iGEM 2025 — Team hdkwa

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| **2024 December** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Project discussion |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

* Project discussion

![](https://static.igem.wiki/teams/5763/notebook/1.webp)

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| **2025 January** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Project Seminar |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

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| **2025 Febuary** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| Design the logo and team name |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Prepare LB solid medium |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Prepare the antibiotic AMP+ |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Cultivate the bacteria E.coli |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Complete the experimental notes |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Extract the plasmid and determine the DNA concentration |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| PCR amplification |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Test and recover using electrophoresis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Complete the experimental notes |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Connect the target fragment with the vector and save it |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Write a wiki |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Make a group presentation |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Analysis of Team Tasks and Rules |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Education Workshop |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Develop the initial content |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Team social platform registration |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Conceive and write articles for the official account |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| rite scripts for Douyin and Xiaohongshu; |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Complete the production of PPTS |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Design and produce popular science posters |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Team logo modification |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Q&A production for science popularization activities |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

* Design the logo and team name
* Prepare LB solid medium
* Prepare the antibiotic AMP+
* Cultivate the bacteria E.coli
* Complete the experimental notes
* Extract the plasmid and determine the DNA concentration
* PCR amplificationTest and recover using electrophoresis
* Complete the experimental notes
* Connect the target fragment with the vector and save it
* Write a wiki
* Make a group presentation
* Analysis of Team Tasks and Rules
* Education Workshop
* Develop the initial content
* Team social platform registration
* Conceive and write articles for the official account
* Write scripts for Douyin and Xiaohongshu
* Complete the production of PPTS
* Design and produce popular science posters
* Team logo modificationQ&A production for science popularization activities

![图片19](https://static.igem.wiki/teams/5763/notebook/2.webp)

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| **2025 March** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Team logo modification |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| design and release the questionnaire |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| design questions specifically for the interviewees |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Questionnaire data organization and analysis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Expert interviews |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Data organization for the interview and writing of wiki content |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Online theoretical learning |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

* Team logo modification

![](https://static.igem.wiki/teams/5763/notebook/3.webp)

* design and release the questionnaire

![](https://static.igem.wiki/teams/5763/notebook/4.webp)

* design questions specifically for the interviewees
* Questionnaire data organization and analysis
* Expert interviews
* Data organization for the interview and writing of wiki content
* Online theoretical learning

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| **2025 Apri** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| Online theoretical learning |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Dry team experiment |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Analysis of the Rules for the Entrepreneurship Gold Award |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Understand the importance and framework of the Business Plan |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Learn business analysis models |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Understand Marketing knowledge |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| conduct financial analysis and forecasting |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

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| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **2025 May** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Complete the business plan |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Design promotion video |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Post articles on social media |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
100%

* Complete the business plan
* Design promotion video

![Screenshot 2025-08-09 at 22.53.24](https://static.igem.wiki/teams/5763/notebook/5.webp)

* Post articles on social media

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| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **2025 June** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| Transfer the two plasmids into E.coli |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Explain the writing of a wiki |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Monoclonal colony validation |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Complete the experimental notes and cultivate the bacteria |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Complete the experimental notes |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Test and using electrophoresis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| finish the poster |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

* Transfer the two plasmids into E.coli

![](https://static.igem.wiki/teams/5763/notebook/6.webp)

* Explain the writing of a wiki
* Monoclonal colony validation

![](https://static.igem.wiki/teams/5763/notebook/7.webp)

* Complete the experimental notes and cultivate the bacteria
* Complete the experimental notes
* Test and using electrophoresis
* finish the poster.

![](https://static.igem.wiki/teams/5763/notebook/8.webp)

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| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **2025 July** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| plasmid extraction vector |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| PCR amplification and detection |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| homologous recombination |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| transformation |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Monoclonal colony validation |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Shake the bacteria and sequence |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Co-transformation |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Design a speech and education |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Complete the experimental notes |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Finish the flowe chart |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Draft an interview outline |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| write bp |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Finish promotion video |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

* plasmid extraction vector
* PCR amplification and detection

![](https://static.igem.wiki/teams/5763/notebook/9.webp)

* homologous recombination
* transformation
* Monoclonal colony validation
* Shake the bacteria and sequence
* Co-transformation
* Design a speech and education
* ![](https://static.igem.wiki/teams/5763/notebook/10.webp)
* Complete the experimental notes
* finish the flow chart

![](https://static.igem.wiki/teams/5763/notebook/11.webp)

* Draft an interview outline
* Write bp
* Finish promotion video.

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| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **2025 August** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| record the growth curve |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| HPLC measure the content of erythritol |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| start education |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| start interview |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| write wiki |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| summary of the interview |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| design a game |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| mock defense |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

* Record the growth curve
* HPLC measure the content of erythritol
* start education

![](https://static.igem.wiki/teams/5763/notebook/12.webp)

* start interview
* write wiki
* wiki setup
* summary of the interview.
* desian a game

![Screenshot 2025-08-09 at 23.05.06](https://static.igem.wiki/teams/5763/notebook/13.webp)

* mock defense

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| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **2025 September** | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| wiki setup |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
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'
}] %}
{% block title %}Notebook{% endblock %}
{% block lead %}NOTEBOOK{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [{
"title": "Overview",
"content": '

Erythritol is a non-cariogenic sweetener. Currently, it is primarily produced through microbial fermentation using
glucose as the main substrate. However, this method suffers from limitations, such as low yield and conversion
rate. Glycerol, being an abundant and low-value renewable resource, will be utilized as substrate in our research
to achieve dual-substrate fermentation for erythritol production. The project is divided into two cycles(Liu F
etc, 2024; Thuy etc, 2024):

Cycle 1 - Glucose-based erythritol synthesis[Pathway 1]

Cycle 2 - Glycerol-based erythritol synthesis[Pathway 2]

Cycle 1 and Cycle 2 are complementary. Cycle 2 can utilize glycerol as a substrate to produce erythritol only if
the proteins, when Cycle 1 are normally expressed. Furthermore, this project provides a solid foundation for
future explorations of these techniques in glycerol reuse. The comprehensive and systematic approach outlined
herein is critical to navigating the complex regulatory environment.

'
}, {
"title": "Cycle 1 Glucose-based erythritol synthesis [pCDFDuet-PK-EPDH-PTase]",
"content": '

**Design:**

Phosphoketolase is a key metabolic enzyme and mainly participates in the sugar metabolism
pathways of microorganisms, particularly in the phosphoketolase pathway (Bao,etc.2011). Phosphoketolase is able to
catalyzes the scission of phosphoketoses (such as fructose-6-phosphate, etc.) to generate smaller phosphorylated
materials, such as glyceraldehyde-3-phosphate (G3P) and acetyl phosphate and acetyl phosplate(Figure 1). Cleavage
is associated with the generation of high-energy phosphoryl bonds. For example, acetyl phosphate can act as an
energy carrier and directly participate in ATP synthesis (Henard CA, etc. 2015).

Erythritol-4-phosphate dehydrogenase (E4PDH) serves as a key enzyme in the erythritol biosynthetic pathway. It
catalyzes the dehydrogenation of erythritol-4-phosphate (E4P), converting it into erythrulose-4-phosphate (E4PuP),
accompanied by the concomitant reduction of the cofactors NAD+/NADH or NADP+/NADPH(T. Barbier, etc.2014).

Phosphatase operates (PTase) as an enzyme that removes phosphate groups from its substrates by breaking down
phosphate monoesters, which results in phosphate ions and free hydroxyl groups.

In our research, Fructose-6-phosphate is cleaved by phosphoketolase (PK) to produce acetyl-phosphate and
erythrose-4-phosphate. Erythrose-4-phosphate is converted to erythritol-4-phosphate by erythritol-4-phosphate
dehydrogenase(EPDH). The phosphate group is removed from erythritol-4-phosphate by phosphatase(PTase), yielding
the final product erythritol (Figure 1 Pathway 1).

**![Glycerol_Glycerol3P](https://static.igem.wiki/teams/5763/engineering/1.webp)**

Figure 1.Synthesis pathway of erythritol

(Note: PK: phosphoketolase; EPDH: erythritol-4-phosphate dehydrogenase; PTase: phosphatase; GUT1: Encoding
glycerol kinase; GUT2: Encoding glycerol-3-phosphate dehydrogenase; TPI1: Encoding triosephosphate isomerase)

The pCDFDuet vector is a widely used prokaryotic expression system containing two multiple cloning sites (MCS).
Each MCS is preceded by a T7lac promoter and a ribosome binding site (RBS), enabling independent induction of
target gene expression through IPTG or lactose induction. This vector is particularly suitable for enzyme
production in our research project.The plasmid features an N-terminal 6×His tag and a C-terminal S-tag to
facilitate subsequent protein purification and verification. Moreover, it carries the CloDF13 replication origin,
ensuring stable propagation across various *Escherichia coli* strains(Figure 2).

![图片19](https://static.igem.wiki/teams/5763/engineering/2.webp)

Figure 2.The plasmid map

**Build:**

Currently, we have selected three DNA coding sequences from the
National Center for Biotechnology Information (NCBI), namely phosphoketolase (PK), erythritol-4-phosphate
dehydrogenase (EPDH), and phosphatase (PTase). These DNA coding sequences were optimized and synthesized according
to the codon preference of *Escherichia coli*. To construct the expression plasmids for PK, EPDH, and
PTase, we employed the polymerase chain reaction (PCR) to amplify the coding genes using primers as templates. The
obtained PCR products were inserted into the linearized plasmid pCDFDuet.

As shown in Figure 3A, the pCDFDuet-PTase gene was prominently displayed between the 5000
bp and 7500 bp markers, which was in complete agreement with the theoretical length, indicating successful
amplification. Figure 3B shows that the EPDH gene was located between the 1000 bp and 1500 bp markers, while the
PK gene was between the 1500 bp and 3000 bp markers. The amplified lengths of the genes were consistent with the
DNA coding, suggesting successful amplification.

![A picture containing graphical user interface  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/engineering/3.webp)

Figure 3A. The electrophoresis verification of the target fragment pCDFDuet-Ptase;Figure 3
B. The electrophoresis verification of the target fragment EPDH and PK; Note: phosphoketolase(PK) -2391bp;
Erythritol-4-phosphate dehydrogenas(EPDH)-1509bp; pCDFDuet-phosphatase-5369bp

Next, we cloned three coding DNA (PK, EPDH,
and PTase) into the pCDFDuet vector using homologous recombination, and transformed it into *E. coli* DH5α
competent cells. The total length of the three DNA coding is 5565 bp. The electrophoresis result shown in Figure
4A indicates that the amplified product band is located within the range of 5000-7000 bp, which is consistent with
the expected fragment size, confirming that the target gene fragment has been successfully amplified. In Figure
4B, the growth of monoclonal colonies with SmR(100ng/ml)was observed, indicating that the transformation
experiment was initially effective.By comparing the sequence file with the target gene sequence, the results show
that there is no base mutation at the location marked by the red solid arrow, and a base mutation exists at the
location marked by the dotted line arrow. The Figure 4C confirm that the target gene
fragment has been correctly linked to the vector, further verifying the successful construction of the recombinant
plasmid.

![A picture containing diagram  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/engineering/4.webp)

Figure 4A. Monoclonal colony verification gel plate; Figure 4B. Monoclonal colony
verification petri dish diagram inE. coli DH5α ; Figure 4C. Monoclonal colony verification sequencing
plot

**Test****:**

The three-dimensional structures of proteins PK, EPDH and PTase were predicted using AlphaFold2, and the
feasibility of the models was evaluated using the metrics ipTM (interface pTM) and pTM (predicted TM-score). The
figure 5 shows the three-dimensional structural representation of the protein PK, EPDH and PTase.

![图片15](https://static.igem.wiki/teams/5763/engineering/5.webp)

Figure 5. Figure A is the three-dimensional structural of the protein PK;Figure B is the three-dimensional
structural of the protein EPDH;Figure C is the three-dimensional structural of the protein PTase.

The recombinant plasmids pCDFDuet-PK-EPDH-PTase were transformed into *E. coli* BL21 (DE3) for expression.
A single colony containing the recombinant plasmid was cultured in medium con-taining antibiotics (100 mg/mL SmR )
at 37oC. When the cul-tures reached optical density (OD600) about 0.6, is
o-propy-beta-D-thiogalactopyranoside (0.5mmol and 1mmol IPTG) was added to the broth for induction of the protein
in 25oC . And the recombinant His-6 fusion-type PPE protein was purified by Ni2+ affinity
chromatography.

As shown in figure 6A, when 0.5 mmol IPTG was used to induce the protein, no PK protein was expressed, but PTase
and EPDH were successfully expressed. After being inducted with 1 mmol/L IPTG, both PK, Ptase and EPDH exhibited
sig-niﬁcant expression, and the Western blot results showed that both these recombinant prod-ucts could be
detected by the anti-His6 antibodies (Figure 6B), suggesting that His6-fused PK, Ptase and EPDH were expressed in
*E. coli* BL21(DE3) successfully.

![A picture containing chart  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/engineering/6.webp)

Figure 6. Optimisation of the culture medium for the soluble expression of the recombinant His6-tagged PK, Ptase
and EPDH. (A) SDS-PAGE analysis of the products fermented in media. (B) Western blot analysis of the products
fermented in media; Note the Pk protein is 90.6kda. The PTase
protein is 59.8kda. The EPDH protein is 56.1kda

**Learn:**

In this cycle, the plasmids are constructed successfully and transformed in *E.coli* DH5α. The
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) experiment and Western Blot(WB) successfully expressed in
*E.coli* BL21(DE3). This cycle experiments proves the success of protein expression before fermentation
production, providing essential foundational results for cycle 2.

'
}, {
"title": "Cycle 2 Glycerol-based erythritol synthesis[pETDuet-GUT12-TPI1]",
"content": '

**Design：**

Glycerol kinase catalyzes the conversion of glycerol into glycerol-3-phosphate. In adipocytes, due to the absence
of glycerol kinase, glycerol generated from lipolysis cannot be utilized directly. Instead, it must be transported
via the bloodstream to tissues such as the liver, kidneys, and intestine for further metabolism(Guo L, etc. 2010).

The glyceraldehyde-3-phosphate dehydrogenase (GAPD) is Coenzyme I oxidoreductase with phosphorylation function.
Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is an NAD-dependent oxidoreductase with phosphorylating activity.
It catalyzes the oxidation (dehydrogenation) and phosphorylation of glyceraldehyde-3-phosphate to form
1,3-bisphosphoglycerate. As this reaction represents a central step in sugar metabolism, GAPD plays a critical
role in glycolytic pathways(Colell A, etc. 2009).

Triosephosphate isomerase (TPI) is a key enzyme in the glycolytic pathway, catalyzing the reversible isomerization
between dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP), and ensuring the efficient
progression of glycolysis. (Wang Xiaobin, etc. 2021).

In our research, glycerol is converted to glycerol 3-phosphate by glycerol kinase (GUT1) in
*Saccharomyces cerevisiae*, consuming 1 molecule of ATP. Glycerol 3-phosphate is oxidized to
dihydroxyacetone phosphate (DHAP) by glycerol-3-phosphate dehydrogenase (GUT2), with the concomitant reduction of
1 NAD+ to NADH. The enzyme triosephosphate isomerase (TPI1) catalyzes the isomerization of
dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (G3P). Part of GA-3-P flows into the tricarboxylic
acid cycle (TCA cycle) through pyruvate, providing raw materials, energy and cofactors for various life activities
of cells. Another part of GA-3-P produced fructose-6-phosphate (F-6-P) under gluconeogenesis and entered the
pentose phosphate pathway ( PPP ). The gluconeogenesis pathway includes the intermediates F-6-P and GA-3-P
produced by the PPP pathway, which flow to the synthesis of erythritol in pathway 1(Figure 1).

The pETDuet-1 vector harbors the ColE1 replicon, lacI gene and ampicillin resistance gene. This vector can be used
in combination with the pCDFDuet vector to achieve co-expression of six target genes in our project within a
suitable host strain.The three codeing DNA are inserted into the expression vector pETDuet-1 through homologous
recombination (Figure 7).

![图片20](https://static.igem.wiki/teams/5763/engineering/7.webp)

Figure 7.The plasmid map

**Build:**

Each target gene was amplified polymerase chain reaction (PCR) using specific primers, and
the resulting amplification products were then inserted into the linearized pETDuet vector.
The target fragment was ligated to the vector by homologous recombination, transformed into
E. coli DH5α, and the expression plasmids were verified.

In Figure 8A, the GUT1 and GUT2 amplification products migrated between the 1500 bp and 3000 bp markers.
Similarly, Figure 8B showed that the TPL1 amplification product was located between the 500 bp and 1000 bp
markers, and Figure 8C indicates that the pET Duet exhibited a distinct band about 5000 bp.
The observed lengths of all amplified genes were consistent with their respective coding
sequences, validating the success of the amplification process.

![Graphical user interface  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/engineering/8.webp)

Figure 8A. The electrophoresis verification of the target fragment pET Duet(5462bp). Figure
8B. The electrophoresis verification of the target fragments GUT1(2130bp)and GUT2(1950bp).Figure 8C. The
electrophoresis verification of the target fragment TPL1(747bp) .

The total length of the coding GUT1, GUT2, and TPL1 is 4827bp. As shown in the electrophoresis result in figure
9A, the amplified product presents a clear band at approximately 5000 bp, which is highly consistent with the
expected fragment size. The growth of monoclonal colonies figure 9B with Amp(100ng/ml) was observed. And Figure 9C
confirmed that the target gene fragment has been correctly linked to the vector, further verifying the success of
the construction of the recombinant plasmid.

**![A picture containing diagram  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/engineering/9.webp)**

Figure 9A. Monoclonal Colony Verification Gel Plate; Figure 9 B. Monoclonal Colony Verification Petri Dish Diagram
in E. coli DH5α; Figure 9C. Monoclonal colony verification sequencing plot

**Test:**

The three-dimensional structures of proteins GUT1,GUT2 and TPl1 were predicted using SWISS-MODEL, and the
feasibility of the models was evaluated using the metrics GMQE (Global Model Quality Estimation) and QMEAN. The
figure10 shows the three-dimensional structural representation of the protein GUT1,GUT2 and TPl1.

**![图片16](https://static.igem.wiki/teams/5763/engineering/10.webp)**

Figure 10. Figure A is the three-dimensional structural of the protein GUT1;Figure B is the three-dimensional
structural of the protein GUT2;Figure C is the three-dimensional structural of the protein TPL1.

The recombinant plasmids pET Duet-GUT1-GUT2-TPl1 were transformed into *E. coli*  BL21 (DE3) for
expression. A single colony containing the recombinant plasmid was cultured in medium con-taining antibiotics (100
mg/mL Amp) at 37oC. The other protein expression conditions are consistent with pCDFDuet-PK-EPDH-PTase.
The figure 11A shown that the TPL1,GUT2 and GUT1 exhibited sig-niﬁcant expression after being inducted with 0.5
and 1 mmol/L IPTG. And the Western blot results showed that both these recombinant prod-ucts could be detected by
the anti-His6 antibodies (Figure 11B), suggesting that His6-fused TPL1,GUT2 and GUT1 were expressed in E. coli
BL21(DE3) successfully.

![Chart  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/engineering/11.webp)

Figure 11. Optimisation of the culture medium for the soluble expression of the recombinant His6-tagged TPL1,GUT2
and GUT1. (A) SDS-PAGE analysis of the products fermented in media. (B) Western blot analysis of the products
fermented in media; NoteThe size of the GUT1 protein is 79.8kda. The size of the GUT2 protein is 72.7kda. The size
of the TPI1 protein is 26.8kda

'
},{
"title": "Fermentation test of Cycle 1 and Cycle 2 co-transfection",
"content": '

**1. Plasmid co-transfection**

The plasmid pCDFDuet-PK-EPDH-PTase are abbreviated as PPE; The
plasmids pET Duet-GUT1-GUT2-TPl1 are abbreviated as GGT. The plasmids PPE and GGT were transfed to
*Escherichia coli* BL21. Monoclonal colonies were screened using 100 mg/ml Ampicillin (Amp+) and
Streptomycin Resistance (SmR) antibiotics. In figure 12, single colonies have grown.

![图片2](https://static.igem.wiki/teams/5763/engineering/12.webp)

Figure 12. Monoclonal colony culture dish diagram in Escherichia coli BL21

As shown in figure 13, lanes 1 to 8 correspond to the amplification products of PPE1, GGT1, PPE2, GGT2, PPE3,
GGT3, PPE4, and GGT4. The results indicate that the amplification bands of PPE4 and GGT4 are present within the
molecular weight range of 2000 bp to 4000 bp. Moreover, their actual sizes are exactly consistent with the
theoretically expected lengths, thereby confirming that these two target fragments have been successfully
amplified.

![](https://static.igem.wiki/teams/5763/engineering/13.webp)

Figure 13. Monoclonal colony verification. note:The length of GGT is 3726 bp, and the length of PPE is 3504 bp

**2. Growth Curve**

The plasmids PPE and GGT were transfed to Escherichia coli BL21. The OD600 values were
measured three times at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, 20 hours, 24 hours, 30 hours, and 48 hours,
respectively. As can be seen from figure 14, from 0 to 12 hours, the OD600 values of both CK(Blank control) and
co-transformation strains increased linearly. From 12 hours to 48 hours, the OD600 values gradually stabilized. In
figure 14, the CK groups were not significant difference to compared with bacterial strain pCDFDuet-PK-EPDH-PTase
and pET Duet-GUT1-GUT2-TPl1 . The coding genes PK, EPDH, PTase, GUT1, GUT2, and TPl1 don’t affect the growth of
the strain.

![共转OD600(1)](https://static.igem.wiki/teams/5763/engineering/14.webp)

Figure 14. The growth curve of pCDF Duet-PK-EPDH-PTase+ pET Duet-GUT1-GUT2-TPl1 and CK.
Note:The plasmid pCDFDuet-PK-EPDH-PTase are abbreviated as PPE; The plasmids pET Duet-GUT1-GUT2-TPl1 are
abbreviated as GGT

**3. Determination of erythritol production by HPLC**

We prepared five different fermentation media with varying substrate compositions and
ratios (fermentation conditions: 37°C, pH 7-8), as follows: Glucose:Glycerol = 10:10 (g/L), Glucose:Glycerol =
20:10 (g/L), Glucose:Glycerol = 10:20 (g/L), Glucose:Glycerol = 20:0 (g/L), Glucose:Glycerol = 0:20 (g/L). Samples
were taken at different time points (5h, 16h, 24h, 48h) to measure the concentrations of glucose, glycerol, and
erythritol.The concentrations of products and substrates were determined by high-performance liquid chromatography
(HPLC). After sampling, centrifugation was performed to collect the supernatant (at 8000
r/min for 5 minutes), and it was filtered through a 0.22 μm membrane. The Aminex HPX-87H analytical column (300 mm
×7.8 mm) (from Bio-Rad, USA), 2414 differential refractive index detector (from Waters, USA). The detection
conditions were column temperature 35 °C, injection volume 10 μL, mobile phase 5 mmol/L sulfuric acid, flow rate
0.6 mL/min. All the diagrams were completed using GraphPad.

According to figure 15A, the glucose concentration gradually declines with time in Glucose: Glycerol = 20:0 g/L.
The concentration of erythritol increased over time, while no erythritol was produced in
the control group(CK-*E.coli* BL21). This indicates that genes PK, EPDH, and PTase were successfully
expressed, and the engineered strain demonstrated the capability to utilize glucose as a carbon substrate for
fermentative production of erythritol.

In figure 15B, The concentration of glycerol gradually decreased, while the concentration of erythritol showed an
upward trend over time in the Glucose: Glycerol = 0:20 g/L. However, no erythritol was produced in the control
group. This indicates that the genes PK, EPDH, PTase, GUT1, GUT2, and TPI1 were successfully co-expressed in the
engineered strain, and the glycerol and glucose metabolic pathways were properly established. Furthermore, it
demonstrates that the strain can utilize a complete glycerol-based medium for fermentative erythritol production.

![图片10](https://static.igem.wiki/teams/5763/engineering/15.webp)

Figure 15. The concentration of erythritol, glucose and glycerol (A). The relationship graph of glucose, glycerol,
and erythritol with time in Glucose: Glycerol = 20:0 g/L. (B) the relationship graph of
glucose, glycerol, and erythritol with time in the Glucose: Glycerol = 0:20 g/L.

The figure 16A and B demonstrate that no erythritol was produced in the CK group (blank control), while the
glucose concentration showed a decreasing trend over time in both the Glucose:Glycerol = 20:10 g/L and
Glucose:Glycerol = 10:20 g/L. The glycerol concentration exhibited no significant change from 5h to 24 h, followed
by a decrease between 24 and 48 hours. At Glucose:Glycerol = 20:10 g/L and Glucose:Glycerol = 10:20 g/L,
erythritol concentration accumulation increased with time.

These results indicate that the strain likely preferentially utilizes glucose as the primary substrate during the
5-24 hour period at glucose and glycerol ratios of 2:1 and 1:2. When the glucose concentration becomes
sufficiently low, the strain further metabolizes glycerol as a secondary substrate for erythritol production. This
confirms that our engineered strain can utilize dual substrates (glucose and glycerol) as carbon sources for
fermentative erythritol biosynthesis.

![图片1](https://static.igem.wiki/teams/5763/engineering/16.webp)

Figure 16. The concentration of erythritol, glucose and glycerol. (A). The relationship graph of glucose,
glycerol, and erythritol with time in Glucose: Glycerol = 20:10 g/L. (B) the relationship graph of glucose,
glycerol, and erythritol with time in the Glucose: Glycerol = 10:20 g/L.

According to figure 17, the concentration of glucose gradually decreases over time in
Glucose: Glycerol = 10:10 g/L, but the concentration of glycerol shows no significant trend from 5 to 24 hours,
and then decreases from 24 to 48 hours in Glucose: Glycerol = 10:10 g/L. The concentration of erythritol increases
over time. These results indicate that glucose is preferentially utilized as the carbon source at glucose and
glycerol ratios of 1:1, 1:2 and 2:1, and erythritol production occurs under all these conditions.

![图片2](https://static.igem.wiki/teams/5763/engineering/17.webp)

Figure 17. The concentration of erythritol, glucose and glycerol in Glucose: Glycerol = 10:10 g/L.

To enhance erythritol production and investigate the effects of varying glucose-to-glycerol
ratios in the culture medium on erythritol yield, we utilized MATLAB software to perform 3D modeling predictions
based on existing data. This model predicts erythritol production levels after fermentation using different
proportional combinations of glucose and glycerol as carbon sources.A three-dimensional model was constructed by
performing surface fitting on each discrete data point, and based on this model, the prediction analysis of the
highest and lowest points was realized.

The Y-axis is labeled as "Time (h)" in the figure 18. The X-axis is labeled as "Glucose: Glycerol", displaying
different ratio values such as 0:20, 10:10, 20:0, etc. The Z-axis is labeled as "Erythritol (g/L)". The figure 17
shown that the highest value is (1.7676, 39.7474, 3.403643), and the lowest value is (0; 0; 0). At the highest
value, the ratio of glucose concentration to glycerol concentration was 17:10. After the initial measurement was
completed, the samples were subjected to a secondary fermentation process, and then the highest values were
measured using high-performance liquid chromatography (HPLC) technology.

![建模](https://static.igem.wiki/teams/5763/engineering/18.webp)

Figure 18. 3D modeling of the production volume of erythritol

In figure 19, the concentration of glucose gradually decreases over time, but the
concentration of glycerol shows no significant trend from 5h to 24h, and then decreases from 24 to 48 hours in
Glucose: Glycerol = 10:10 g/L. The concentration of erythritol increases over time. These results indicate that
glucose is preferentially utilized as the carbon source at glucose and glycerol ratios of 1:1, 1:2, 2:1and 1.7:1,
and erythritol production occurs under all these conditions.

![图片13](https://static.igem.wiki/teams/5763/engineering/19.webp)

Figure 19. the relationship graph of glucose, glycerol, and erythritol with time atGlucose: Glycerol = 17:10 g/L

After 48 hours of fermentation in media with varying ratios of glucose to glycerol, the
consumption of glucose and glycerol, as well as the erythritol production yield and conversion rate, were
calculated based on data determined by HPLC. After 48 hours, the strain consumed 13.13g glucose , and produced
2.33g erythritol with a conversion rate of 17.74% at Glucose: Glycerol = 20:0 g/L. At Glucose: Glycerol = 20:10
g/L, the strain consumed18.73g glucose and5.03g of glycerol, and produced 2.82g erythritol with a conversion rate
of 11.85% . The experimental data for other groups are presented in Table 1. The table 1 shows that the
experimental group with the highest erythritol yield was Glucose:Glycerol = 17:10 g/L, followed by
Glucose:Glycerol = 10:20 g/L. The group with the lowest erythritol yield was Glucose:Glycerol = 20:0 g/L. In Table
1, the highest erythritol conversion rate was 20.99% in the Glucose:Glycerol = 0:20 g/L, while the lowest was
11.85% in the Glucose:Glycerol = 20:10 g/L .

Table 1. Under different ratios of glucose and glycerol, the concentrations and conversion rates of glucose,
glycerol, and erythritol

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Glucose: Glycerol(g/L)** | **ΔGlucose** | **Δglycerol** | **Erythritol(g/L)** | **Percent conversion** |
| 20:0 | 13.13 | 0.00 | 2.33 | 17.74% |
| 20:10 | 18.73 | 5.03 | 2.82 | 11.85% |
| 17:10 | 16.37 | 6.63 | 3.42 | 14.86% |
| 10:10 | 9.04 | 6.75 | 2.61 | 16.55% |
| 10:20 | 8.92 | 10.01 | 3.11 | 16.41% |
| 0:20 | 0.00 | 12.20 | 2.56 | 20.99% |

**Learn:**

We have developed a dual-substrate production system by employing molecular biological techniques to enhance and
integrate the glucose and glycerol metabolic pathways within the bacterial. This innovative approach offers a
novel production paradigm characterized by environmental sustainability, operational safety, and reduced by
product formation, thereby enabling efficient utilization of glycerol resources.

The experimental results demonstrated that the engineered strain can utilize both glucose and glycerol as mixed
substrates for erythritol production. We further investigated the effects of varying glucose-to-glycerol ratios on
erythritol yield, which revealed the strain\'s preferential utilization of glucose over glycerol. However, the
optimal substrate ratio for maximizing production yield was not identified in this study.Overall, our study
provides both technical and data support for the industrial production of erythritol.

'
}, {
"title": 'References',
"content": '

Bao GWang RZhu YDong HMao S, Zhang Y, Chen Z, Li Y, Ma Y2011.Complete Genome Sequence of Clostridium
acetobutylicum DSM 1731, a Solvent-Producing Strain with Multireplicon Genome Architecture . J
Bacteriol193:.https://doi.org/10.1128/jb.05596-11

Colell A, Green DR, Ricci JE (2009).Novel roles for GAPDH in cell death and carcinogenesis.Cell Death
Differ.Dec;16(12):1573-81.

Guo L, Jiang K, Du L Q, et al. Knockout of glycerol kinase gene (glpK) and glycerol dehydrogenase gene (gldA) in
Escherichia coli and its effect on glycerol production[J]. Industrial Microbiology, 2010(3):5.
DOI:10.3969/j.issn.1001-6678.2010.03.008.

Henard CA, Freed EF, Guarnieri MT. Phosphoketolase pathway engineering for carbon-efficient biocatalysis. Curr
Opin Biotechnol. 2015 Dec;36:183-8. doi: 10.1016/j.copbio.2015.08.018. Epub 2015 Sep 8. PMID: 26360872.

Liu F, Tian JT, Wang YT, Zhao L, Liu Z, Chen J, Wei LJ, Fickers P, Hua Q. Improving an Alternative Glycerol
Catabolism Pathway in Yarrowia lipolytica to Enhance Erythritol Production. Yeast. 2024 Oct;41(10):605-614. doi:
10.1002/yea.3980. Epub 2024 Sep 11. PMID: 39262092.

 Retraction Note to: HHIP gene overexpression inhibits the growth, migration and invasion of human liver
cancer cells.. Wang Xiaobin;Ma Wenjie;Yin Jun;Chen Meizhu;Jin Hong.Journal of B.U.ON. : official journal of the
Balkan Union of Oncology.2021

T. Barbier, F. Collard, A. Zúñiga-Ripa, I. Moriyón, T. Godard, J. Becker, C. Wittmann, E. Van Schaftingen, &
J. Letesson, Erythritol feeds the pentose phosphate pathway via three new isomerases leading to
D-erythrose-4-phosphate in Brucella, Proc. Natl. Acad. Sci. U.S.A. 111 (50) 17815-17820,
https://doi.org/10.1073/pnas.1414622111 (2014).

Thuy Le H, Vu YT, Duong GH, Le TK, Dang MK, Pham DD, Pham NK, Sichaem J, Nguyen NH, Duong TH. Bio-Guided Isolation
of Alpha-Glucosidase Inhibitory Compounds from Vietnamese Lichen Roccella Montagnei. Chem Biodivers. 2024
Jul;21(7):e202400438. doi: 10.1002/cbdv.202400438. Epub 2024 May 28. PMID: 38581153.

'
}] %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}ENGINEERING{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [{
"title": "1. The construction of a plasmid",
"content": '

**1.1 Prepare solid LB medium**

Main experimental instrument:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Balance | Tryptone |
| Weighing paper | Yeast extract |
| Volumetric flask | NaCl |
| Cylinder | Agar |
| Autoclave | DDwater |

Process:

1. Turn on the balance
2. Put the weighing paper.
3. Use the balance to measure out 2.5g of Tryptone, 1.25g of Yeast extract, 2.5g of NaCl, and 3.75g of Agar. Place them in a
   volumetric flask.
4. Measure out 250ml of ddwater using a cylinder.
5. Add the material above together.
6. Align the air hole on the seal with the mouth of the bottle and tie it up with a rubber band.
7. Put it in the Autoclave at 121℃ for 20min
8. Let it cool for 1 hour

**1.2 Allocation of antibiotics Amp+ and SmR**

Main experimental instruments:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Balance | DDwater |
| Cylinder | Amp+ |
| Centrifuge tube |  |
| Sterilizing table |
| UV light |
| Culture tube |
| Sterilizing filter |

Process:

1. Measure out 1g of Amp+ using the balance.
2. Measure 10ml of water using the cylinder
3. Add them together into the centrifuge tube
4. Shake them and wait until fully mixed.
5. Put the equipment on the sterilizing table.
6. Turn on the UV light and wait for 10 minutes.
7. Take out a cell culture tube
8. Place a sterilizing filter on it
9. Use the syringe to suck up the Amp+ solution
10. transfer it into the cell culture tube through the sterilizing filter
11. Close the lid
12. Seal it with sealing film

**1.3 Culture bacteria—****PK, EPDH, and PTase**

Main experimental instruments:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Bechtop | LB medium |
| UV lamp | PK, EPDH, and PTase |
| Shaker |  |

Process:

1. Put the materials on the sterilizing table.
2. Turn on the UV light and wait for 10 minutes.
3. Take out the culture tube and add in Enzyme PK, Pet-Ptac-T7, PTase, EPDH, and liquid LB using the syringe for 3ml.
4. Add 5 μL of any of the solutions into the culture tube
5. Seal the lid of the culture tube with the sealing film.
6. Exchange the head of the pipette.
7. Repeat the pattern until all bacteria have been used
8. Put all into the shaker at 37℃ for 12-16 hours.

**1.4 Extract the plasmid**

Main experimental instrument:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Centrifugal machine | PTase bacterial liquid |
| Centrifuge tube | SP1、SP2、SP3 |
| Adsorption column | Wash Solution |
|  | Elution Buffer |

Process:

1. Fill the centrifuge tube with PTase.
2. Put the centrifuge tube into the centrifuge at a speed of 8000xg for 2 min.
3. Take out the centrifuge tube and drop the supernatant from the top, only keeping the sediment below.
4. Use the micropipette to take 250 μL SP1 and put it into the centrifuge tube. And put the SP2 250ml and the 350ml SP3 into
   the tube and slowly shake it.
5. Then put it into the centrifuge at a speed of 8000xg for 2min.
6. Take the centrifuge out and use the micropipette into the adsorption column, which has a collection tube below.
7. Mix 96ul of absolute ethanol into the wash solution
8. Take out the centrifuge tube and transfer the clarified solution to the spin column (yellow tube) using a pipette set to
   750 µL.
9. Place the spin column and collection tube into the centrifuge, set the speed to 8000 × g, and centrifuge for 30 seconds.
   Discard the liquid from the bottom of the collection tube.
10. Add 500 µL of Wash Solution to the spin column. Place the spin column and collection tube into the centrifuge, set the
    speed to 8000 × g, and centrifuge for 30 seconds. Discard the liquid from the bottom of the collection tube.
11. Repeat step 10.
12. Place the empty spin column into the centrifuge and centrifuge at 9000 × g for 1 minute to dry the membrane.
13. Prepare a new microcentrifuge tube and label it with the strain name. Insert the yellow spin column into the new tube. Add
    75 µL of Elution Buffer directly onto the membrane, avoiding contact with the sides. Centrifuge at 9000 × g for 1 minute
    to elute the DNA.

**1.5 PCR Procedure**

Main experimental instrument

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| PCR Tubes | Primer solution |
| PCR machine | DDwater |
| Centrifugal machine | Primestar mix |
| Turbine mixer | DNA template |

Process:

1. If precipitation is present in the primer solution, place the spin column in a centrifuge, set to 9000 × g, and centrifuge
   for 1 minute.
2. After centrifugation, add 100 µL dd water to the primer solution.
3. Take a PCR tube and label it with the primer name.
4. Add 25 µL Primestar Mix to the PCR tube.
5. Add 19 µL dd water to the PCR tube.
6. Add 2 µL forward primer (F-primer) to the PCR tube.
7. Add 2 µL reverse primer (R-primer) to the PCR tube.
8. Add 2 µL template DNA to the PCR tube.
9. Place the PCR tube on a vortex mixer and mix for 30 seconds.
10. Place the PCR tube in a centrifuge, set to 9000 × g, and centrifuge for 1 minute.
11. Load the prepared reaction system into the PCR machine for amplification.

PCR System Setup (Configuration)

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (µL) | Component | Volume (µL) | Component | Volume (µL) | Component | Volume (µL) | Component | Volume (µL) | Component | Volume (µL) | Component | Volume (µL) |
| Mix | 25 | Mix | 25 | Mix | 25 | Mix | 25 | Mix | 25 | Mix | 25 | Mix | 25 |
| Gut1-F | 2 | Gut2-F | 2 | PTase-F | 2 | EPDH-F | 2 | pET-R | 2 | PK-F | 2 | TPL1-F | 2 |
| Gut1- R | 2 | Gut2- R | 2 | PTase-R | 2 | EPDH-R | 2 | pET-F | 2 | PK-R | 2 | TPL1-R | 2 |
| GUT1 | 2 | GUT2 | 2 | PTase | 2 | EPDH | 2 | PET | 2 | PK | 2 | TPL1 | 2 |
| DDwater | 19 | DDwater | 19 | DDwater | 19 | DDwater | 19 | DDwater | 19 | DDwater | 19 | DDwater | 19 |

PCR Program (Temperature Settings):

|  |  |
| --- | --- |
| Temperature | Time |
| ··98°C | 10min |
| 98°C | 30ses |
| 60°C | 30ses |
| 72°C | 2min30sec |
| 72°C | 10min |

Repeat steps 2–4 for 30 cycles.

**1.6 Measure the concentrations of EPDH, Ptase, and PK using Nanadrop**

Main experimental instrument

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Nanodrop measurement cuvette | DDwater |
|  | PK、 EPDH、PTase、PET |

Process:

1. Turn on the Nanodrop spectrophotometer and calibrate the instrument.
2. Place the prepared sample into the Nanodrop measurement cuvette to measure the DNA concentration.
3. Use 2 µL of water to precisely contact the sample measurement area. Place the cuvette in the instrument and click to
   measure.
4. Here are the results:

EPDH: 57.1 ng/μL

PET: 81.8 ng/μL

PTase: 51.2 ng/μL

PTase: 57.3 ng/μL

PK: 20.1, 18.3 ng/μL

**1.7 Gel Preparation**

Main experimental instruments

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Balance | Agarose |
| Erlenmeyer flask | TAE Buffer Solution |
| Microwave oven | Nucleic acid dye |
| Plastic mould manufacturing | Loading Buffer |
| Agarose gel electrophoresis tank and power supply | 15K Marker |
| Turbine mixer | Buffer B2 |
| Gel imaging system | Wash Solution |
| Knife | Elution Solution |
| Water bath | Homologous recombinase buffer |
| Centrifugal machine |  |
| Spin column |
| PCR Tubes |

Process:

1. Turn on the balance, weigh 1g of agarose, and prepare two conical flasks.
2. Add 1 L of water and one packet of TAE powder into one conical flask, then mix the agarose with the TAE solution in the
   flask.
3. Take 100ml of the mixture and heat it in the microwave for 3 minutes at high temperature.
4. After heating, allow the mixture to cool to room temperature.
5. Add 4µL of nucleic acid dye to turn the solution pale pink for easier observation.
6. Pour the mixture into the prepared mold and let it cool for 15 minutes.
7. Prepare the agarose gel electrophoresis apparatus. Add 8 μL of loading buffer into the prepared PCR tubes.
8. Place the prepared materials in a vortex mixer, turn on the machine, and mix for 30 seconds.
9. Remove the prepared gel from the mold.
10. Place the gel into the electrophoresis tank with the sample wells facing the negative electrode.
11. Add TAE buffer until it covers the surface of the gel.
12. Load 20 μL of 15K marker into the sample wells, pipetting vertically.
13. Loading Order ⬇️
14. 20 μL marker
15. 50 μL GUT1
16. 50 μL GUT2
17. 50 μL TPL1
18. 50 μL PK
19. 50 μL EPDH
20. 50 μL PTase
21. 50 μL Bacteria P
22. Close the lid, set to 180V for 20 minutes.
23. Use a blade to cut out the successful bands.
24. Transfer the cut bands into a centrifuge tube and add 400 μL Buffer B2.
25. Place the gel under a gel imaging system to check if the results are successful.

Repeat the failed groups until imaging is successful.

1. Once all samples are successfully imaged, use a water bath to melt the gel.
2. Add the gel solution to a spin column, then centrifuge at 9000 × g for 30 seconds.
3. Discard the flow-through, add Wash Solution, centrifuge at 9000 × g for 30 seconds, and discard the flow-through again.
4. Repeat step 19 six times.
5. Dry the spin column by centrifuging at 9000 × g for 1 minute.
6. Transfer the spin column into a clean 1.5 mL microcentrifuge tube, then add 30 μL Elution Solution directly onto the
   center of the membrane.
7. Let the sample sit at room temperature for 1 minute, then centrifuge at 9000 × g for 1 minute. Store the eluted DNA
   solution.
8. Take 1 μL of the prepared solution and add it to the Nanodrop spectrophotometer for concentration measurement; for samples
   with a volume less than 1 μL, treat them uniformly as 1 μL during the operation.
9. Prepare two PCR tubes and label them with the names of the two plasmids.
10. Add 4 μL of homologous recombination enzyme buffer and 4 μL of the target gene fragment contained in the corresponding
    plasmid to each PCR tube. Vortex the mixture for 30 seconds, then incubate at 37°C for 30 minutes.

**1.8 Transferring the PPE and GGT plasmid into *Escherichia coli***

Main experimental instruments:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Centrifuge tube | *E. coli* competent cells |
| Thermostat water bath | PPE、GGT |
| Centrifugal machine | LB medium |
| Temperature incubator | Spectinomycin hydrochloride |
| Culture dish | Ampicillin |

Process:

1. Take two centrifuge tubes and add 100 μL of competent E. coli culture to each.
2. Add 10 μL of the target plasmid to each centrifuge tube and gently mix.
3. Incubate the centrifuge tubes in an ice bath for 30 minutes.
4. Perform a 45-second heat shock treatment on the centrifuge tubes to initiate the transformation process.
5. Immediately transfer the centrifuge tubes to an ice bath and cool for 3–5 minutes.
6. Add 900 μL of LB liquid medium to each centrifuge tube and mix thoroughly.
7. Incubate the centrifuge tubes at 37°C for 2 hours to complete bacterial recovery.
8. Pre-treatment of bacterial cultures: Centrifuge the bacterial cultures at 8000×g for 5 minutes to allow the bacterial
   cells to settle at the bottom of the centrifuge tube.
9. Concentration and resuspension of bacterial suspension: Remove the supernatant, retain 100 μL of LB liquid medium to
   maintain bacterial cell viability, and gently resuspend the precipitate to form a uniform bacterial suspension.
10. Preparation of solid selective medium:
11. Prepare LB solid medium containing the corresponding selection antibiotic (containing 1.5% agar powder): Mix LB liquid
    medium with the specific antibiotic, add 1.5% agar powder, heat to dissolve and mix thoroughly, then pour into 4 petri
    dishes and allow to cool and solidify naturally.
12. Antibiotic selection criteria: For the PK plasmid, spectinomycin hydrochloride is selected, matching its SmR resistance
    gene; for the GGT plasmid, ampicillin is selected, corresponding to its AmPt resistance gene.
13. Spread the bacterial suspension evenly on the surface of the prepared selective medium, let it sit, then put it in an
    incubator. The next day, check the colony growth to see if the transformation was successful.

**1.9 Extract PPE and GGT plasmids from single colonies on agar plates and perform PCR amplification**

Main experimental instrument:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Biohazard safety equipment | Single colony |
| Culture dish | DDwater |
| PCR Tubes | Primer |
| Centrifugal machine |  |
| PCR machine |

Process:

1. Transfer the petri dishes with single colonies into a biosafety cabinet and start sterile
   operation.
2. Use a micropipette to pick single colonies (10 colonies in total, covering 2 types of plasmids from 4 dishes), and
   transfer each into one of 14 PCR tubes.
3. Add 8 μl distilled water to each PCR tube to dissolve the colony.
4. Add 1 μl FR primers (upstream and downstream mixture), mix thoroughly by pipetting.
5. Place PCR tubes in a centrifuge and spin for 1 min (at standard speed, e.g., 5000×g, to collect liquid at the tube
   bottom).
6. Place PCR tubes into the PCR machine and run for 2.5 hours.

**2.0 The target fragments PPE and GGT were verified by agarose gel electrophoresis**

Main experimental instruments:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Balance | Agarose |
| Plastic mould manufacturing | TAE Buffer Solution |
| Electrophoresis tank and power supply | Nucleic acid dye |
| Ultraviolet imaging system | Loading Buffer |
| Metal bath | PCR amplification product |
| Culture tube | LB liquid medium |
| Biohazard safety equipment | SmR |
| Shaker | Amp⁺ |

Process:

1. Weigh 1 g of agarose and add to 100 ml of TAE buffer, heat until fully transparent (agarose dissolved).
2. Cool to 50–60℃, then add 3–5 μl nucleic acid dye and mix gently.
3. Pour the gel solution into an electrophoresis mold, let sit for 15 min until the gel is fully solidified.
4. Add 3 μl Loading Buffer into each PCR-amplified sample tube, mix thoroughly by pipetting.
5. Heat-treat for 3 min (e.g., boiling at 95℃ to denature nucleic acids, facilitating electrophoretic separation).
6. Load the treated samples into the wells of the agarose gel.
7. Start the electrophoresis apparatus, set the voltage to 180V, and run for 20 min.
8. After electrophoresis, transfer the gel to the UV imaging system and take photos to record band distribution.
9. Label culture tubes (distinguish between smr and ampt resistance groups), ensuring each group corresponds to the correct
   colony on the petri dish.
10. Add 3 mL LB liquid medium to each culture tube (operate under sterile conditions to avoid contamination).
11. According to a 1/1000 volume ratio, add 3 μl of SmR antibiotic to SmR group tubes and 3 μl of Amp+ antibiotic
    to Amp+ group tubes, gently invert 3 times to mix.
12. In the biosafety cabinet, use a micropipette to pick single colonies with the corresponding resistance.
13. Immerse the colony clump just below the surface of the liquid in the culture tube, gently pipette 3 times to disperse the
    colony and avoid floating.
14. Place culture tubes vertically in a 37℃ constant temperature shaker and begin shaking (if the device has a default speed,
    record as “220 rpm”; if no parameters are given, omit), incubate for 12–16 hours.

',
}, {
"title": "2. Protein expression",
"content": '

**2.1 Culture Expansion and Protein Induction**

To convert PK EPDH PTase and GUT1, GUT2, and TPl1 into *E. coli* BL21(DE3):

Main experimental instruments:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Bechtop | LB medium |
| Balance | SmR |
| Centrifuge tube | Amp+ |
| Shaker | IPTG |
| Centrifugal machine | Lysis buffer |
| Turbine | Nickel column |
| Ultrasonic cell disrupto | Separation gel |
| Metal bath | Spacer gel |
| Spin Columns | Tris-Glycine-SDS buffer |
| Rubber sheets and combs | Loading Buffer |
| Electrophoresis tank and power supply | Marker |
| Temperature incubato |  |

Process:

1. In the aseptic operation table, add 100 milliliters of LB medium to a conical flask, then add 100 microliters of SmR and
   PPE. Finally, seal the flask and write a label.
2. In the aseptic operation table, place 100 milliliters of LB medium into another conical flask, add 100 microliters of GGT
   and Amp+, seal it, and write a label.
3. In the aseptic operating table, place 20 milliliters of LB medium into another centrifuge tube, add 20 milliliters of PPE
   and SmR, seal it, and write a label.
4. In the aseptic operating table, place 20 milliliters of LB medium into another centrifuge tube, add 20 milliliters of PPE
   and SmR, seal it, and write a label.
5. Put the above-mentioned container into a 37-degree shaker and shake.
6. Take 4 large-capacity centrifuge tubes. Transfer the 4 bacterial cultures (cultured to OD600 = 0.6-0.8, then
   add 100 μL 0.5mmol and 1mmol IPTG to induce protein expression, and incubate at 25°C with shaking for 12–16 hours) to the
   centrifuge tubes and fill them.

**2.2 Protein purification and identification**

Process:

1. Then centrifuge at 4°C and 12,000×g for 10 minutes.
2. After centrifugation, discard the supernatant, add 15 mL of lysis buffer to each centrifuge tube, gently resuspend the
   bacterial pellet using a 1 mL pipette, transfer the suspension to a vortex mixer, and then place it in an ice bath for 20
   minutes.
3. Transfer the above-mentioned processed bacterial suspension to the ultrasonic cell disruptor for cell disruption.
4. Prepare 4 purification columns, labeled “Ppe 1mM,” “Ppe 0.5mM,” “Ggt 1mM,” and “Ggt 0.5mM,” respectively. Add 1 mL of
   nickel affinity resin to each purification column (to purify proteins by utilizing the specific binding of His tags to
   nickel ions).
5. Remove the sealing plug at the bottom of the purification column, wait for the liquid inside the column to flow out
   naturally, and retain the blue nickel affinity resin portion.
6. Take 4 new microcentrifuge tubes, label them according to the same rules as above, and set them aside for later use.
7. Transfer the ultrasonically disrupted bacterial suspension to a centrifuge tube and centrifuge at 10,000×g for 15 minutes.
   Collect the supernatant and transfer it to the corresponding microcentrifuge tube labeled in step 6.
8. Mix the supernatant (containing the target protein) from each microcentrifuge tube with the corresponding labeled
   purification column nickel affinity resin, and incubate at 4°C on a shaking incubator for 1.5-2 hours to remove
   impurities.
9. Prepare the separating gel: Mix 8.1 mL of ddH₂O, 6.7 μL of Acr-Bis (acrylamide-bisacrylamide), 5 mL of Gel Buffer A, 0.2
   mL of APS (ammonium persulfate), and 12 μL of TEMED in sequence. After thorough mixing, inject the mixture into the gel
   casting apparatus and cover the upper layer with deionized water.
10. Prepare the concentrated gel: Mix 1.33 mL ddH₂O, 0.67 mL Acr-Bis, 2 mL Gel buffer B, 0.04 mL APS, and 0.004 mL TEMED in
    sequence, mix well, pour out the water on the upper layer of the separating gel in the gel preparation device, inject the
    concentrated gel, and insert the comb.
11. Once the gel has completely solidified, place it in the electrophoresis chamber and add Tris-Glycine-SDS buffer.
12. Prepare 20 microcentrifuge tubes, labeled as “Ppe 1mM 1-5,” “Ppe 0.5mM 1-5,” “Ggt 1 mM 1-5,” and “Ggt 0.5 mM 1-5.” Add the
    corresponding protein samples and loading buffer to each centrifuge tube, then heat in a 95°C metal bath for 10 minutes.
13. SDS-PAGE (Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis)
    analysis (for protein separation and identification)：
14. Add 15 μL of protein marker to the first sample well of the electrophoresis chamber, and add 20 μL of the above-treated
    protein samples (Ggt and Ppe series) to the remaining sample wells in sequence.
15. Cover and perform electrophoresis at a constant voltage of 120 V for 20 minutes.
16. After electrophoresis, remove the gel and place it on A4 paper for later use.

**2.3 Convert SDS-PAGE to WB**

Main experimental instruments:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Film transfer device | Transfer buffer solution |
| Sponge, filter paper | Lichun Red staining solution |
| Shaker | confining liquid |
| Chemiluminescence imaging system | TBST buffer solution |
| PVDF membrane | Chemiluminescence detection substrate |
|  | primary antibodies |
| second antibody |

Process:

1. Place the cut 0.22 μm PVDF membrane in methanol and soak for 10-15 seconds (until it becomes translucent). Remove the blue
   protective film and rinse with deionized water, keeping the membrane wet throughout the process.
2. Prepare the transfer device: The transfer device has black and white sides. Place the gel in the “black adhesive, white
   membrane” direction to construct a sandwich structure (from the white edge to the black edge, in order: sponge → filter
   paper → PVDF membrane → gel → filter paper → sponge).
3. Remove the gel plate, take out the gel, and cut off the excess parts (retain the target band area, and avoid touching the
   gel surface with your hands during the operation); pre-soak the sponge in the pre-cooled transfer buffer solution, and
   place it on the transfer device blackboard (pay attention to the direction of the clamp).
4. Transfer the gel to the sponge, adjust the position to ensure that the area containing the bands completely covers the
   PVDF membrane (discard the part without bands), and avoid creating bubbles. Place the PVDF membrane (pay attention to the
   direction), flatten it, and remove any bubbles, then cover it with another layer of sponge and a lid, and clamp the film
   transfer device.
5. Place the sandwich structure into the transfer chamber (orientation: black side facing the black side of the chamber,
   white side facing the red side of the chamber), and fill with pre-chilled transfer buffer; place the transfer chamber in
   an ice box (maintain low temperature to prevent heat generated during transfer from affecting the results), and perform
   the transfer under one of the following conditions: Constant current 400 mA for 20 minutes, or constant voltage 100 V for
   1 hour, or constant current 350 mA for 1.5 hours (adjust specific parameters based on buffer type and protein molecular
   weight).
6. After membrane transfer, remove the PVDF membrane (or nitrocellulose membrane, cellulose acetate membrane), immerse it in
   the Lichunhong staining solution, and agitate for 3 minutes (or until the bands are clear). Record the staining results
   and recover the staining solution. Rinse the membrane with distilled water or PBS 2–3 times, each for 1 minute, until the
   Lichun Red is mostly removed (if subsequent Western blot analysis is required, rinse until the bands disappear).
7. Immerse the PVDF membrane face up in blocking solution (5% skim milk powder, prepared with TBST) and incubate on a shaking
   incubator at room temperature for 1-2 hours; remove the blocking solution (can be recycled and reused).
8. Wash the membrane 2-3 times with TBST for 10 minutes each time; cut the target fragment from the membrane, transfer it to
   a container containing the primary antibody, incubate overnight at 4°C with slow shaking, and recover the primary
   antibody.
9. Wash the membrane three times with TBST for 10 minutes each time; add the secondary antibody, incubate on a shaking
   incubator at room temperature for 1.5 hours, and recover the secondary antibody.
10. Wash the membrane three times with TBST for 10 minutes each time; mix the two developers in a 1:1 ratio to prepare the
    chemiluminescence detection substrate working solution, immerse the membrane in it for 7-10 seconds, perform the entire
    process in the dark, and then perform exposure detection.

',
}, {
"title": "3. Functional test",
"content": '

**3.1 Conjugate the PPE plasmid and the GGT plasmid together into the competent cells of *Escherichia coli***

Main experimental instruments:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| centrifuge tube | competent cell |
| thermostat water bath | LB medium |
| constant temperature shaker | SmR |
| centrifugal machine | Amp+ |
| culture dish | PCR reaction buffer |
| temperature incubator | DDwater |
| PCR Tubes | primer |
| PCR machine | agarose |
| Agarose gel electrophoresis tank and power supply | TAE Buffer Solution |
| Plastic mould manufacturing | nucleic acid dye |
| Ultraviolet and visible spectrophotometer | Loading Buffer |

Process:

1. Take the competent cells out of the ice.
2. Add 5 microliters of PPE and GGT plasmids, respectively, to the competent cells.
3. Place two small centrifuge tubes in a water bath at 42 degrees Celsius and heat for 45 seconds.
4. After heating, take it out quickly and let it stand in ice for four minutes
5. Add 900 microliters of LB medium to two small centrifuge tubes and gently shake.
6. Put it in a 37-degree shaker and shake for one and a half hours
7. After shaking, take it out and place it in a centrifuge for centrifugation at 8000xg for ten minutes
8. 8. Add 100 microliters of SmR and Amp+, respectively, to 100 milliliters of LB solution
9. 9. Pour an appropriate amount of the LB culture medium with antibiotics added into the culture dish
10. Repeat the above steps until all 500 milliliters of LB culture medium have been poured out.
11. After the centrifugation is completed in the centrifuge, discard the clear liquid on top, leaving 100 microliters. Blow up
    the colonies until the liquid becomes turbid.
12. 12. Take 30 microliters and evenly apply them to the petri dishes just used. Place them in a constant temperature
    incubator at 37 degrees Celsius and incubate upside down for 12 to 16 hours
13. Preparation of PCR reaction system: Take 8 PCR tubes, 4 of which are used for PPE detection and 4 for GGT detection. Add
    10 μL of reaction buffer, 8 μL of ddH₂O, and 2 μL of the corresponding primers (PPE-specific primers or GGT-specific
    primers) to each PCR tube in sequence, then vortex mix.
14. Template bacterial suspension preparation: Select a single colony from the center of the culture plate. Divide one
    PPE-positive colony into two portions and add each to two PPE PCR tubes; divide one GGT-positive colony into two portions
    and add each to two GGT PCR tubes (with two replicates per group). Gently pipette to ensure the bacterial cells are evenly
    distributed throughout the reaction system.
15. PCR amplification verification: Place the above PCR tubes in a PCR machine to perform amplification reactions to verify
    the presence of the target gene.
16. Agarose gel preparation: Accurately weigh 1 g of agarose, add 100 mL of TAE buffer, heat and dissolve for 3 minutes, add 5
    μL of nucleic acid dye and mix evenly, pour into the gel casting tray, and allow to solidify.
17. Gel electrophoresis analysis: After the PCR reaction is complete, add 3 μL of loading buffer to each reaction tube and
    centrifuge for 30 seconds to mix thoroughly. Place the solidified agarose gel into the electrophoresis chamber, add 20 μL
    of PCR product to each sample well, and perform electrophoresis at a constant voltage of 140 V for 20 minutes.
18. Culture and monitoring of bacterial suspension: Prepare 100 mL of LB culture medium, add 100 μL of a mixture of
    streptomycin sulfate (SMR) and ampicillin (AMP⁺) antibiotics, inoculate with 500 μL of co-transformed bacterial
    suspension, and take 1 mL of bacterial suspension as the initial sample. At regular intervals, take 1 mL of bacterial
    culture from different growth stages and measure its OD600 value using a UV-visible spectrophotometer.
19. Carbon source regulation: Pre-prepare five groups of glucose-glycerol mixed carbon sources with different proportions.
    When thOD600 value of the bacterial suspension reaches the preset threshold, add the corresponding proportions
    of glucose and glycerol to the culture system.

**3.2 Growing curve**

Main experimental instruments:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Ultraviolet and visible spectrophotometer | Culture medium of co-transformed strains |

Process:

1. OD600 values were measured at 0 h, 2 h, 4 h, 8 h, 12 h, 20 h, 24 h, 30 h, and 48 h of cultivation, with three
   replicates at each time point.
2. Based on the obtained data, a line chart was plotted (with cultivation time on the x-axis and erythritol concentration on
   the y-axis) to visually present the growth dynamics of the co-transformed strains.

**3.3 HPLC identification of erythritol,Glycerol and Glucose**

Main experimental instruments:

|  |  |
| --- | --- |
| Experiment equipment | Experiment reagent |
| Centrifugal machine | 5 mmol/L sulfuric acid solution |
| Filter plant |  |
| Binary High Pressure HPLC System |
| Glycerol |  |
| Glucose |  |

Process:

1. We prepared five different fermentation media with varying substrate compositions and ratios (fermentation conditions:
   37°C, pH 7-8), as follows: Glucose:Glycerol = 10:10 (g/L), Glucose:Glycerol = 20:10 (g/L), Glucose:Glycerol = 10:20 (g/L),
   Glucose:Glycerol = 20:0 (g/L), Glucose:Glycerol = 0:20 (g/L).
2. The bacteria were inoculated at 1% (v/v) in medium containing ampicillin (Amp) and streptomycin (SmR), and cultured until
   OD₆₀₀ reached 0.6-0.8, followed by induction with IPTG.
3. Samples were taken at different time points (5h, 16h, 24h, 48h) to measure the concentrations of glucose, glycerol, and
   erythritol.
4. After sampling, centrifuge the sample at 8000 r/min for 5 minutes to collect the supernatant.
5. Filter the collected supernatant through a 0.22 μm membrane.
6. Use an Aminex HPX-87H analytical column (300 mm × 7.8 mm, Bio-Rad, USA) coupled with a 2414 differential refractive index
   detector (Waters, USA) for analysis.
7. Set the detection conditions as follows: column temperature 35 °C, injection volume 10 μL, mobile phase 5 mmol/L sulfuric
   acid, and flow rate 0.6 mL/min.

',
}] %}
{% block title %}Experiments{% endblock %}
{% block lead %}EXPERIMENTS{% endblock %}

{% extends "layout.html" %}
{% set info\_data = [{
"title": "Overview",
"content": '

Erythritol, a polyol sweetener, exhibits non-cariogenic properties, hypoglycemic effects, and
negligible caloric value. This functional sweetener serves as an ideal sucrose alternative for food product development. In
current manufacturing methodologies, chemical synthesis prove economically unviable, whereas microbial biosynthesis
predominates in industrial production(Liu F etc, 2024; Thuy etc, 2024).We aim to develop a novel and eco-efficient
bioprocess for erythritol production while enhancing its yield. Our project involves the synthesis of erythritol using two
substrates, which are glycerol and glucose. The pathway for erythritol synthesis is as follows:

Pathway1(Thuy etc, 2024): Synthesizes erythritol using glucose as the substrate, utilizing phosphoketolase(PK),
erythritol-4-phosphate dehydrogenase(EPDH) and phosphatase(PTase). Pathway 2: Synthesizes erythritol using glycerol as the
substrate. It first utilizes encoding glycerol kinase(GUT1), Encoding glycerol-3-phosphate dehydrogenase(GUT2) and Encoding
triosephosphate isomerase(TPI1) to produce fructose-6-phosphate. This fructose-6-phosphate is then further converted into
erythritol by the enzymes of Pathway 1(Figure 1).

![图片1](https://static.igem.wiki/teams/5763/result/1.webp)

Figure 1. Erythritol Biosynthetic Pathway

We first designed expression elements containing the T7 promoter, divided them into two vectors, named
as pCDF Duet-PK-EPDH-PTase and pET Duet-GUT1-GUT2-TPl1, and constructed them into *Escherichia coli* BL21. Then, we
used Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE) and Western Blot(WB) to verify the expression of
functional proteins, and High Performance Liquid Chromatography(HPLC) to detect the yield of erythritol (Figure 2). Based on
the test results, we explored the influence of cultivation conditions on the yield and iteratively optimized the process to
improve the production efficiency of erythritol. So the purpose of our research is to increase the yield of erythritol and
improve the utilization rate of glycerol.

![Diagram  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/result/2-1.webp)

Figure 2. The engineering design schematic diagram.

**Results of your work**

1. **The construction of a plasmid**

**1.1** **pCDFDuet-****PK-EPDH-PTase**

Currently, we have selected three DNA coding sequences
from the National Center for Biotechnology Information (NCBI), namely phosphoketolase (PK), erythritol-4-phosphate
dehydrogenase (EPDH), and phosphatase (PTase). These DNA coding sequences were optimized and synthesized according to the
codon preference of *Escherichia coli*. To construct the expression plasmids for PK, EPDH, and PTase, we employed the
polymerase chain reaction (PCR) to amplify the coding genes using primers as templates. The obtained PCR products were
inserted into the linearized plasmid pCDFDuet.

As shown in Figure 3A, the pCDFDuet-PTase gene was prominently displayed between the 5000 bp and 7500
bp markers, which was in complete agreement with the theoretical length, indicating successful amplification. Figure 3B
shows that the EPDH gene was located between the 1000 bp and 1500 bp markers, while the PK gene was between the 1500 bp and
3000 bp markers. The amplified lengths of the genes were consistent with the DNA coding, suggesting successful
amplification.

![A picture containing graphical user interface  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/result/3.webp)

Figure 3A. The electrophoresis verification of the target fragment pCDFDuet-Ptase;Figure 3 B. The
electrophoresis verification of the target fragment EPDH and PK; Note: phosphoketolase(PK) -2391bp; Erythritol-4-phosphate
dehydrogenas(EPDH)-1509bp; pCDFDuet-phosphatase-5369bp

Next, we cloned three coding DNA (PK, EPDH, and PTase)
into the pCDFDuet vector using homologous recombination, and transformed it into *E. coli* DH5α competent cells. The
total length of the three DNA coding is 5565 bp. The electrophoresis result shown in Figure 4A indicates that the amplified
product band is located within the range of 5000-7000 bp, which is consistent with the expected fragment size, confirming
that the target gene fragment has been successfully amplified. In Figure 4B, the growth of monoclonal colonies with
SmR(100ng/ml)was observed, indicating that the transformation experiment was initially effective. Figure 4C is the
sequencing report returned by the sequencing company. By comparing the sequence file with the target gene sequence, the
results show that there is no base mutation at the location marked by the red solid arrow, and a base mutation exists at the
location marked by the dotted line arrow. The sequencing results confirm that the target gene
fragment has been correctly linked to the vector, further verifying the successful construction of the recombinant plasmid.

![A picture containing diagram  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/result/4.webp)

Figure 4A. Monoclonal colony verification gel plate; Figure 4B. Monoclonal colony verification petri
dish diagram inE. coli DH5α ; Figure 4C. Monoclonal colony verification sequencing plot

**1.2** **pET Duet-****GUT1-GUT2-TPl1**

Three DNA sequences corresponding to GUT1, GUT2, and TPL1, respectively, were
selected from the NCBI database for this study. These sequences were codon-optimized based on the codon preference of
*Escherichia coli* before synthesis. To construct an expression plasmid containing glycerol kinase (GUT1),
glyceraldehyde-3-phosphate dehydrogenase (GUT2), and isomerase (TPL1), each target gene was amplified polymerase chain
reaction (PCR) using specific primers, and the resulting amplification products were then inserted into the linearized pET
Duet vector. The target fragment was ligated to the vector by homologous recombination, transformed
into E. coli DH5α, and the expression plasmids were verified.

In Figure 5A, the GUT1 and GUT2 amplification products migrated between the 1500 bp and 3000 bp markers. Similarly, Figure
5B showed that showed that the TPL1 amplification product was located between the 500 bp and 1000 bp markers, and Figure 5C
indicates that the pET Duet exhibited a distinct band about 5000 bp. The observed lengths of all
amplified genes were consistent with their respective coding sequences, validating the success of the amplification process.

![Graphical user interface  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/result/5.webp)

Figure 5A. The electrophoresis verification of the target fragment pET Duet(5462bp). Figure 5B. The
electrophoresis verification of the target fragments GUT1(2130bp)and GUT2(1950bp).Figure 5C. The electrophoresis
verification of the target fragment TPL1(747bp) .

The total length of the coding GUT1, GUT2, and TPL1 is 4827bp. As shown in the electrophoresis result in figure 6A, the
amplified product presents a clear band at approximately 5000 bp, which is highly consistent with the expected fragment
size. The growth of monoclonal colonies figure 6B with Amp(100ng/ml) was observed. And Figure 6C showed that no base
mutation occurred at the position marked by the solid red arrow, while a base mutation was present at the position marked by
comparing the sequencing file with the target gene sequence. All results confirmed that the target gene fragment has been
correctly linked to the vector, further verifying the success of the construction of the recombinant plasmid.

**![A picture containing diagram  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/result/6.webp)**

Figure 6A. Monoclonal Colony Verification Gel Plate; Figure 6B. Monoclonal Colony Verification Petri Dish Diagram in E.
coli DH5α; Figure 6C. Monoclonal colony verification sequencing plot

1. **Protein expression**

**2.1 pCDFDuet-PK-EPDH-PTase**

The three-dimensional structures of proteins PK, EPDH, and PTase were predicted using AlphaFold2, and the feasibility of the
models was evaluated using the metrics ipTM (interface pTM) and pTM (predicted TM-score). Thefigure 7 shows the
three-dimensional structural representation of the protein PK, EPDH, and PTase.

![图片15](https://static.igem.wiki/teams/5763/result/7.webp)

Figure 7. Figure A is the three-dimensional structural of the protein PK;Figure B is the three-dimensional structural of the
protein EPDH;Figure C is the three-dimensional structural of the protein PTase.

The recombinant plasmids pCDFDuet-PK-EPDH-PTase were transformed into *E. coli*  BL21 (DE3) for expression. A single
colony containing the recombinant plasmid was cultured in medium con-taining antibiotics (100 mg/mL SmR ) at
37oC. When the cul-tures reached optical density (OD600) about 0.6, iso-propy-beta-D-thiogalactopyranoside
(0.5mmol and 1mmol IPTG) was added to the broth for induction of the protein in 25oC . And the recombinant His-6
fusion-type PPE protein was purified by Ni2+ affinity chromatography.

As shown in figure 8A, when 0.5 mmol IPTG was used to induce the protein, no PK protein was expressed, but PTase and EPDH
were successfully expressed. After being inducted with 1 mmol/L IPTG, both PK, Ptase and EPDH exhibited sig-niﬁcant
expression, and the Western blot results showed that both these recombinant prod-ucts could be detected by the anti-His6
antibodies (Figure 8B), suggesting that His6-fused PK, Ptase and EPDH were expressed in E. coli BL21(DE3) successfully.

![A picture containing chart  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/result/8.webp)

Figure 8. Optimisation of the culture medium for the soluble expression of the recombinant His6-tagged PK, Ptase and EPDH.
(A) SDS-PAGE analysis of the products fermented in media. (B) Western blot analysis of the products fermented in media; Note the Pk protein is 90.6kda. The PTase protein is 59.8kda. The EPDH protein is 56.1kda

**2.2pET Duet-GUT1-GUT2-TPl1**

The three-dimensional structures of proteins GUT1,GUT2 and TPl1 were predicted using SWISS-MODEL, and the feasibility of the
models was evaluated using the metrics GMQE (Global Model Quality Estimation) and QMEAN. The figure 9 shows the
three-dimensional structural representation of the protein GUT1,GUT2 and TPl1.

**![图片16](https://static.igem.wiki/teams/5763/result/9.webp)**

Figure 9. Figure A is the three-dimensional structural of the protein GUT1;Figure B is the three-dimensional structural of
the protein GUT2;Figure C is the three-dimensional structural of the protein TPL1.

The recombinant plasmids pET Duet-GUT1-GUT2-TPl1 were transformed into *E. coli*  BL21 (DE3) for expression. A single
colony containing the recombinant plasmid was cultured in medium con-taining antibiotics (100 mg/mL Amp) at 37oC.
The other protein expression conditions are consistent with pCDFDuet-PK-EPDH-PTase. The figure 10A shown that the TPL1,GUT2
and GUT1 exhibited sig-niﬁcant expression after being inducted with 0.5 and 1 mmol/L IPTG. And the Western blot results
showed that both these recombinant prod-ucts could be detected by the anti-His6 antibodies (Figure 10B), suggesting that
His6-fused TPL1,GUT2 and GUT1 were expressed in E. coli BL21(DE3) successfully.

![Chart  AI-generated content may be incorrect.](https://static.igem.wiki/teams/5763/result/10.webp)

Figure 10. Optimisation of the culture medium for the soluble expression of the recombinant His6-tagged TPL1,GUT2 and GUT1.
(A) SDS-PAGE analysis of the products fermented in media. (B) Western blot analysis of the products fermented in media;
NoteThe size of the GUT1 protein is 79.8kda. The size of the GUT2 protein is 72.7kda. The size of the TPI1 protein is
26.8kda

**3．Functional test**

**3.1** **Plasmid co-transfection**

The plasmid pCDFDuet-PK-EPDH-PTase are abbreviated as PPE; The plasmids pET
Duet-GUT1-GUT2-TPl1 are abbreviated as GGT. The plasmids PPE and GGT were transfed to *Escherichia coli* BL21.
Monoclonal colonies were screened using 100 mg/ml Ampicillin (Amp+) and Streptomycin Resistance (SmR)
antibiotics. In figure 11, single colonies have grown.

![图片2](https://static.igem.wiki/teams/5763/result/11.webp)

Figure 11. Monoclonal colony culture dish diagram in Escherichia coli BL21

As shown in figure 12, lanes 1 to 8 correspond to the amplification products of PPE1, GGT1, PPE2, GGT2, PPE3, GGT3, PPE4,
and GGT4. The results indicate that the amplification bands of PPE4 and GGT4 are present within the molecular weight range
of 2000 bp to 4000 bp. Moreover, their actual sizes are exactly consistent with the theoretically expected lengths, thereby
confirming that these two target fragments have been successfully amplified.

![](https://static.igem.wiki/teams/5763/result/12.webp)

Figure 12. Monoclonal colony verification. note:The length of GGT is 3726 bp, and the length of PPE is 3504 bp

**3.2 Growth Curve**

The plasmids PPE and GGT were transfed to Escherichia coli BL21. The OD600 values were measured three
times at 0 hours, 2 hours, 4 hours, 8 hours, 12 hours, 20 hours, 24 hours, 30 hours, and 48 hours, respectively. As can be
seen from figure 13, from 0 to 12 hours, the OD600 values of both CK(Blank control) and co-transformation strains increased
linearly. From 12 hours to 48 hours, the OD600 values gradually stabilized. In figure 13, the CK groups were not significant
difference to compared with bacterial strain pCDFDuet-PK-EPDH-PTase and pET Duet-GUT1-GUT2-TPl1 . The coding genes PK, EPDH,
PTase, GUT1, GUT2, and TPl1 don’t affect the growth of the strain.

![共转OD600(1)](https://static.igem.wiki/teams/5763/result/13.webp)

Figure 13. The growth curve of pCDF Duet-PK-EPDH-PTase+ pET Duet-GUT1-GUT2-TPl1 and CK. Note:The
plasmid pCDFDuet-PK-EPDH-PTase are abbreviated as PPE; The plasmids pET Duet-GUT1-GUT2-TPl1 are abbreviated as GGT

**3.3 Determination of erythritol production by HPLC**

We prepared five different fermentation media with varying substrate compositions and ratios
(fermentation conditions: 37°C, pH 7-8), as follows: Glucose:Glycerol = 10:10 (g/L), Glucose:Glycerol = 20:10 (g/L),
Glucose:Glycerol = 10:20 (g/L), Glucose:Glycerol = 20:0 (g/L), Glucose:Glycerol = 0:20 (g/L). Samples were taken at
different time points (5h, 16h, 24h, 48h) to measure the concentrations of glucose, glycerol, and erythritol.The
concentrations of products and substrates were determined by high-performance liquid chromatography (HPLC).
After sampling, centrifugation was performed to collect the supernatant (at 8000 r/min for 5
minutes), and it was filtered through a 0.22 μm membrane. The Aminex HPX-87H analytical column (300 mm ×7.8 mm) (from
Bio-Rad, USA), 2414 differential refractive index detector (from Waters, USA). The detection conditions were column
temperature 35 °C, injection volume 10 μL, mobile phase 5 mmol/L sulfuric acid, flow rate 0.6 mL/min. All the diagrams were
completed using GraphPad.

According to figure 14A, the glucose concentration gradually declines with time in Glucose: Glycerol = 20:0 g/L. The
concentration of erythritol increased over time, while no erythritol was produced in the control
group(CK-*E.coli* BL21). This indicates that genes PK, EPDH, and PTase were successfully expressed, and the
engineered strain demonstrated the capability to utilize glucose as a carbon substrate for fermentative production of
erythritol.

In figure 14B, The concentration of glycerol gradually decreased, while the concentration of erythritol showed an upward
trend over time in the Glucose: Glycerol = 0:20 g/L. However, no erythritol was produced in the control group. This
indicates that the genes PK, EPDH, PTase, GUT1, GUT2, and TPI1 were successfully co-expressed in the engineered strain, and
the glycerol and glucose metabolic pathways were properly established. Furthermore, it demonstrates that the strain can
utilize a complete glycerol-based medium for fermentative erythritol production.

![图片10](https://static.igem.wiki/teams/5763/result/14.webp)

Figure 14. The concentration of erythritol, glucose and glycerol (A). The relationship graph of glucose, glycerol, and
erythritol with time in Glucose: Glycerol = 20:0 g/L. (B) the relationship graph of glucose,
glycerol, and erythritol with time in the Glucose: Glycerol = 0:20 g/L.

The figure 15A and B demonstrate that no erythritol was produced in the CK group (blank control), while the glucose
concentration showed a decreasing trend over time in both the Glucose:Glycerol = 20:10 g/L and Glucose:Glycerol = 10:20 g/L.
The glycerol concentration exhibited no significant change from 5h to 24 h, followed by a decrease between 24 and 48 hours.
At Glucose:Glycerol = 20:10 g/L and Glucose:Glycerol = 10:20 g/L, erythritol concentration accumulation increased with time.

These results indicate that the strain likely preferentially utilizes glucose as the primary substrate during the 5-24 hour
period at glucose and glycerol ratios of 2:1 and 1:2. When the glucose concentration becomes sufficiently low, the strain
further metabolizes glycerol as a secondary substrate for erythritol production. This confirms that our engineered strain
can utilize dual substrates (glucose and glycerol) as carbon sources for fermentative erythritol biosynthesis.

![图片1](https://static.igem.wiki/teams/5763/result/15.webp)

Figure 15. The concentration of erythritol, glucose and glycerol. (A). The relationship graph of glucose, glycerol, and
erythritol with time in Glucose: Glycerol = 20:10 g/L. (B) the relationship graph of glucose, glycerol, and erythritol with
time in the Glucose: Glycerol = 10:20 g/L.

According to figure 16, the concentration of glucose gradually decreases over time in Glucose:
Glycerol = 10:10 g/L, but the concentration of glycerol shows no significant trend from 5 to 24 hours, and then decreases
from 24 to 48 hours in Glucose: Glycerol = 10:10 g/L. The concentration of erythritol increases over time. These results
indicate that glucose is preferentially utilized as the carbon source at glucose and glycerol ratios of 1:1, 1:2 and 2:1,
and erythritol production occurs under all these conditions.

![图片2](https://static.igem.wiki/teams/5763/result/16.webp)

Figure 16. The concentration of erythritol, glucose and glycerol in Glucose: Glycerol = 10:10 g/L.

To enhance erythritol production and investigate the effects of varying glucose-to-glycerol ratios in
the culture medium on erythritol yield, we utilized MATLAB software to perform 3D modeling predictions based on existing
data. This model predicts erythritol production levels after fermentation using different proportional combinations of
glucose and glycerol as carbon sources.A three-dimensional model was constructed by performing surface fitting on each
discrete data point, and based on this model, the prediction analysis of the highest and lowest points was realized.

The Y-axis is labeled as "Time (h)" in the figure 17. The X-axis is labeled as "Glucose: Glycerol", displaying different
ratio values such as 0:20, 10:10, 20:0, etc. The Z-axis is labeled as "Erythritol (g/L)". The figure 17 shown that the
highest value is (1.7676, 39.7474, 3.403643), and the lowest value is (0; 0; 0). At the highest value, the ratio of glucose
concentration to glycerol concentration was 17:10. After the initial measurement was completed, the samples were subjected
to a secondary fermentation process, and then the highest values were measured using high-performance liquid chromatography
(HPLC) technology.

![建模](https://static.igem.wiki/teams/5763/result/17.webp)

Figure 17. 3D modeling of the production volume of erythritol

In figure 18, the concentration of glucose gradually decreases over time, but the concentration of
glycerol shows no significant trend from 5h to 24h, and then decreases from 24 to 48 hours in Glucose: Glycerol = 10:10 g/L.
The concentration of erythritol increases over time. These results indicate that glucose is preferentially utilized as the
carbon source at glucose and glycerol ratios of 1:1, 1:2, 2:1and 1.7:1, and erythritol production occurs under all these
conditions.

![图片13](https://static.igem.wiki/teams/5763/result/18.webp)

Figure 18. the relationship graph of glucose, glycerol, and erythritol with time atGlucose: Glycerol = 17:10 g/L

After 48 hours of fermentation in media with varying ratios of glucose to glycerol, the consumption
of glucose and glycerol, as well as the erythritol production yield and conversion rate, were calculated based on data
determined by HPLC. After 48 hours, the strain consumed 13.13g glucose , and produced 2.33g erythritol with a conversion
rate of 17.74% at Glucose: Glycerol = 20:0 g/L. At Glucose: Glycerol = 20:10 g/L, the strain consumed18.73g glucose and5.03g
of glycerol, and produced 2.82g erythritol with a conversion rate of 11.85% . The experimental data for other groups are
presented in Table 1. The table 1 shows that the experimental group with the highest erythritol yield was Glucose:Glycerol =
17:10 g/L, followed by Glucose:Glycerol = 10:20 g/L. The group with the lowest erythritol yield was Glucose:Glycerol = 20:0
g/L. In Table 1, the highest erythritol conversion rate was 20.99% in the Glucose:Glycerol = 0:20 g/L, while the lowest was
11.85% in the Glucose:Glycerol = 20:10 g/L .

Table 1. Under different ratios of glucose and glycerol, the concentrations and conversion rates of glucose, glycerol, and
erythritol

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Glucose: Glycerol(g/L)** | **ΔGlucose** | **Δglycerol** | **Erythritol(g/L)** | **Percent conversion** |
| 20:0 | 13.13 | 0.00 | 2.33 | 17.74% |
| 20:10 | 18.73 | 5.03 | 2.82 | 11.85% |
| 17:10 | 16.37 | 6.63 | 3.42 | 14.86% |
| 10:10 | 9.04 | 6.75 | 2.61 | 16.55% |
| 10:20 | 8.92 | 10.01 | 3.11 | 16.41% |
| 0:20 | 0.00 | 12.20 | 2.56 | 20.99% |

**Further Discussion**

In Table 1, the highest erythritol conversion rate was 20.99% in the Glucose:Glycerol = 0:20 g/L, while the lowest was
11.85% in the Glucose:Glycerol = 20:10 g/L . The erythritol conversion rate was not significant difference in the
Glucose:Glycerol = 10:10 g/L and 10:20 g/L. According to figure 16, the concentration of glucose gradually decreases over
time in Glucose: Glycerol = 10:10 g/L, but the concentration of glycerol shows no significant trend from 5 to 24 hours, and
then decreases from 24 to 48 hours in Glucose: Glycerol = 10:10 g/L. The concentration of erythritol increases over time.
These results indicate that glucose is preferentially utilized as the carbon source at glucose and glycerol ratios of 1:1,
1:2 and 2:1, and erythritol production occurs under all these conditions. Based on our experimental data, the strain
preferentially utilizes glucose and continues to consume glycerol when glucose concentration becomes low. When glucose is
present, it inhibits the ability of Escherichia coli to utilize other secondary carbon sources (such as glycerol, lactose,
arabinose, etc.).The metabolism of glucose reduces the intracellular concentration of a key signaling molecule—cAMP (cyclic
adenosine monophosphate). cAMP must bind to CRP (cAMP receptor protein) to form a complex (cAMP-CRP)l(Jacqueline, etc,
2022). This complex is an essential transcription factor required for activating the expression of many operons involved in
the metabolism of other carbon sources, such as those for lactose, arabinose, and glyceol(Jacqueline, etc; 2022).

Therefore, subsequent optimization can focus on testing ratios ranging from 10:20 to 0:20 (Glucose:Glycerol) to identify a
fermentation medium that achieves both higher conversion rate and higher yield(Kaika, etc,2022; Thuy,etc; 2024 ).
Additionally, the effects of varying fermentation temperatures and pH levels on erythritol production yield should be
investigated(Heghine, etc; 2022).

The current 3D model is limited by insufficient data points, which compromises its ability to accurately capture overall
trends and increases susceptibility to prediction deviations. Moreover, the model does not include the erythritol conversion
rate as a key metric. Converting predicted outputs into percentage format would significantly enhance its practicality for
observational analysis and decision-making.

**Reference**

Heghine G ,Satenik K ,Anait V , et al.Metabolic pathways and ΔpH regulation in Escherichia coli during fermentation of
glucose and glycerol in the presence of formate at pH the role of FhlA transcriptional activator.[J].FEMS microbiology
letters,2022,369(1):

Liu F, Tian JT, Wang YT, Zhao L, Liu Z, Chen J, Wei LJ, Fickers P, Hua Q. Improving an Alternative Glycerol Catabolism
Pathway in Yarrowia lipolytica to Enhance Erythritol Production. Yeast. 2024 Oct;41(10):605-614. doi: 10.1002/yea.3980. Epub
2024 Sep 11. PMID: 39262092.

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'
}] %}
{% block title %}Results{% endblock %}
{% block lead %}RESULTS{% endblock %}