix the two cultures at a 1:1 ratio.
3. Spot 10 &mu;L of the mixture onto a solid agar plate.

> Yeasts mate more efficiently on solid medium.

4. Allow the spot to air-dry for ~10 min, then incubate the plate inverted at 30°C for ~24 h.
5. Following incubation, pick a small portion from the mating spot and streak onto selective medium (containing both antibiotics, Nat and Hyg in our case) to isolate single colonies.

## Other Experimental Methods

[Bateria transformation](https://2023.igem.wiki/fudan/experiments/#plasmid-transformation) was conducted as described on our 2023 page.

[Plasmid miniprep](https://2022.igem.wiki/fudan/experiments#plasmid-miniprep) was conducted as described on our 2022 page.

[Bacteria colony PCR](https://2022.igem.wiki/fudan/experiments#colony-pcr) was conducted as described on our 2022 page.

[Agarose gel electrophoresis](https://2022.igem.wiki/fudan/experiments#agarose-gel-electrophoresis) was conducted as described on our 2022 page.

## References

[^1]: Shaw, W. M., Khalil, A. S., & Ellis, T. (2023). A Multiplex MoClo Toolkit for Extensive and Flexible Engineering of *Saccharomyces cerevisiae*. *ACS synthetic biology*, *12*(11), 3393–3405. DOI: 10.1021/acssynbio.3c00423
[^2]: Sorida, M., & Bonasio, R. (2023). An efficient cloning method to expand vector and restriction site compatibility of Golden Gate Assembly. *Cell reports methods*, *3*(8), 100564. DOI: 10.1016/j.crmeth.2023.100564
[^3]: Liu, H., & Naismith, J. H. (2008). An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. *BMC biotechnology*, *8*, 91. DOI: 10.1186/1472-6750-8-91
[^4]: Zheng, L., Baumann, U., & Reymond, J. L. (2004). An efficient one-step site-directed and site-saturation mutagenesis protocol. *Nucleic acids research*, *32*(14), e115. DOI: 10.1093/nar/gnh110
[^5]: Lõoke, M., Kristjuhan, K., & Kristjuhan, A. (2011). Extraction of genomic DNA from yeasts for PCR-based applications. *BioTechniques*, *50*(5), 325–328. DOI: 10.2144/000113672
[^6]: Tong, K., Datta, S., Cheng, V., Haas, D. J., Gourisetti, S., Yopp, H. L., Day, T. C., Lac, D. T., Khalil, A. S., Conlin, P. L., Bozdag, G. O., & Ratcliff, W. C. (2025). Genome duplication in a long-term multicellularity evolution experiment. *Nature*, *639*(8055), 691–699. DOI: 10.1038/s41586-025-08689-6
[^7]: Sakai, K., Kondo, Y., Fujioka, H., Kamiya, M., Aoki, K., & Goto, Y. (2021). Near-infrared imaging in fission yeast using a genetically encoded phycocyanobilin biosynthesis system. *Journal of cell science*, *134*(24), jcs259315. DOI: 10.1242/jcs.259315
[^8]: Hodgins-Davis, A., Duveau, F., Walker, E. A., & Wittkopp, P. J. (2019). Empirical measures of mutational effects define neutral models of regulatory evolution in *Saccharomyces cerevisiae*. *Proceedings of the National Academy of Sciences of the United States of America*, *116*(42), 21085–21093. DOI: 10.1073/pnas.1902823116

---
title: Results
authors:
  - name: Zhiqin Wang
    url: /fudan/team/#Zhiqin
    avatar: https://static.igem.wiki/teams/5643/pageimage/team/wzq-a.webp
  - name: Yixuan Lu
    url: /fudan/team/#Yixuan
    avatar: https://static.igem.wiki/teams/5643/pageimage/team/lyx-a.webp
  - name: Yue Yue
    url: /fudan/team/#Yue
    avatar: https://static.igem.wiki/teams/5643/pageimage/team/yy-a.webp
layout: igem
heroImage: https://static.igem.wiki/teams/5643/header/screenshot-2025-10-08-at-23-13-36.avif
description: On this page, we show the results of our experiments.
---

Updated on 2025-10-31: we won the <a href="https://competition.igem.org/results/2025?tab=medals" target="_blank">Gold Medal</a>, and our project was nominated for the <a href="https://competition.igem.org/results/2025?tab=special-prizes#inclusivity-award" target="_blank">Inclusivity Award</a>.

## Characteristics of Grape Yeast

### Chitin Imaging Reveals the Morphology of Multicellularity

<div style="text-align:center;" id="fig1">
  <img src="https://static.igem.wiki/teams/5643/pageimage/results/single-vs-multi.avif" style="width:50%">
  <div style="width:50%;margin-left:25%"><span style="text-align:left; margin:0; color:gray;">Figure 1. Chitin Imaging of Single vs Grape Yeast.
    <br>Morphological comparison of unicellular and multicellular yeast under bright-field (BF) and calcofluor white fluorescence (FL) microscopy. Scale bar = 10 &mu;m.
  </span></div>
</div>

We first confirmed the multicellular morphology of the *ACE2*-knockout grape yeast. As shown in Figure 1, the yeast cell wall was [stained](/experiemnts/#chitin-staining) with a chitin-specific dye calcofluor white and immediately imaged under both bright-field and fluorescence microscopy. The multicellular cells grew in grape-like clusters, and at the junctions—where the walls were markedly thicker—the corresponding fluorescence intensity was higher. This pattern allowed us to delineate familial clusters. Next we compared multicellular pattern between 2N and 4N Grape Yeast. As shown in Figure 2, blue arrows highlight regions where cells appeared to contact each other in bright-field images but showed no connecting fluorescent signal, indicating that these were not true cellular junctions.

To determine the temporal sequence of growth and identify mother–daughter relationships, the culture was resumed for an additional 24 h after staining. Under bright-field illumination, several cells lacked detectable fluorescence (orange arrows), demonstrating that the fluorophore had not yet incorporated into these nascent cell structures.

<div style="text-align:center;" id="fig2">
  <img src="https://static.igem.wiki/teams/5643/pageimage/results/chitin-imaging-2.webp" style="width:100%;max-width:100%">
  <div  style="width:66%;margin-left:17%"><span style="text-align:left; margin:0; color:gray;">Figure 2. Chitin Imaging of 2N and 4N Grape Yeast.
    <br>Morphological comparison of diploid and tetraploid yeast under bright-field (BF) and fluorescence (FL) microscopy. Fix: cells were fixed and stained immediately before imaging; Fix-Grow: cells were stained and then allowed to grow for an additional 24 hours prior to fixation and imaging. Blue arrows: regions that appear as cell junctions or buds in the bright-field channel but lack corresponding fluorescence signals; Orange arrows: newly formed cells (buds) that emerged after the staining procedure and display minimal or no fluorescence signal. Scale bar = 10 &mu;m.
  </span></div>
</div>

However, a subset of cells in the Fix group were found outside the focal plane during image acquisition, making their fluorescence undetectable and potentially confounding the identification of newborn cells in the Fix-and-Grow cohort. To overcome this limitation, we engineered a brighter and more definitive reporter [TU Timer](/part-collection/#fluorescent-timer) (BBa_25AT6YR4) to resolve the temporal relationships between adjacent cells.

### Settling Selection Leads to Ploidy Abnormalities in Tetraploid Yeast

It is indicated that during gravity-based selection, multicellular yeast undergoes ploidy doubling[^1], which is much similar to the change some pathogenic fungi undergo under the pressure of drug[^2]. Hence, we subjected both diploid (2N) and tetraploid (4N) Grape Yeast under [settling selection](/experiments/#grape-yeast-settling-selection) over successive generations to simulate the ploidy shift experienced by pathogenic fungi under drug-induced stress. We performed Propidium Iodide (PI) staining on the cell nuclei and subsequently acquired fluorescence images. The log-transformed PI fluorescence intensity is known to be linearly correlated with the number of chromosomes[^1].

While the ploidy of diploid grape yeast remained relatively stable in short time (9 generations), we unexpectedly discovered a distinct abnormality in the ploidy of the tetraploid (4N) strain. As shown in Figure 3A, the blue arrow points to a single cell containing two nuclei—the nucleus on the right appears to be in a state of division—suggesting a higher ploidy state, potentially hexaploid (6N).

Our quantitative statistical data (Figure 3B, C) further supports this observation. The distribution of PI fluorescence intensity for the 4N strain was significantly more dispersed than the 2N strain, and a distinct minor peak corresponding to a putative hexaploid (6N) population was visible in the histogram (Figure 3B).

These results collectively suggest that, under the pressure of settling selection, higher-ploidy strains of *S. cerevisiae* possess a comparatively less stable genome and exhibit a pronounced tendency toward increased ploidy. We hypothesize that the underlying cause involves a failure in the ploidy sensing and maintenance machinery. This defect may lead to chromosome missegregation events, resulting in an unstable state where the cell is unable to correctly regulate its division cycle based on its current chromosome complement.

<div style="text-align:center;" id="fig3">
  <img src="https://static.igem.wiki/teams/5643/pageimage/results/ploid-abnormality-3.webp" style="width:100%;max-width:100%">
  <div style="width:66%;margin-left:17%"><span style="text-align:left; margin:0; color:gray;">Figure 3. Microscopy images and statistical analysis of yeast ploidy changes.
    <br>(A) Merged bright-field and fluorescence images of PI-stained diploid (left) and tetraploid (right) yeast after 9 generations of settling selection. Blue arrow: a single cell containing two nuclei, suggestive of an abnormal karyotype. Scale bar = 10 &mu;m. <br>
(B) Population-level distribution of fluorescence intensity in diploid and tetraploid yeast. <br>
(C) Fluorescence intensity distribution across individual biological replicates of diploid (n = 7) and tetraploid (n = 9) yeast samples.
  </span></div>
</div>

### Polyploid Yeast Exhibits a Faster Sedimentation Rate

Throughout the settling selection experiment, we noticed a gradual increase in yeast settling speed with successive passages. As illustrated in Fig.1 & Fig.2(A), tetraploid cells displayed noticeably larger cell volumes compared to diploids. Together with our earlier observation that chromosome number tends to rise during long-term passaging, this led us to hypothesize that higher passage number and increased ploidy may both contribute to larger cell size and faster sedimentation.

To test this idea, we measured the settling rates of yeast at different passages (d15 vs. d45) and ploidy levels (2N vs. 4N). Consistent with our expectation, the 4N strain in d45 settled the fastest.

<div style="text-align: center;" id="vid1">
    <img src="https://static.igem.wiki/teams/5643/pageimage/results/od5.webp" style="width:80%">
    <div>
        <span style="color:gray">Video 1. Comparison of sedimentation rate OD600 values for four strains.</span>
        <br><br>
    </div>
</div>

### Dynamics of Grape Yeast Branching

As microscopic images of fixed yeast cells fail to capture the real-time, authentic dynamics of Grape Yeast branching, we employed [live-cell imaging](/experiments/#yeast-live-cell-imaging-in-agarose) over a 3-hour span to track the yeast growth process.

<div style="text-align:center;" id="fig4">
  <img src="https://gitlab.igem.org/2025/fudan/-/raw/main/microscopy.webp?ref_type=heads" style="width:100%;margin-bottom:0"><br>
  <img src="https://static.igem.wiki/teams/5643/pageimage/results/pos5-1-montage-new.webp" style="width:100%;margin-top:0">
  <div style="width:66%;margin-left:17%"><span style="text-align:left; margin:0; color:gray;">Figure 4. Live-cell imaging of Grape Yeast.
    <br>Grape Yeast was embedded in 0.4% low-melting agarose in SC media and presented on a glass-botton dish. Live-cell growth was imaged using a microscope at a maintained temperature of 30°C. Red arrows indicate the buddiing process of Grape Yeast in 3 h. The time-lapse movie at the top left corner was used to generate the montage image.
  </span></div>
</div>

## Visualize the Evolution: The Timer & Recorder Extension

Having obtained the multicellular [Grape Yeast](/design/) and gained familiarity with the its properties, we initiated an evolutionary tracing study.

In this section, we evaluated various combination of promoters and fluorescent protein from different perspectives, including [fluoresence imaging](/measurement/#microscopy-qualitative-observation-of-signal-variation), [flow cytometry](/measurement/#flow-cytometry-data-processing-and-composite-score-calculating) and [deep sequencing](/measurement/#deep-sequencing-molecular-validation-of-the-mechanism). Through this thorough assessment, we eventually come up with a optimized fluorescent reporter, pSTM1 driven EMSfp499 (BBa_255T0PHY), that can function effectively under stress conditions. For more details, please refer to our [Measurement](/measurement/) page.

## What we learn

### Grape Yeast Characteristics

- The multicellular Grape Yeast grows in grape-like clusters and was confirmed using chitin staining, which provides a great chassis for us to trace the growth and mutation of yeast.
- Higher-ploidy tetraploid yeast exhibits a less stable genome than diploid yeast under gravity-based settling selection. This characteristic shows the potential for grape yeast to mimic pathogenic fungus under pressure.
- Meanwhile, as some studies have reported that certain fungi develop drug resistance by downregulating the expression of drug targets—potentially through mechanisms such as gene modification or genome ploidy reduction—we employed IME1 to induce meiosis in grape yeast as a model for this phenomenon. The corresponding results is presented in [our presentation video](https://video.igem.org/w/iRsWyoY68ey3E83McFmNf2).
- Both higher ploidy and increased passage number lead to faster sedimentation rates.
Future teams may easily sort out grape yeast based on this characteristic.

### Evolution Tracing

- The live-cell images give us confidence to track mother-daughter sequence utilizing our [TU timer](/part-collection/#fluorescent-timer).
- An optimized fluorescent reporter, [pSTM1 driven EMSfp499](https://registry.igem.org/parts/bba-255t0phy) was developed to effectively trace the evolutionary dynamics of the Grape Yeast under selective pressure.
- On the next stage of our project, we will integrate [pSTM1 driven EMSfp499](https://registry.igem.org/parts/bba-255t0phy) into sixteen chromosomes of yeast to find out the most unstable chromosome and even gene loci. This work would lay the foundation for advancing the understanding of fungal drug resistance mechanisms as well as the development of fungal drug targets.

## Reference

[^1]: Tong, K., Datta, S., Cheng, V., Haas, D. J., Gourisetti, S., Yopp, H. L., Day, T. C., Lac, D. T., Khalil, A. S., Conlin, P. L., Bozdag, G. O., & Ratcliff, W. C. (2025). Genome duplication in a long-term multicellularity evolution experiment. *Nature*, *639*(8055), 691–699. DOI: 10.1038/s41586-025-08689-6
[^2]: Khateb, A., Gago, S., Bromley, M., Richardson, M., & Bowyer, P. (2023). Aneuploidy Is Associated with Azole Resistance in Aspergillus fumigatus. *Antimicrobial agents and chemotherapy*, *67*(4), e0125322. DOI: 10.1128/aac.01253-22