iGEM 2024 — Team izju-china

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{% block title %}Notebook{% endblock %}
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February

• February 29

March

• March 10

• March 27–April 1

April

• April 9

• April 12

• April 24

• April 28

May

• May 10–17

• May 17–31

• May 31

June

• June 11

• June 24

July

• July 3

• July 20

August

• August 5

• August 13

September

• September 13

October

# February

## • February 29

### Agenda

- Conducted a brainstorming session based on the specific track.

### Notes

- Wu Di and Kong Kexuan: Focused on biomanufacturing using genetic oscillators to produce ethanol and isobutanol.

- Wang Chen, Cao Xiaoran, He Zihang, Xiao Mingyang, Cao Yue, Lv Siyuan, Zhang Xiaohan, Yu Haoyang, and Chen Jinpeng: Explored new therapies for Alzheimer's disease, pancreatic cancer, etc.

- Prof. Huang’s suggestion: Consulted Prof. Chen Ming and voted for the preferred project.

# March

## • March 10

### Agenda

- Selected the project topic: "Production of ethanol and isobutanol based on gene oscillators."

- Brainstormed project details.

- Divided writing tasks for the iGEM project application.

### Notes

- Based on the input from Prof. Chen Ming and team voted, the topic "Production of ethanol and isobutanol based on gene oscillators" was selected.

Writing task allocation:

- Background: HP group

- Project design: Dry lab

- Theoretical basis: Wet lab

## • March 27–April 1

### Agenda

- Wet lab, dry lab, and HP groups conducted interviews and recruited new team members.

### Notes

- Each group prepared questions and conducted interviews.

# April

## • April 9

### Agenda

- Full team meeting.

### Notes

- iGEM overview (Wang Chen):

1. Timeline & process for iGEM defense.

2. Medals, Villages, and individual awards.

3. Introduction to the offline Grand Jamboree in Paris.

4. Competition fees.

- Introduction of advisors:

1. Prof. Huang Wenwen, Prof. Jin Mengdi, and advisor Zheng Ximeng share their experiences.

2. Prof. Huang emphasized the importance of planning and adhering to deadlines.

3. Prof. Jin highlighted the availability of lab space.

4. Advisor Zheng Ximeng shared insights into building a complete project narrative and leveraging domestic and international resources.

## • April 12

### Agenda

- Consulted teacher Lian regarding the experimental scheme for longevity yeast.

### Notes

- Explored advanced knowledge of ethanol and isobutanol production.

- Acknowledged the challenges in building the genetic oscillator.

- Considered collaborating with Zhouzou’s lab for yeast strains containing oscillators.

## • April 24

### Agenda

- Switched to the Fashion & Cosmetics Village.

### Notes

- The initial idea of ethanol and isobutanol production was derived from an optogenetics paper. However, combining two papers without significant innovation led us to consider alternatives.

## • April 28

### Agenda

- Meeting with cosmetic expert Dr. Qiao.

### Notes

- Ethanol and isobutanol were unsuitable for cosmetics. Dr. Qiao suggested exploring glycolipids and preservatives as alternatives.

# May

## • May 10–17

### Agenda

- Set up the laboratory and conduct access training.

- Began culturing yeast containing the oscillator.

## • May 17–31

- Prepared for exams

## • May 31

### Agenda

- Student leaders' meeting.

- Planned summer vacation tasks for the wet lab, dry lab, and HP groups.

# June

## • June 11

### Agenda

- Student leaders' meeting.

- Reviewed recent work and discuss project issues.

### Notes

- Consulted Prof. Chen for feedback on production choices and project specifics.

- Prof. Chen’s suggestions:

1. If uncertain, write vaguely for now.

2. Use well-researched products as a base.

## • June 24

### Agenda

- Student leaders' meeting.

- Reviewed recent work and discussed project issues.

### Notes

- Defined specific schedules for HP and wet lab groups.

- Discussed the hardware design—microfluidic chip.

# July

## • July 3

### Agenda

- Student leaders' meeting.

### Notes

- Discussed issues with ceramide AP (sensitive skin) and NP (exfoliation).

- Planned visits to Evonik and BASF for product exchanges.

- Outlined QC, safety, and toxicology testing for products.

- Planned a visit to Guanchen Biotech in August.

- Finalized the story logic for the project and cosmetic product development.

## • July 20

### Agenda

- Student leaders' meeting.

- Reviewed recent work and discussed project issues.

### Notes

- Finalized details for promotion video shooting and script design.

- Discussed microscope use for microfluidic chip observation.

- Wiki group showcased progress.

- Wet lab updated on MrBBS plasmid construction.

- Shifted modeling focused to longevity effects.

# August

## • August 5

### Agenda

- Student leaders' meeting.

- Reviewed recent work and discussed project issues.

### Notes

- Discussed French visa requirements.

- Prepared for visits to Guanchen Biotech (August 7) and Ashland (August 9).

- Prof. Chen Di’s feedback:

1. Emphasized the connection between yeast senescence and production.

2. Clarified the necessity of the microfluidic system.

## • August 13

### Agenda

- Student leaders' meeting.

- Reviewed suggestions from advisors.

- Discussed medal criteria.

# September

## • September 13

### Agenda

- Student leaders' meeting.

- Reviewed recent work and discussed project issues.

### Notes

- Wiki group presented the final wiki design.

- Wet lab encountered difficulties in PCR of the specific region, might require a new oscillator yeast strain.

- Modeling and software updated discussed Fast Fourier Transform and LOWESS fit.

# October

- Wiki was frozen.

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{%
endblock %}
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![](https://static.igem.wiki/teams/5371/project/engineering/engineering.svg)

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Overview

Project Design

• Cycle 1 - production of ceramides and bisabolol

• Cycle 2 - introduction of genome longevity oscillator

• Cycle 3 - modular design integrates production and oscillation

Building pRS42H-TSC10 Plasmid

• Cycle 1 - original experiment design

• Cycle 2 - improvement on plasmid design and experiments

Microfluidic Chip & Accessories

• Cycle 1 - microfluidic chip with accessories

• Cycle 2 - improved chip, accessories, and operating procedures

• Cycle 3 - jig of the chip

# Overview

![](https://static.igem.wiki/teams/5371/project/engineering/1.png)

Figure 1 Design-Build-Test-Learn Engineering cycle

On this page we describe the engineering design cycle for the following subprojects: project design,
building of TSC10 plasmid, and hardware. The engineering cycle consists of four stages: Design, Build,
Test, and Learn.

# Project Design

## • Cycle 1 - production of ceramides and bisabolol

### Design

Given the prevalence of skin problems caused by environmental irritants, we aim to design an
eco-friendly, effective, and affordable skincare product. To achieve this, we decided to modify
metabolic pathways in yeast to produce key cosmetic ingredients using synthetic biology
strategies. After thorough research, we selected ceramides and bisabolol as the primary active
components for our product.

### Build

Our design involves overexpressing the tsc10 gene to produce excess ceramides and introducing
the fused protein ERG20-MrBBS into yeast. We can monitor the expression of TSC10 and ERG20-MrBBS
through the green and blue fluorescence emitted by EGFP and BFP, respectively[1][2].

![](https://static.igem.wiki/teams/5371/project/engineering/2.png)

Figure 2 Overexpressing MrBBS-ERG20 and TSC10 to produce bisabolol and ceramide

### Test

Upon further literature review, we discovered that excess ceramides and bisabolol can have
adverse effects on yeast[3][4].

![](https://static.igem.wiki/teams/5371/project/engineering/3.png)

Figure 3 Adverse effects of excess bisabolol and ceramide

Therefore, we assessed the feasibility and limitations of our initial design.

Feasibility

-All components are readily available and listed in the [Parts](part)
section.

-Both TSC10 and ERG20-MrBBS are driven by the CYC1 promoter, which is effective and
commonly used in yeast.

-TSC10 is tagged with EGFP, and ERG20-MrBBS is tagged with BFP, allowing easy detection
of gene expression through green and blue fluorescence.

Shortcomings

-Both bisabolol and ceramides can negatively impact yeast viability.

-Excess ceramides can also cause skin issues, necessitating careful control of ceramide
levels in skincare products.

### Learn

We decided to proceed with overexpressing tsc10 and introducing ERG20-MrBBS, but with a greater focus on
protecting yeast to enhance ingredient production. We conducted further literature reviews to develop
strategies for controlling production within safe and effective ranges.

## • Cycle 2 - introduction of genome longevity oscillator

### Design

Inspired by Zhou et al. (2023), we explored the use of a genome oscillator, specifically the SIR2-HAP4
oscillator, to extend the lifespan of yeast. We realized that this system could be adapted to protect yeast
and enhance the production of our target ingredients.

### Build

Our design involves inserting tsc10 and ERG20-MrBBS downstream of the sir2 and hap4 genes, respectively.

![](https://static.igem.wiki/teams/5371/project/engineering/4.png)

Figure 4 Inserting ERG20-MrBBS and TSC10 into yeast genome to regulate bisabolol-ceramide oscillation

This setup allows the oscillation of sir2 and hap4 to cause fluctuations in the production of ceramides and
bisabolol.

### Test

After consulting with our instructors, we evaluated the feasibility and limitations of this approach:

Feasibility

-All components are readily available and listed in the [Parts](part)
section.

-ERG20-MrBBS will be inserted downstream of sir2 and will oscillate with the SIR2
protein.

-TSC10 will be inserted into the 35S rDNA region and will oscillate with the HAP4
protein.

-The oscillating system can effectively protect yeast from the negative effects of
bisabolol and ceramides, enhancing production by increasing yeast viability.

Shortcomings

-Gene insertions in yeast can be challenging and prone to failure.

-Insertions limit the flexibility of yeast to produce additional cosmetic products.

-Insertions restrict the copy number of tsc10 and ERG20-MrBBS, limiting the maximum
production of ceramides and bisabolol.

### Learn

We recognize the genome longevity oscillator as an effective method to enhance yeast production in the
presence of toxic products. However, we decided to redesign the system to better integrate cosmetic
ingredient production into the yeast genome oscillator.

## • Cycle 3 - modular design integrates production and oscillation

### Design

We adopted a modular design approach to rebuild our production system, achieving oscillation without
inserting genes into the yeast genome. This modular design facilitates the parallel assembly of multiple
plasmids during experiments, allows for easy modification in case of issues, and enables the future
expansion of cosmetic ingredient production.

### Build

Instead of directly inserting tsc10 and ERG20-MrBBS into the genome, we maintained these genes on plasmids
and swapped the promoters to link the production system with SIR2 and HAP4.

![](https://static.igem.wiki/teams/5371/project/engineering/5.png)

Figure 5 Modular design to separate oscillation and production.

### Test

We successfully transferred the pRS42H-TSC10 plasmid and the pESC-ERG20-MrBBS plasmid into yeast with the
genome longevity oscillator, significantly enhancing the production of ceramides and bisabolol. For more
details, please refer to our [Results](https://2024.igem.wiki/izju-china/results) section.

### Learn

Our future work will focus on the following aspects:

1. Yeast Suicide Module: To further ensure the safety of our product, we plan to introduce a promoter
(activated by small molecules) - SIR2 plasmid into yeast. This will allow us to control yeast quantity by
adding small molecules to induce cell senescence when there are excess yeasts.

2. Precise Manipulation of Cosmetic Ingredient Production: After simulating an improved model, we will
modify the promoters and add other regulatory elements accordingly. This will enable precise control over
the production of cosmetic ingredients, ensuring that yeast produces ceramides and bisabolol at levels that
meet industry standards.

# Building pRS42H-TSC10 Plasmid

## • Cycle 1 - original experiment design

### Design

First, we decided to use a restriction enzyme to cut the vector, preparing it for subsequent insertion
steps. Next, we utilized PCR to obtain the CYC1 promoter and the TSC10 gene fragment. We then connected
these fragments using Gibson assembly, ensuring they were correctly integrated into the vector. This process
will help us construct the desired recombinant vector for future experiments.

### Build

(1) To build the vector, we used BplⅠ and BamHⅠ to cut the vector and disrupt the TEF promoter, allowing us
to insert the CYC1 promoter and TSC10 into the plasmid.

![](https://static.igem.wiki/teams/5371/project/engineering/6-1.png)

Figure 6 BplⅠ and BamHⅠare used to cut the vector, but did not delete the whole TEF promoter

(2) To obtain CYC1 promoter and TSC10, we designed relevant primers and conducted colony PCR (click on [protocol](https://2024.igem.wiki/izju-china/protocol) for more information)

### Test

(1) After cutting the vector, we realized that using BplⅠ and BamHⅠ might not completely disrupt the TEF
promoter, potentially overriding the regulative function of CYC1 promoter. Thus, we decided to look for
other restriction enzymes and cutting sites.

(2) Using colony PCR, we observed PCYC1 electrophoretic band, but no TSC10 bands.

![](https://static.igem.wiki/teams/5371/project/engineering/7.png)

Figure 7 There are PCYC bands but no TSC10 bands in colony PCR results

### Learn

(1) Since the TEF promoter is stronger than the CYC1 promoter, it is crucial to completely remove the TEF
promoter sequence. Therefore, we need to find better cutting sites.

(2) To determine the cause of the experiment's failure, we checked our primers, PCR programs, and DNA
templates, and decided to change our DNA extraction method and improve the PCR programs.

## • Cycle 2 - improvement on plasmid design and experiments

### Design

Following the last iteration of the Design-Test-Build-Learn cycle, we revised our design and tried a
different PCR program. We decided to continue using restriction enzymes to linearize the vector due to its
simplicity and speed. However, we switched to different restriction enzymes to ensure the TEF promoter was
fully disrupted. To address the poor TSC10 PCR results, we adopted touchdown PCR and extracted yeast genomic
DNA instead of using colony PCR.

### Build

(1) After consulting our instructors, we used NaeⅠ and BamHⅠ this time. Although NaeⅠ also cuts the f1 ori
region on the plasmid, it completely removes the TEF promoter.

![](https://static.igem.wiki/teams/5371/project/engineering/8-1.png)

Figure 8 NaeⅠ and BamHⅠare used to cut the vector and deleted the whole TEF promoter

(2) We used commercial kits to extract yeast genome DNA instead of colony PCR. Also, we changed the PCR
program into touchdown PCR. For more information, please click on [protocol](https://2024.igem.wiki/izju-china/protocol).

### Test

After using NaeⅠ and BamHⅠto cut the vector and connect to CYC1 promoter and TSC10, we successfully built
pRS42H-TSC10 plasmid:

![](https://static.igem.wiki/teams/5371/project/engineering/9.png)

Figure 9 After updating vector-cutting and PCR programs, our PCR achieved positive results.

### Learn

Moving forward, we need to measure the level of ceramides after transferring the pRS42H-TSC10 plasmid and
adjust promoters or other regulatory elements of gene expression to ensure that yeast produces ceramides at
levels that meet industry standards.

# Microfluidic Chip & Accessories

## • Cycle 1 - microfluidic chip with accessories

### Design

To meet our need of observing fluorescent intensity of yeast cells in single-cell resolution and a
real-time manner, a microfluidic chip was designed. The microstructures anchoring yeast cells and
dispatching cell suspension and culturing media were designed referring to (5). PDMS was chosen as
the material for the chip for transparency and stability. The outer profile of the chip was designed to
fit into the slot of culturing chamber of the confocal microscope that we chose for observation.

![](https://static.igem.wiki/teams/5371/project/engineering/10.svg)

Figure 10. The microfluidic chip structure.

### Build

The design was sent to Zhongxinhengqi Co. for manufacture. The chip was bond to 1mm thick glass slide as
carrying plate. The media supplying tubes were inserted into the chip via straight stainless-steel tubes.
Culturing media were contained in a 50ml syringe with piston removed raised to 90cm above the chip for
gravity feeding. Outlet tube was inserted in the same way as the supplying tube, with its end placed at
same level as the chip to avoid excess positive/negative pressure. Check [hardware](https://2024.igem.wiki/izju-china/hardware) for detailed design.

### Test

The chip with accessories were installed as designed. Yeast cells suspension was filled into the 50ml
syringe and raised to 60mm above the chip, trying to load cells through gravity. No cells were observed to
be loaded into chip. So it was attempted to be forced into the chip by pushing the piston of the syringe.
Though successful, it was met with significant resistance. The 60x lens that was planned for observation
could not properly focus, so it was switched to 20x lens. The tubes connected to the chip were forced to
bend to side to enable the cover of culturing chamber to close up. After testing, when disassembling and
cleaning the chip, we discovered cracks on the PDMS chip around the pores with reduced tightness under
hydraulic pressure, which was most likely caused by stress from stainless-steel tubes inserted. We also
discovered clogs in channels in the chip, making it unable to be reused.

### Learn

The resistance was caused by air in the chip, which should be expelled from the whole system in further
tests, from the inlet tubes, chip, to the outlet tube.

Inability of 60x lens to properly focus was caused by their limited working distance. Thinner carrying
glass plate should be applied to address this issue.

The clogging of the chip could be avoided by filtering all acellular fluids through 0.22μm filters and
washing all tubes with filtered ddH2O.

The stress on PDMS chip by stainless-steel tubes was mainly caused by the height limit imposed by
culturing chamber. Design choices like 90 degrees bended tubes and supports of the tubes could be adopted.

![](https://static.igem.wiki/teams/5371/project/engineering/11.svg)

Figure 11. The microfluidic with. Red box indicates the stress from the stainless steel tube.

## • Cycle 2 - improved chip, accessories, and operating procedures

### Design

Based on what were learned from the last cycle, the carrying glass plate was switched to a 0.17mm thick
cover slip. The stainless-steel tubes connecting the chip and inlet and outlet tubes changed from straight
ones to 90 degrees bended ones.

![](https://static.igem.wiki/teams/5371/project/engineering/12.png)

Figure 12. The stainless steel was changed to the 90 degree bended stainless-steel tube.

### Build

The chip and tubes were pre perfused with filtered ddH2O. The chip was perfused in vacuum, with 0.0075%
Tween-20 dropped on pores of the chip. The tubes were perfused with syringe. The whole chip and accessories
assembly was built according to design.

### Test

The YPD media were filtered before usage. A time-lapse microscopy was set up to test the system in real
observation. Focus shift that was not compensated by auto focus was discovered when inspecting pictures taken.

![](https://static.igem.wiki/teams/5371/project/engineering/13.svg)

Figure 13. The 0.17mm coverslip enable us to test cell under the 60x microscope.

### Learn

The focus shift was caused by deformation of chip significant enough to be uncorrectable by auto focus. This could be caused
by two factors. One was shifting to thinner carrying glass plates, which were not rigid enough to resist minor
forces imposed on them. Another was the stainless-steel tubes inserted into the chip, which were likely to be
the sources of subtle forces. A support or jig for both the chip itself and the stainless-steel tubes could
probably solve the problems of focus shift.

## • Cycle 3 - jig of the chip

### Design

A jig to support the chip itself and the stainless-steel tubes inserted into the chip was designed. The bottom
piece and the mainframe supported the chip, while a piece of drilled acrylic screwed to mainframe supported
the
stainless-steel tubes without significantly affecting observation. Check [hardware](https://2024.igem.wiki/izju-china/hardware) for detailed design.

![](https://static.igem.wiki/teams/5371/project/engineering/14.svg)

Figure 14. The jig of the chip.

### Build

The bottom plate and midframe were 3D printed in light curing resin. The piece of acrylic was hand drilled to
ensure matching precision to the chip. The acrylic piece was screwed to the midframe with M2 screws. The jig
and the chip were secured using paper tapes. Check [hardware](https://2024.igem.wiki/izju-china/hardware)
for detailed information.

### Test

The chip-jig assembly with accessory plumbing systems were tested with time-lapse microscopy under 60x lens.
No obvious focus shift was observed.

### Learn

This design was effective. It helped support the jig and reduce deformation of the chip, hence less focus shift.

![](https://static.igem.wiki/teams/5371/project/engineering/11.jpg)

Figure 15. The focus shift was largely reduced by using the chip jig.

# References

1. Jiang, Y. et al. Engineering Saccharomyces cerevisiae for enhanced (-)-α-bisabolol production. Synth Syst
Biotechnol 8, 187–195 (2023).

2. Morimoto, Y. & Tani, M. Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of
cell integrity of yeast Saccharomyces cerevisiae. Mol Microbiol 95, 706–722 (2015).

3. Boo, Y. C. Human Skin Lightening Efficacy of Resveratrol and Its Analogs: From in Vitro Studies to
Cosmetic Applications. Antioxidants 8, 332 (2019).

4. Uchida, Y. & Park, K. Ceramides in Skin Health and Disease: An Update. Am J Clin Dermatol 22, 853–866
(2021).

5. Li Y, Jiang Y, Paxman J, O'Laughlin R, Klepin S, Zhu Y, et al. A programmable fate decision landscape underlies single-cell aging in yeast. Science. 2020 Jul 17;369(6501):325–9.

{% endblock %}


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

![](https://static.igem.wiki/teams/5371/wet-lab/result/results.svg)

![Scrollbar Bar](https://static.igem.wiki/teams/5371/menu/curveagain.svg)

![Scrollbar Bar](https://static.igem.wiki/teams/5371/menu/curveagaingrey.svg)

![YEAST](https://static.igem.wiki/teams/5371/menu/yeast-nobg.svg)

Production of Ceramides and α-Bisabolol

• Ceramide production

• α-bisabolol production

Introduction of Ceramide-Bisabolol Oscillator

• Connecting longevity oscillator with production

• Measuring ceramide-bisabolol oscillation

Oscillation Increases Production and Fibroblast Growth

Biosafety

# Production of Ceramides and α-Bisabolol

## • Ceramide production

Previous research [1] indicated that the tsc10 gene ([BBa\_K5371001](https://parts.igem.org/Part%3ABBa_K5371001)) acts as a key enzyme in the synthesis of ceramides. Therefore, overexpressing the tsc10 gene can activate the production of dihydrosphingosine, ultimately increasing ceramide production (Fig. 1A). The pRS42H-TSC10-EGFP plasmid ([BBa\_K5371205](https://parts.igem.org/Part%3ABBa_K5371205), Fig. 1B) was transfected into yeast cells. To visualize the expression of TSC10, EGFP was linked to TSC10 using a P2A sequence ([BBa\_K5371002](https://parts.igem.org/Part%3ABBa_K5371002)). Green fluorescence was observed, indicating successful expression (Fig. 1C).

![](https://static.igem.wiki/teams/5371/wet-lab/result/1.png)

Figure 1 Ceramide production. (A) The mechanism of TSC10 enhancing ceramide production. (B) Structure of pRS42H-TSC10-EGFP plasmid. (C) Green fluorescence of EGFP showing successful expression of tsc10, shot under Nikon CWU-1 Spinning Disk Microscope.

## • α-bisabolol production

To produce α-bisabolol in yeast, it is necessary to increase the transformation from farnesyl pyrophosphate and decrease squalene production (Fig. 2B). We designed two plasmids for this purpose: pESC-URA-MrBBS-BFP ([BBa\_K5371203](https://parts.igem.org/Part%3ABBa_K5371203)) for producing the fused protein ERG20-MrBBS ([BBa\_K5371101](https://parts.igem.org/Part%3ABBa_K5371101)), and pRS42B-erg9 ([BBa\_K5371204](https://parts.igem.org/Part%3ABBa_K5371204)) to switch the original strong promoter of ERG9 to a weak promoter, pHXT1 ([BBa\_K5371010](https://parts.igem.org/Part%3ABBa_K5371010), Fig. 2C). ERG20 ([BBa\_K5371007](https://parts.igem.org/Part%3ABBa_K5371007)) and MrBBS ([BBa\_K5371011](https://parts.igem.org/Part%3ABBa_K5371011)) are both key enzymes involved in α-bisabolol synthesis. By fusing them, substrate diffusion effects can be reduced and upregulate α-bisabolol production [2]. BFP ([BBa\_K5371009](https://parts.igem.org/Part%3ABBa_K5371009)) was connected to ERG20-MrBBS via a P2A sequence in this plasmid. After transfection, blue fluorescence emitted by BFP confirmed the expression of ERG20-MrBBS in yeast (Fig. 2D).

![](https://static.igem.wiki/teams/5371/wet-lab/result/2.png)

Figure 2 α-bisabolol production. (A) The mechanism of ERG20-MrBBS enhancing and ERG9 competing with α-bisabolol production. (B) The structure of pESC-URA-MrBBS-BFP. (C) The structure of pRS42B-erg9. (D) The blue florescence of BFP showing successful expression of ERG20-MrBBS, shot under Nikon CWU-1 Spinning Disk Microscope.

# Introduction of Ceramide-Bisabolol Oscillator

Zhou et al. [3] designed a yeast longevity oscillator involving the genes sir2 and hap4 by inserting a hap4-sensitive CYC promoter onto sir2 and relocating the hap4 gene to the 35S rDNA region controlled by sir2. As sir2 expression increases, SIR2 protein lowers hap4 expression by inhibiting transcription of the 35S rDNA region. When hap4 is inhibited, its ability to activate the CYC promoter of sir2 decreases, affecting SIR2 expression. This creates a periodic expression pattern for SIR2 and HAP4, which was shown to increase yeast replicative lifespan by 82%. This long-live yeast strain was named NH1574, derived from BY4741, and the oscillation can be measured with mCherry (Fig. 4A ).

After overexpressing tsc10, ERG20-MrBBS, and downregulating ERG9, we successfully constructed production yeast strains, BY4741 transfected with pRS42H-TSC10-EGFP, pESC-URA-MrBBS-BFP, GAL4 ([BBa\_K5371202](https://parts.igem.org/Part%3ABBa_K5371202)), and pRS42B-erg9 plasmids (Fig. 3B). However, these production yeasts experienced significant metabolic stress. Excess ceramides can induce apoptosis [4], and α-bisabolol is toxic to yeast cells [2]. Therefore, using these production yeasts to produce ceramides and α-bisabolol is inefficient. We hypothesize that yeast strains with oscillators can alleviate metabolic and apoptotic stress, thereby enhancing production.

![](https://static.igem.wiki/teams/5371/wet-lab/result/3.png)

Figure 3 Three strains of yeasts used. (A) WT strain. (B) BY4741 transfected with pRS42H-TSC10-EGFP, pESC-URA-MrBBS-BFP, GAL4, and pRS42B-erg9 plasmids. (C) NH1574 transfected with pRS42H-TSC10-EGFP, pESC-URA-MrBBS-BFP, pAbAi-GAL4-NH, and pRS42B-erg9 plasmids

## • Connecting longevity oscillator with production

To mitigate the negative effects of excess ceramides and α-bisabolol, we connected the production systems with the longevity oscillator to regulate production. We used the CYC1 promoter ([BBa\_K5371000](https://parts.igem.org/Part%3ABBa_K5371000)) to allow hap4 to control tsc10 expression. Additionally, we inserted the gal4 gene ([BBa\_K5371006](https://parts.igem.org/Part%3ABBa_K5371006)) downstream of sir2 ([BBa\_K5371005](https://parts.igem.org/Part%3ABBa_K5371005)) via homologous recombination (Fig. 4B) and selected the gal10 promoter for ERG20-MrBBS, synchronizing its expression with sir2. This alignment ensures that the expression of tsc10 and ERG20-MrBBS, and consequently the production of ceramides and α-bisabolol, follows the expression patterns of hap4 and sir2. We then constructed production & oscillation yeast strains, NH1574 transfected with pRS42H-TSC10-EGFP, pESC-URA-MrBBS-BFP, pAbAi-GAL4-NH ([BBa\_K5371201](https://parts.igem.org/Part%3ABBa_K5371201)), and pRS42B-erg9 plasmids (Fig. 3C).

![](https://static.igem.wiki/teams/5371/wet-lab/result/4.png)

Figure 4 Connecting longevity oscillator with production. (A) Longevity oscillator can result in the oscillation of mCherry ([BBa\_K5371004](https://parts.igem.org/Part%3ABBa_K5371004), inserted downstream of sir2) expression and red fluorescence. (B) By transfecting pAbAi-GAL4-NH into yeasts, we connected GAL4 with SIR2.

## • Measuring ceramide-bisabolol oscillation

It is important to isolate individual cells to measure the oscillation of tsc10 and ERG20-MrBBS, as different cells may be in different phases of the oscillation cycle. If fluorescence is measured in populations, the different phases can cancel each other out, making oscillations unobservable. Microfluidic chips allow us to independently observe up to seven single cells simultaneously in a single field of view for more than 20 hours, while providing necessary nutrients to the yeast cells (Fig. 5A). Therefore, we used EGFP connected to tsc10 as a marker and recorded the relative fluorescence intensity of each single cell in the microfluidic chip for 22 hours using a Nikon CWU-1 Spinning Disk Microscope.

In production & oscillation yeasts, the oscillation of EGFP was evident (Fig. 5B) compared to production yeasts (Fig. 5C). The power spectrum of an individual time trace was calculated as P(period)=|FFT(D(t)) |2/N. Based on the fluorescence curve, the period less than 1h or magnitude less than 100 (arbitrary unit) was not considered. The measured oscillating period is almost the same in mCherry, BFP, and GFP, indicating that the ceramide-bisabolol oscillation shown by BFP and GFP is synchronized with the original longevity oscillation shown by mCherry (Fig. 5D), as we expected. Based on the level of magnitude, we estimated that the peak of oscillation period should be within the range of 263-788 minutes. Higher sampling frequency will be applied to further narrow the period range.

![](https://static.igem.wiki/teams/5371/wet-lab/result/pic5-2.png)

Figure 5 Measuring ceramide-bisabolol oscillation. (A) Observing single yeast in microfluidic chip under Nikon CWU-1 Spinning Disk Microscope. (B) Three representative cells show the oscillation of TSC10/EGFP in Production & Oscillation cells. (C) Three representative cells show no oscillation of TSC10/EGFP in Production cells. (D) The Boxplots show the period-dependent distributions of power spectrum of mCherry, BFP, and GFP. The bottom and top of the box are first and third quartiles, respectively. The band inside is the median. The x axis stands for the oscillation period (minute) while the y axis stands for the magnitude (arbitrary unit).

# Oscillation Increases Production and Fibroblast Growth

Having tested the stability of the oscillation, we focused on how the ceramide-bisabolol oscillator can enhance the efficacy of our product. Compared with production yeasts, production & oscillation yeasts showed significantly higher cell numbers at the stationary phase and relatively higher growth rates in growth curves (Fig. 6A). Additionally, ELISA showed that production & oscillation yeasts had significantly higher ceramide levels compared with both WT and production yeasts (Fig. 6B). These results indicate that the oscillator can mitigate the negative effects of producing ceramide and α-bisabolol. The oscillator endows yeasts with stronger resistance to adverse conditions. Therefore, production & oscillation yeasts can withstand harsh environments, such as toxic products or crowded conditions, resulting in higher production per unit time in a real production environment due to their advantage in cell number.

To validate whether our product can promote fibroblast growth, 1.25%, 2.5%, 5%, and 10% yeast lysate was added into cell culture medium and tested cell viability after 12 hours. Except at 5%, the other groups of production & oscillation cells all showed higher cell viability than the control group, indicating that our product can be beneficial to fibroblasts. Production & oscillation cells demonstrated much better effects in promoting fibroblast growth compared to production yeasts at 2.5% (Fig. 6C). The results above indicate that production & oscillation yeasts are not only more effective in ceramide and bisabolol production but also have the potential to be competitive cosmetic products in the future.

![](https://static.igem.wiki/teams/5371/wet-lab/result/6.png)

Figure 6 Oscillation Increases Production and Fibroblast Growth. n.s. (not significant): The p-value is greater than 0.05. \* (single asterisk): p-value less than 0.05. \*\* (double asterisk): p-value less than 0.01. \*\*\* (triple asterisk): p-value less than 0.001. \*\*\*\* (quadruple asterisk): p-value less than 0.0001. (A) The growth curve of the three strains, showing original dots and fitted Logistic curves. n=3. (B) Ceramide level is measured using ELISA, n=3. (C) 1.25%, 2.5%, 5%, and 10% yeast lysate was added into cell culture medium and tested cell viability after 12 hours, n=7, control = 5.

# Biosafety

1. **Non-pathogenic strains**: We use two strains of yeast (Saccharomyces cerevisiae) that are generally recognized as safe (GRAS) for genetic modifications: BY4741 and NH1574 (derived from BY4741).

2. **Gene containment**: To prevent the accidental release of genetically modified organisms (GMOs), all strains used do not have functional met gene, ensuring they cannot survive outside the laboratory environment without specific supplements (Fig. 3).

3. **Horizontal gene transfer**: We have minimized the risk of horizontal gene transfer by using plasmids that are not easily transferable to other organisms.

4. **Cell toxicity test**: We added lysate of our strains into fibroblast culture medium (Fig. 5C). Most groups demonstrated positive effects in fibroblast growth.

# References

1. Morimoto Y, Tani M. Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of cell integrity of yeast Saccharomyces cerevisiae. Mol Microbiol. 2015 Feb;95(4):706–22.

2. Jiang Y, Xia L, Gao S, Li N, Yu S, Zhou J. Engineering Saccharomyces cerevisiae for enhanced (–)-α-bisabolol production. Synthetic and Systems Biotechnology. 2023 Jun 1;8(2):187–95.

3. Zhou Z, Liu Y, Feng Y, Klepin S, Tsimring LS, Pillus L, et al. Engineering longevity-design of a synthetic gene oscillator to slow cellular aging. Science. 2023 Apr 28;380(6643):376–81.

4. Kim SK, Noh YH, Koo JR, Yun HS. Effect of expression of genes in the sphingolipid synthesis pathway on the biosynthesis of ceramide in Saccharomyces cerevisiae. J Microbiol Biotechnol. 2010 Feb;20(2):356–62.

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