iGEM 2024 — Team ouc-haide

{% extends "testlayout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

# Notebook

**2023**

2023.05.01 Establishment of 2024 OUC-Haide Recruitment Group.
2023.05.09 Full-scale launch of 2024 OUC-Haide recruitment work.
2023.06.03 Start of interviews for 2024 OUC-Haide reserve members.
2023.06.17 Confirmation of 2024 OUC-Haide reserve members.
2023.07.05 Start of reserve members meeting.
2023.07.10 Study of protein composition and structure, basic concepts of enzymes.
2023.07.17 Study of nucleic acid structure and DNA replication, reading "A One Pot, One Step, Precision Cloning Method with High Throughput Capability".
2023.07.24 Study of RNA synthesis, processing, and regulation, reading "Capture-SELEX for aptamer selection: A short review".
2023.07.31 Study of protein synthesis and recombinant DNA technology, reading "The Biology of CRISPR-Cas: Backward and Forward".

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| ![](https://static.igem.wiki/teams/5314/notebook/new-summertime-schedule-2024-igem-00-new.png) |
| Figure 1: Summer School Task List |

2023.08.24 Completion of English and Chinese test papers for the assessment of 2024 OUC-Haide official members.
2023.08.30 Completion of laboratory equipment safety training and passing the laboratory access exam.
2023.09.02 Printing of English and Chinese test papers for the assessment of 2024 OUC-Haide official members.
2023.09.02 Conducting the assessment of 2024 OUC-Haide official members.
2023.09.13 Completion of grading the test papers for the assessment of 2024 OUC-Haide official members.
2023.09.13 Confirmation of 2024 OUC-Haide official core members, 2024 OUC-Haide was officially established today!
2023.09.18 Meeting between 2023 & 2024 OUC-Haide members.

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| ![](https://static.igem.wiki/teams/5314/notebook/2.png) |
| Figure 2: OUC-Haide 2023 & 2024 OUC-Haide Meeting |

2023.11.05 2023 OUC-Haide won the Gold Medal.

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| ![](https://static.igem.wiki/teams/5314/notebook/3.png) |
| Figure 3: 2023 OUC-Haide Award Ceremony |

2023.12.07 Group meeting, learning about the specific tasks required for iGEM, determining the first round of excellent Wiki shares for 2024 OUC-Haide core members.
2023.12.14 Group meeting, sharing the first round of Wiki readings, determining the second round of Wiki readings.
2023.12.21 Group meeting, sharing the second round of Wiki readings, determining the third round of Wiki readings.

**2024**

**the First Quarter**

2024.02.02 Each member registers an iGEM account.
2024.02.04 Group meeting, sharing the third round of Wiki readings, determining the fourth round of Wiki readings.
2024.02.06 Group meeting, sharing readings on Golden and Gibson technologies.
2024.02.08 Group meeting, sharing the fourth round of Wiki readings.
2024.02.21 Joining the 2024 China iGEM group chat.
2024.02.22 2024 OUC-Haide officially starts the project.
2024.03.08 Start recruiting graphic designers.
2024.03.10 First round of project initiation for 2024 OUC-Haide.
2024.03.14 Officially forming 2024 OUC-Haide on the iGEM website.
2024.03.17 Second round of project initiation for 2024 OUC-Haide.
2024.03.20 Confirming two graphic designers.

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| ![](https://static.igem.wiki/teams/5314/notebook/4.png) |
| Figure 4: 2023 OUC-Haide Award Ceremony at Ocean University of China |

2024.03.21 2024 OUC-Haide volunteers at the "Children of the Sea" exemplary student award ceremony, presenting flowers to 2023 OUC-Haide
2024.03.24 Third round of project initiation for 2024 OUC-Haide.
2024.03.24 Core members share the registration fee.
2024.03.25 Planning to host the 11th CCiC.
2024.03.26 Writing application materials for hosting the 11th CCiC.
2024.03.27 Determining the project theme of β-glucan, starting to organize materials.
2024.03.28 Studying the feasibility of improving Saccharomyces cerevisiae for efficient production of β-glucan.

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| ![](https://static.igem.wiki/teams/5314/notebook/5.png) |
| Figure 5: β-glucan |

**the Second Quarter**

2024.04.01 Core members attempt to pay the registration fee.
2024.04.02 The team communicates with the advisor and summarizes multiple project initiation experiences, confirming project objectives and indicators.
2024.04.02 Confirming the advisor and having in-depth discussions to determine the next steps.
2024.04.05 Changing the chassis organism to marine yeast.
2024.04.05 Preliminary design of a toxin gene suicide switch.
2024.04.05 Core members successfully pay the registration fee.
2024.04.05 Filling out the funding application form.
2024.04.06 Completing the materials for hosting CCiC and submitting them to the college for approval.
2024.04.07 Borrowing GEMs from the related species Aspergillus niger.
2024.04.08 Establishing the basic TPNR framework for HP.
2024.04.09 Confirming communication with SDU-China.
2024.04.09 Using CarveMe and Cobrapy for analysis.

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| ![](https://static.igem.wiki/teams/5314/notebook/6.png) |
| Figure 6: Metabolic Network |

2024.04.12 Completing the funding application and submitting it.
2024.04.13 Starting to culture marine yeast.
2024.04.14 Several core members go to apply for passports together.
2024.04.15 Submitting CCiC materials for school approval, delayed by the sports meeting.
2024.04.15 Lysing and extracting total DNA from marine yeast.
2024.04.16 Using the OptForce algorithm to identify key reactions and determine secondary metabolites affecting β-glucan synthesis.
2024.04.18 Using Simbiology to confirm gene knockout targets.
2024.04.19 Starting to amplify the melanin gene using PCR.
2024.04.21 Learning that CCiC will be held in Suzhou, abandoning the application process.
2024.04.24 Discussing with the Youth League teacher whether to rely on the college's official account or create our own team account for publicity.
2024.04.25 Finding the melanin gene to be long, designing three pairs of primers for segmented amplification.
2024.04.29 Completing the PCR of the melanin gene.
2024.04.30 Confirming the initial EDU concept.
2024.04.30 Agarose gel electrophoresis identification of PCR products, with many nonspecific bands.
2024.05.02 Re-amplifying the melanin gene in segments using PCR.
2024.05.03 Agarose gel electrophoresis identification of PCR products and recovery.
2024.05.03 Proposing standardized recruitment.
2024.05.04 Starting to establish the sugar metabolism pathway.
2024.05.06 Non-laboratory work officially begins in full swing, with tasks assigned to individuals.
2024.05.08 Connecting the PCR product to the T vector.
2024.05.10 Proposing to conduct HP in conjunction with mangroves.
2024.05.11 Preparing materials for communication with SDU-China.
2024.05.11 Transforming E. coli.
2024.05.12 Screening positive clones and verifying with PCR.
2024.05.12 Communicating with SDU-China.
2024.05.14 Extracting, sequencing, and comparing plasmids.
2024.05.14 Drawing the initial version of the team logo.
2024.05.14 Proposing a team logo based on the Ascension of Christ.
2024.05.16 Confirming participation in CCiC, starting to make team uniforms.
2024.05.19 Verifying the upstream sequence of the melanin gene.
2024.05.20 Starting modeling exploration and optimizing culture conditions.
2024.05.21 Starting to make the CCiC poster.
2024.05.21 Writing the push for communication with SDU-China.
2024.05.21 Filling out the Safety Form and Check-in Form.
2024.05.23 Reflecting on the long road ahead.
2024.05.24 Collecting the list of high schools and universities that have participated in iGEM in the past for EDU preparation.
2024.05.26 Taking group photos for the CCiC brochure.

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| ![](https://static.igem.wiki/teams/5314/notebook/8.png) |
| Figure 8: Group photo for the CCiC brochure |

2024.05.26 Completing the push for communication with SDU-China.
2024.05.27 Deciding to hold a conference in Wuhan with Wuhan University, Huazhong University of Science and Technology, and HZAU-China.
2024.05.27 The Ascension of Christ logo element is incorporated into the new team logo.
2024.05.27 Starting to construct the melanin gene knockout vector.
2024.05.29 Modifying the Safety Form based on feedback.
2024.05.30 Attending the Math conference.
2024.05.31 Confirming the track is agriculture.
2024.05.31 PCR amplification of the 5’ and 3’ homologous regions of the melanin gene.

**the Third Quarter**

2024.02.02 Each member registers an iGEM account.
20242024.06.01 Agarose gel electrophoresis identification of PCR products and recovery.
2024.06.02 Connecting the PCR product to the vector.
2024.06.03 Proposing practical agricultural HP directions for β-glucan.
2024.06.04 Submitting the CCiC reimbursement application.
2024.06.04 Transforming E. coli with the vector.
2024.06.06 Verifying positive transformants.
2024.06.06 Proposing the initial version of the team uniform.
2024.06.09 Lysing E. coli and extracting plasmids.
2024.06.09 Conducting HP at Beihang University.
2024.06.10 Conducting HP at Peking University Cardiovascular Research Institute.
2024.06.10 Conducting HP at HZAU-China.
2024.06.13 Starting to make the CCiC poster.
2024.06.14 Proposing improvements to the team uniform, which eventually becomes the official team uniform.
2024.06.14 Starting to design Hardware.
2024.06.14 Performing double digestion and using DNA ligase to connect the 5’ and 3’ homologous regions, completing the construction of the knockout vector.
2024.06.17 PCR amplification of the knockout vector to obtain linear knockout fragments.
2024.06.18 Agarose gel electrophoresis identification of PCR products.
2024.06.22 Picking single colonies and inoculating into HC medium.
2024.06.22 Filling out various CCiC materials.
2024.06.23 Completing the CCiC poster.
2024.06.23 Reaching an OD600nm of 1.1.
2024.06.23 Performing PEG transformation and culturing on antibiotic medium.
2024.06.24 Obtaining transformants.
2024.06.24 Completing the final team logo and team uniform.

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| ![](https://static.igem.wiki/teams/5314/notebook/9-new.png) |
| Figure 8: Team logo |

2024.06.25 Re-screening the transformants.
2024.06.28 Visible transformant colonies are significantly whiter than wild-type colonies.

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| ![](https://static.igem.wiki/teams/5314/notebook/10.png) |
| Figure 10: Melanin knockout experiment results |

2024.06.28 Wuhan University comes to the school for HP.
2024.06.30 Extracting DNA from marine yeast for genotyping.
2024.07.01 Comparing the relative expression levels of the melanin gene in knockout and wild-type strains.
2024.07.04 Performing RT-qPCR to confirm the decreased relative expression of the melanin gene in the knockout strain.
2024.07.05 Completing the CCiC presentation PPT.
2024.07.06 Discussing culture medium optimization plans.
2024.07.11 Performing infrared spectroscopy analysis of melanin content changes.
2024.07.11 Arriving in Suzhou to attend CCiC.

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| ![](https://static.igem.wiki/teams/5314/notebook/11.png) |
| Figure 11: Team in CCiC |

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| ![](https://static.igem.wiki/teams/5314/notebook/12.png) |
| Figure 12: Team in CCiC |

2024.07.12 Performing NMR analysis of melanin content changes.
2024.07.12 Proposing HP can create antibiotic distribution data analysis.
2024.07.14 Conducting stress resistance experiments on knockout and wild-type strains.
2024.07.14 Reporting at CCiC

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| ![](https://static.igem.wiki/teams/5314/notebook/14.png) |
| Figure 13: Reporing in CCiC |

2024.07.14 Inspired to pay attention to cultural and conceptual differences at home and abroad.
2024.07.14 Proposing the wiki homepage should reflect livestock and feed.
2024.07.15 Starting to construct the pullulan gene knockout vector.
2024.07.17 PCR amplification of the 5’ and 3’ homologous regions of the pullulan gene.
2024.07.18 Agarose gel electrophoresis identification of PCR products and recovery.
2024.07.18 Proposing to participate in the CJUH-JLU-China ethics white paper compilation independently.
2024.07.18 Completing the initial model of the oxygen supply protein.
2024.07.19 Establishing the summer work schedule.
2024.07.19 Starting to summarize and write experimental results.
2024.07.19 Connecting the PCR product to the vector.
2024.07.20 Starting to construct 3D printing models and display diagrams.
2024.07.21 Participating in the JLU-NBBMS ethics white paper compilation.
2024.07.22 Establishing the Wiki production plan.
2024.07.23 Transforming E. coli with the vector.
2024.07.23 Verifying positive transformants.
2024.07.24 Confirming participation in the NNU-CHINA The Light of Biology, Building Future Together podcast.
2024.07.24 Discovering the push for SDU-China was not sent out, finally realizing it was not reported to the college, deciding to send it together with the CCiC push.
2024.07.25 Improving the oxygen supply protein model.
2024.07.25 Going to the Tengzhou Animal Quarantine Declaration Point for HP.
2024.07.25 Completing the antibiotic distribution map

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| ![](https://static.igem.wiki/teams/5314/notebook/15.png) |
| Figure 14: Antibiotic distribution map |

2024.07.26 Completing the CCiC push draft.
2024.07.26 Lysing E. coli and extracting plasmids.
2024.07.27 Going to the farm for HP.
2024.07.30 Publishing the SDU-China push draft and the CCiC push draft.
2024.07.30 Going to the farm for HP.
2024.08.01 Performing double digestion and using DNA ligase to connect the 5’ and 3’ homologous regions, completing the construction of the knockout vector.
2024.08.02 Completing the initial model of the anchor protein.
2024.08.03 Modifying the Safety form based on feedback.
2024.08.03 Accepting SDU-China's request to come to the school for HP.
2024.08.05 PCR amplification of the knockout vector to obtain linear knockout fragments.
2024.08.06 Agarose gel electrophoresis identification of PCR products.
2024.08.06 Publishing the first mangrove podcast.
2024.08.07 Publishing the second mangrove podcast.
2024.08.08 Optimizing the carbon and nitrogen sources in the culture medium.
2024.08.09 Starting to make the Project Promotion Video.
2024.08.09 Project name confirmed as BEST-ABfree (β-glucan efficient synthesis technology to reduce antibiotic utilization).
2024.08.10 Improving the anchor protein model.
2024.08.10 Picking single colonies and inoculating into HC medium.

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| ![](https://static.igem.wiki/teams/5314/notebook/16.png) |
| Figure 15: Anchor Protein |

2024.08.11 Wiki production with clickable bubble effect.
2024.08.12 Publishing the antibiotic history podcast.
2024.08.12 Confirming iGEM's participation in the Central China Regional Exchange Conference.
2024.08.14 Discussing modeling with CAU-China, NJTECH-China, and SDU-China.
2024.08.15 Completing the compilation of The Biosecurity and Bioethics Whitepaper.

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| ![](https://static.igem.wiki/teams/5314/notebook/17.png) |
| Figure 16: Biosecurity and Bioethics Whitepaper |

2024.08.16 Reaching an OD600nm of 1.1.
2024.08.16 Performing PEG transformation and culturing on antibiotic medium.
2024.08.17 Obtaining transformants.
2024.08.18 Communicating with the advisor, deciding to further develop the Art based on Nezha.
2024.08.18 Re-screening the transformants.
2024.08.20 Receiving a named invitation letter.
2024.08.20 Extracting DNA from marine yeast for genotyping.
2024.08.21 Comparing the relative expression levels of the pullulan gene in knockout and wild-type strains.
2024.08.21 Establishing the 2025 OUC-Haide Recruitment Group.
2024.08.22 Performing RT-qPCR to confirm the decreased relative expression of the pullulan gene in the knockout strain.
2024.08.23 Performing HPLC analysis of pullulan content changes.
2024.08.24 Using cre-loxp technology for antibiotic resistance removal to ensure safety.
2024.08.26 PCR identification of successful antibiotic resistance removal.
2024.08.26 Establishing a fusion model of fluorescent protein and anchor protein.
2024.08.27 Starting to write various parts of the text.
2024.09.03 Establishing a model of the interaction between fusion protein and cell membrane.
2024.09.05 Sending the recruitment push to the freshmen group.
2024.09.05 Sending the recruitment push to the school's official account.
2024.09.08 Designing the Home Page.
2024.09.11 Completing the optimization of the carbon and nitrogen sources in the culture medium.
2024.09.13 Testing and improving the Hardware.
2024.09.17 Starting to construct the wiki.
2024.09.20 Completing the Hardware.

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| ![](https://static.igem.wiki/teams/5314/notebook/18-new.png) |
| Figure 17: Hardware Diagram |

2024.09.22 Completing the writing of various texts.
2024.09.27 Applying for a French visa.

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| ![](https://static.igem.wiki/teams/5314/notebook/19.png) |
| Figure 18: Group photo of some members |

**2024.09.27 Completing the NoteBook.**

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{% extends "testlayout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

# Engineering

Catalog

## Project Design Cycle

#### CYCLE 1: Preventing Gene Leakage

1. Design

With the emergence and development of synthetic biology, genetically modified microorganisms are increasingly used in biomedical, industrial, and environmental applications. To prevent engineered microorganisms from leaking into the natural environment and to stop engineered genetic material from spreading to unintended host cells, it is essential to develop safe and reliable methods to inhibit their proliferation. Methods to prevent leakage of engineered microorganisms include utilizing engineered nutrient-deficient strains for containment, inducing cell death through suicide switches for active containment, and barriers to gene flow, among others [1]. The most commonly used method is the toxin gene suicide switch. Therefore, we initially designed and incorporated a suicide switch into our engineered strains to ensure biosafety.

2. Build

We drafted a gene construct containing a standard suicide switch, where the toxin gene will be expressed in non-laboratory environments to induce cell death.

3. Test

Based on our design, we predicted that our suicide switch would function normally in BZ-11. Before conducting actual laboratory experiments, our Human Practice (HP) team communicated with food regulatory agencies, feed manufacturers, and consumers to assess the acceptability of this system in animal feed production.

4. Learn

During HP interviews with food regulatory agencies, feed manufacturers, and consumers, we were informed that the use of toxin genes and antibiotic resistance markers in food-related products is unacceptable. Regulatory agencies emphasized that strains containing toxin proteins or resistance genes do not meet food safety approval standards. Moreover, consumers expressed significant concerns about the presence of such elements in food.

In light of this feedback, we contacted our instructor to seek possible solutions and ultimately decided to abandon the use of the suicide switch before conducting any practical experiments. We shifted to a "Resistance Removal" method, which ensures that the antibiotic resistance marker disappears in the final engineered strains through the Cre-loxP system, thereby avoiding the leakage of antibiotic resistance genes into the environment. This approach maintains the necessary control and safety requirements while addressing regulatory and consumer concerns, allowing us to proceed without using toxin genes.

## Dry Lab & Wet Lab Cycle

#### CYCLE 1: Constructing the Global Metabolic Network

1. Design

Metabolic networks are interconnected biochemical pathways within living cells that assemble the components or compounds required for cellular functions or generate energy and materials through the breakdown of biomolecules. Enzymes act as catalysts in the vast majority of reactions, thus playing a central role in metabolic networks, and the interactions of their components determine the dynamic behavior of the entire network [2]. The presence of numerous uncertainties in biological systems complicates the study of biological metabolic networks, making it a complex and vast undertaking. To better understand our chassis organism, *Aureobasidium melanogenum* BZ-11, modeling in systems biology aids our comprehension of metabolic mechanisms and provides essential insights for our genetic modifications.

Genome-scale metabolic models (GEMs) are formed using gene-protein-reaction (GPR) associations based on genomic annotation data and experimental information. These models encompass system information such as metabolites, reactions, genes, and compartments, describing the metabolic network of an entire biological system and predicting metabolic fluxes of system metabolites through optimization techniques [3].

2. Build

Since *Aureobasidium melanogenum* BZ-11 currently lacks a genome-scale metabolic network, we compared it with the closest species available in databases based on the phylogenetic tree of Ascomycota species [4], identifying *Aspergillus niger*’s GEMs network.

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| ![](https://static.igem.wiki/teams/5314/engineering/1.jpg) |
| Figure 1: The phylogenetic tree of Ascomycota species, adapted from previous work [4]. |

3. Test

We analyzed the GEMs network of *Aspergillus niger* and attempted to fit the known pathways of *Aureobasidium melanogenum* BZ-11 one by one.

4. Learn

We found that many pathways in the genome-scale metabolic network of *Aspergillus niger* do not match those known for Aureobasidium melanogenum. For example, in melanin synthesis, *Aureobasidium melanogenum* employs the dihydroxynaphthalene (DHN) pathway, while *Aspergillus niger* utilizes the L-3,4-dihydroxyphenylalanine (L-DOPA) pathway [5]. This significantly impacted our analysis. Additionally, we realized that this method of searching pathways one by one is inefficient and undesirable. Therefore, we decided to construct the metabolic network ourselves. After gathering information, we discovered that building a GEM-level metabolic network requires whole-genome sequencing of the strain. However, this posed a challenge for us. Given the current situation and the problems encountered, we consulted our instructor, who agreed with our solution and generously provided us with the whole-genome sequencing information for the *Aureobasidium melanogenum* P16 strain, which greatly assisted us.

#### CYCLE 2: Predicting the Relationship Between β-glucan Yield and BZ-11 Growth

1. Design

Since the *Aureobasidium melanogenum* BZ-11 strain lacks a genome-scale metabolic network and it's difficult to use the metabolic networks of closely related species, we chose to use the whole-genome sequencing information of the *Aureobasidium melanogenum* P16 strain to construct a GEM for facilitating the prediction of the relationship between β-glucan yield and its growth.

After reviewing literature and gathering information, we decided to use the CarveMe reconstruction tool. CarveMe is an automated reconstruction tool for genome-scale metabolic models that uses the Diamond sequence aligner to compare sequences in databases. It can infer an organism's uptake/secretion capabilities solely from genetic evidence, thereby constructing metabolic networks, making it particularly suitable for organisms that cannot be cultured under well-defined media [6].

2. Build

We constructed a genome-scale metabolic network using the CarveMe reconstruction tool by running it in Linux Ubuntu.

3. Test

We analyzed the constructed GEMs network using flux balance analysis (FBA) methods, simulating it with COBRApy, and performed two optimizations with β-glucan and growth generation as objective functions.

4. Learn

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| ![](https://static.igem.wiki/teams/5314/model/fig3-new.png) |
| Figure 2: Relationship Between β-glucan and Growth Flux |

From the analysis results, we found that the β-glucan flux does not affect growth until it reaches 175 mmol/gDW/h. After reaching a maximum yield of 667 mmol/gDW/h, growth decreases by about 87%. Therefore, to maximize β-glucan production, it is necessary to minimize the impact on cell growth and reduce system load. Additionally, we still could not determine which specific secondary metabolites' synthesis would affect β-glucan synthesis. To gain insights, we reviewed past iGEM teams' wikis and ultimately found further analysis methods in the 2023 iGEM Leide team's wiki [7].

#### CYCLE 3: Analyzing Secondary Metabolites That Significantly Affect β-glucan Synthesis

1. Design

The determination and study of in vivo metabolic flux are known as flux analysis, which plays a central role in metabolic engineering and is a fundamental determinant of cellular physiology, providing key parameters for various pathways in metabolism [8]. OptForce is a computational optimization framework based on flux variability analysis (FVA) that aims to identify all potential genetic modifications leading to the overproduction of desired metabolites in microorganisms through flux balance analysis (FBA). A primary challenge in metabolic engineering is to maximize the conversion of renewable resources into useful products. OptForce improves these methods by integrating experimental flux data from wild-type strains and focusing on which fluxes must change to achieve overproduction goals. It compares the range of metabolic fluxes between wild-type and overproducing strains to determine which reactions must be increased (MUSTU) or decreased (MUSTL) to meet target yields. Once all necessary flux changes are identified, OptForce employs a dual-layer optimization program to determine the minimal set of genetic interventions required for the expected overproduction (FORCE set). This method ensures efficient strain engineering by precisely identifying key modifications while minimizing the number of genetic changes needed for the target phenotype [9].

2. Build

Inspired by the 2023 iGEM Leide team, we used the OptForce algorithm based on the MathWorks COBRA Toolbox for our construction [7].

3. Test

Through reasonable constraints and calculations, we obtained the first-order Must and second-order Must sets, and simulated the OptForce algorithm, returning the top 10 single reaction force sets.

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| ![](https://static.igem.wiki/teams/5314/model/fig5-new.png) |
| Figure 3: OptForce result |

4. Learn

We ultimately focused on the pullulan\_01 pathway for the synthesis of the secondary metabolite pullulan. We also assessed the preceding reaction, pullulan\_00, considering it to significantly affect β-glucan synthesis as well. Meanwhile, our wet experiments revealed the presence of pullulan through the analysis of extracellular polysaccharide components, indicating that knocking out this pathway could enhance β-glucan yield while avoiding impurity issues during the purification of extracellular polysaccharides. To improve the quality of the final product, we decided to knock out the melanin synthesis pathway; however, upon performing FBA simulations and gene knockout simulations again, we found that controlling these sub-reactions was not sensitive, making it difficult to draw conclusions from the simulations.

#### CYCLE 4: Construction of Local Metabolic Network (LMN)

1. Design

After analyzing the GEMs metabolic network, we narrowed our focus to the metabolic pathways of β-glucan, melanin, and pullulan. Based on this, we decided to use traditional kinetics for modeling to conduct a more refined analysis and further identify the key reactions we want to knock out.

Simbiology is widely used for pharmacokinetic simulations and serves as an efficient tool for simulating small metabolic systems. It allows observation of the network's dynamic behavior and provides more refined control and optimization strategies [10]. Biochemical network simulation can dynamically observe the changes of various substances over a certain time scale, aiding our understanding of the network's dynamic behavior. Considering that there are not many synergistic effects in the network, we employed a lightweight local sensitivity analysis to identify the parts of the system with high sensitivity.

2. Build

Using MathWorks-based Simbiology for kinetic modeling, we ignored enzyme inhibition and activation effects, focusing on which reactions enzymes mainly participate in to further simplify the model load.

3. Test

We observed the dynamic changes of system substances at low concentrations, analyzing the yield of the target product under different parameters. We iterated the parameters of the first three reactions that showed high sensitivity in the synthesis of pullulan and melanin, observing the corresponding yields and the degree of fluctuation.

4. Learn

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| ![](https://static.igem.wiki/teams/5314/model/fig9-new.png) |
| Figure 4: Analysis of the Melanin Synthesis Pathway (LSA) |

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| ![](https://static.igem.wiki/teams/5314/model/fig10-new.png) |
| Figure 5: Analysis of the Pullulan Synthesis Pathway (LSA) |

The sensitivity values for Kf\_26 involved in pullulan synthesis and Kf\_25 involved in melanin synthesis were the highest. Our experiments will focus on the melain\_01 and pullulan\_01 reactions for gene knockout. Ultimately, we decided to target the Ags2 and PKS genes for knockout in our wet experiments. However, due to the compact nature of our experimental section, some parameters were not fitted, and we currently cannot achieve complete quantitative analysis. Subsequently, we compared the results of the LMN back to the GEMs, seeking to visualize and analyze the deficiencies in GMN analysis, discovering many key pathways that were overlooked. This indicates the need for further model optimization and the addition of constraints for in-depth exploration in the future.

#### CYCLE 5: Screening of Anchor Proteins

1. Design

*Vitreoscilla* Hemoglobin (VHb) is a type of hemoglobin derived from *Vitreoscilla*, which serves as a terminal electron transport chain component, enhances the survival of microorganisms under low oxygen conditions, and promotes total protein synthesis in cells. It has been applied in industrial fermentation [11]. To accurately localize and attach VHb to the outer side of the cell wall of *A. melanogenum* BZ-11 to enhance oxygen uptake, we designed to combine the cell wall anchoring protein with VHb to ensure its localization at the cell wall.

Cell wall protein is a mannan protein consisting of an N-terminal signal peptide and a C-terminal anchoring sequence, where the N-terminal is responsible for protein secretion, and the C-terminal anchors the protein to the cell wall. This protein can bind to the structural 1,6-β-glucan present in the cell wall [12]. We also identified a cell wall protein anchoring sequence in *A. melanogenum* BZ-11. Additionally, we found cell wall proteins from other species, namely *Yarrowia lipolytica* (CWP) and *Ogataea angusta* (Sed, Tip), and compared their sequences with those from our species to score and find the most suitable sequence copy for *A. melanogenum* BZ-11 as the final sequence.

2. Build

We retrieved the GPI-CWP sequences of these species from NCBI and successfully predicted the N-terminal signal peptide and C-terminal anchoring region of these sequences using the SignalP and big-PI Fungal Predictor tools. We then fused the predicted N-terminal and C-terminal sequences with the target protein.

3. Test

We used SignalP to simulate the cleavage site and determine if any changes occurred. By gradually adding amino acids and comparing with the recombined sequence of the target protein, we identified the sequence with the least polarity change.

4. Learn

Based on the sequence alignment results, we preliminarily determined to use the AM.CWP protein from *A. melanogenum* BZ-11 as the anchoring protein. However, the sequence alignment data alone lacked sufficient convincing power and required further validation. Next, we plan to connect the cell wall protein sequence with EGFP to construct a plasmid for introduction into *A. melanogenum* BZ-11 and check the fluorescence intensity on the cell wall. To ensure the stability and functionality of the protein for better validation of EGFP, we also predicted the EGFP sequence by gradually adding amino acids, resulting in the addition of three amino acids (AYI) compared to the original sequence.

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| ![](https://static.igem.wiki/teams/5314/engineering/6.png) |
| Figure 6: EGFP Signal Peptide Prediction |

#### CYCLE 6: Knockout of *PKS* Gene and *AGS2-1*, *AGS2-2* Genes

1. Design

In previous work, we confirmed that the *PKS* gene is a key gene for melanin synthesis in *A. melanogenum* BZ-11, while the *AGS2-1* and *AGS2-2* genes are essential for pullulan synthesis in the same organism.

We amplified the 5' arm and 3' arm sequences of the *PKS*, *AGS2-1*, and *AGS2-2* genes, and constructed linearized gene knockout vectors pFL4A-NAT-loxp-PKS, pFL4A-NAT-loxp-△AGS2-1, and pFL4A-NAT-loxp-△AGS2-2 using the *NAT* gene as a marker. These vectors were then sequentially introduced into the organism.

2. Build

The strains containing the knockout vectors were able to grow on media containing nourseothricin

3. Test

We verified the knockout effects using colony PCR.

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| ![](https://static.igem.wiki/teams/5314/engineering/7-new.jpg) |
| Figure 7: Electrophoresis to detect knockout results |

4. Learn

From the colony PCR results, the final BZW△ags2-1/2 strains showed that the *PKS*, *AGS2-1*, and *AGS2-2* genes were all knocked out. However, the knockout of these genes does not necessarily mean that the metabolic pathways have been completely blocked; further validation is needed using high-performance liquid chromatography and infrared spectroscopy analysis.

## Harware Cycle

#### CYCLE 1: More Useful Hardware

1. Design

During our Human Practices activities, we identified a widespread issue through conversations with aquaculture farmers: feed sinking to the bottom leads to significant waste, as much of the feed is not efficiently utilized, potentially limiting the growth of aquatic animals. Moreover, feed sinking and overfeeding can cause severe water pollution, sometimes even resulting in the mass death of aquatic species. Thus, "precision feeding" plays a crucial and indispensable role in improving both production efficiency and economic outcomes in aquaculture.

To prevent feed from sinking to the bottom, we designed a feed-holding dispenser composed of three parts: a floater, a connecting rod, and a feed holder. Since most aquatic animals do not deliberately seek food at the bottom and prefer fresh feed, we can place the feed directly on the holder, ensuring that it remains available to the animals before sinking. The aquatic animals can easily consume the feed from the holder (Fig. 8).

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| ![](https://static.igem.wiki/teams/5314/hardware/image3-new.png) |
| Figure 8: Schematic diagram of hardware usage |

2. Build

Using SolidWorks, we created a hardware model where the floater is a sphere with a diameter of 3 cm and a thickness of 2 mm, the connecting rod is 8 cm long with a 3 mm diameter, and the feed holder is a curved disc with a diameter of 10 cm and a thickness of 3 mm. We then created physical models using 3D printing methods (Fig. 9)

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| ![](https://static.igem.wiki/teams/5314/hardware/img45.png) |
| Figure 9: Feeder modeling and 3D-printed model |

3. Test

We simulated a water environment and then tested the hardware to determine the effectiveness of the hardware in use (Vedio 1).

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| Video 1: Feed tossing demonstration by the feeder |

4. Learn

During our feed placement experiments, we encountered an issue: it was challenging to accurately determine where to feed in an open water surface, resulting in a significant portion of the feed missing the holder and sinking to the bottom. To address this, we sought to design a method that would restrict the feeding area, ensuring that all feed could be precisely placed onto the feed holder, rather than sinking to the bottom again.

As a solution, we added a ring around the floater with a diameter smaller than that of the feed holder (8cm), to delineate the feeding area (Fig. 10). This ensures that the feed remains within the designated range and does not sink, effectively mitigating the issue of feed waste (Vedio 2).

In this Engineering process, we realized the importance of taking a comprehensive approach to hardware design to ensure it can be effectively applied in real-world scenarios.

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| ![](https://static.igem.wiki/teams/5314/hardware/image678new.png) |
| Figure 10: Feeder modeling and 3D-printed model |

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| Video 2: Feed tossing demonstration by the feeder |

#### CYCLE 2: More reliable hardware Design

1. Design

When feeding we ah find that the upper floater takes up a lot of space on the surface of the water and is a significant hindrance to feeding. Therefore, we improved the floater design by combining the range-defining ring and the floating sphere into a single floating ring (Fig. 11). This modification significantly reduces the surface area occupied by the floater while maintaining its functional effectiveness.

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| ![](https://static.igem.wiki/teams/5314/hardware/image910.png) |
| Figure 11: Feeder modeling and 3D-printed model |

2. Build

We then created physical models using 3D printing methods (Fig. 11).

3. Test

To better reflect real-world conditions, we introduced a water flow simulator to replicate water movement in aquaculture environments and assess the effectiveness of our device. Following this, we conducted a feeding simulation.

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| Video 3: Feed tossing demonstration by the automatic feeder |

4. Learn

During the simulation, we observed that without being anchored, the feeder would drift with the water current, resulting in unstable feeding positions (Video 3). This instability could make it difficult for aquatic animals to locate the feeding area, or cause the feeder to drift away from the group, leading to inefficiencies in feeding.

To address this issue, we attached a stabilizing rod to the feeder, ensuring that it remains stationary in flowing water. Furthermore, we replaced the stabilizing rod with an adjustable rope, allowing for flexibility in various aquaculture environments while maintaining the feeder’s stability and precision (Fig. 13 and Video 4).

We recognized that when conducting hardware simulations, it is essential to closely mimic real-world conditions. This approach helps us more easily identify potential issues that may arise during application, thereby increasing the feasibility of its use.

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| ![](https://static.igem.wiki/teams/5314/hardware/image1213.png) |
| Figure 13: Feeder modeling and 3D-printed model |

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| Video 4: Feed tossing demonstration by the automatic feeder |

## References

[1] Moe-Behrens, G. H. G., Davis, R., & Haynes, K. A. (2013). Preparing synthetic biology for the world. *Frontiers in Microbiology*, 4. https://doi.org/10.3389/fmicb.2013.00005
[2] Dräger, A., & Planatscher, H. (2013). Metabolic Networks. *Encyclopedia of Systems Biology*, 1249–1251. https://doi.org/10.1007/978-1-4419-9863-7\_1277
[3] Gu, C., Kim, G. B., Kim, W. J., Kim, H. U., & Lee, S. Y. (2019). Current status and applications of genome-scale metabolic models. *Genome Biology*, 20(1), 121. https://doi.org/10.1186/s13059-019-1730-3
[4] Xiao, D., Driller, M., Dielentheis‐Frenken, M., Haala, F., Kohl, P., Stein, K., Blank, L. M., & Tiso, T. (2024). Advances in *Aureobasidium* research: Paving the path to industrial utilization. *Microbial Biotechnology*, 17(8). https://doi.org/10.1111/1751-7915.14535
[5] Jia, S.-L., Chi, Z., Chen, L., Liu, G.-L., Hu, Z., & Chi, Z.-M. (2021). Molecular evolution and regulation of DHN melanin-related gene clusters are closely related to adaptation of different melanin-producing fungi. *Genomics*, 113(4), 1962–1975. https://doi.org/10.1016/j.ygeno.2021.04.034
[6] Machado, D., Andrejev, S., Tramontano, M., & Patil, K. R. (2018). Fast automated reconstruction of genome-scale metabolic models for microbial species and communities. *Nucleic Acids Research*, 46(15), 7542–7553. https://doi.org/10.1093/nar/gky537
[7] Modeling | Leiden - iGEM 2023. (2023). Igem.wiki. https://2023.igem.wiki/leiden/model
[8] Stephanopoulos, G. (1999). Metabolic Fluxes and Metabolic Engineering. *Metabolic Engineering*, 1(1), 1–11. https://doi.org/10.1006/mben.1998.0101
[9] Ranganathan, S., Suthers, P. F., & Maranas, C. D. (2010). OptForce: An Optimization Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions. *PLoS Computational Biology*, 6(4), e1000744. https://doi.org/10.1371/journal.pcbi.1000744
[10] SimBiology. (2024). Mathworks.cn. https://ww2.mathworks.cn/products/simbiology.html
[11] Zhang, L., Li, Y., Wang, Z., Xia, Y., Chen, W., & Tang, K. (2007). Recent developments and future prospects of *Vitreoscilla* hemoglobin application in metabolic engineering. *Biotechnology Advances*, 25(2), 123–136. https://doi.org/10.1016/j.biotechadv.2006.11.001
[12] Jaafar, L., & Zueco, J. (2004). Characterization of a glycosylphosphatidylinositol-bound cell-wall protein (GPI-CWP) in *Yarrowia lipolytica*. *Microbiolog*, 150(1), 53–60. https://doi.org/10.1099/mic.0.26430-0

{% endblock %}

{% extends "testlayout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# Experiments

Catalog

## Medium Preparation

## Plasmid Establishment

## *E.coli* Experiments

## Yeast Experiments

## Source of Experimental Materials

{% endblock %}

{% extends "testlayout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Results

Catalog

## Characterisation of strain BZ-11

We ultimately selected *Aureobasidium melanogenum* BZ-11 as our chassis strain. During the cultivation of the strain, certain characteristics of BZ-11 demonstrated its potential to serve as a chassis strain for the synthetic β-glucan pathway, while other traits also presented challenges for its utilization.

#### Exopolysaccharides and melanin

Colonies of the *Aureobasidium melanogenum* BZ-11 strain were cultivated on both YPD (Yeast Peptone Dextrose) and PDA (Potato Dextrose Agar) media. The colony structure and microscopic observation are depicted in the figures below.

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| ![](https://static.igem.wiki/teams/5314/result/1-new.png) |
| Figure 1: Colony Morphology and Microscopic Observations of BZ-11. (a) Colony morphology of BZ-11 on YPD medium. (b) Colony morphology of BZ-11 on PDA medium. (c) Microscopic observation of BZ-11. |

Upon examination of the images, it is evident that BZ-11 secretes a substantial amount of extracellular polysaccharides when cultured on YPD medium, classifying it as a high-yielding strain for extracellular polysaccharide production. After cultivation on PDA plates, a pronounced brown-yellow halo at the colony's edge is observed, leading to the inference that BZ-11 is capable of secreting melanin extracellularly. This hypothesis was subsequently confirmed by a review of the relevant literature, which showed that *Aureobasidium melanogenum* not only produces melanin, but has even been used to synthesise it efficiently[1][2]. The incorporation of melanin into the extracellular polysaccharides poses a challenge for separation, thus becoming a limiting factor for the BZ-11 strain. Microscopic examination revealed the morphological characteristics and reproductive modes of individual cells.

#### Extracellular β-glucan

The type strain 110.374 of *Aureobasidium spp*. and the strain BZ-11 were cultivated at the same situation. At the same time, the extracellular β-glucan, extracellular polymeric substances (EPS), cell wall β-glucan, and biomass titer of both strains were measured. The results are depicted in the figure below.

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| ![](https://static.igem.wiki/teams/5314/result/2-new.png) |
| Figure 2: Comparative Analysis of Strains 110.374 and BZ-11. |

Upon comparison of the biomass titers of the two strains, no significant differences were observed, indicating that their growth conditions and metabolic activities are fundamentally similar. This implies that the differences in biomass have a negligible impact on the experimental conclusions.

Under these premises, the EPS titer of BZ-11 was found to be significantly higher than that of 110.374, suggesting that BZ-11 possesses a stronger capacity for extracellular polysaccharide synthesis under the same conditions. The cell wall β-glucan content of both strains is similar, but BZ-11's EPS contains β-glucan similar in amount to that in the cell wall, whereas 110.374 does not secrete extracellular β-glucan. This indicates that BZ-11 has an inherent advantage in β-glucan production. Moreover, the extraction of extracellular β-glucan is simpler, more environmentally friendly, and BZ-11 has a clear potential for β-glucan production.

It is worth noting that the yield of extracellular polysaccharides in BZ-11 is greater than the content of extracellular β-glucan, indicating that the extracellular polysaccharides contain other types of polysaccharides.

#### Pullulan

Through the study of the manuscript, we observed that the fermentation process of *Aureobasidium spp*. often accompanies the production of pullulan[3]. Therefore, an enzymatic hydrolysis method was employed to detect the pullulan in the extracellular polysaccharides of the BZ-11 strain. Utilizing pullulanase, pullulan can be decomposed into maltotriose.

High-performance liquid chromatography (HPLC) analysis was conducted on maltotriose standards, pullulan standards, the supernatant of polysaccharide fermentation from the BZ-11 strain, and β-glucan standards, with the results depicted in the figure below.

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| ![](https://static.igem.wiki/teams/5314/result/2d-new.png) |
| Figure 3: HPLC profiles of pullulanase hydrolysates of pullulan standard, glucan standard, exopolysaccharides produced by BZ-11, as well as maltotriose standard without pullulanase hydrolysates. |

Analysis of the maltotriose standard revealed a peak at approximately 9 minutes, which corresponds to maltotriose. Under these conditions, the enzymatic hydrolysis of pullulan standard also yielded a peak at the same retention time, indicating that the pullulanase used in the experiment is active and effective. The β-glucan standard solution did not exhibit a peak after enzymatic hydrolysis, suggesting that the presence of β-glucan does not generate maltotriose that could interfere with the experimental results. Consequently, the appearance of a maltotriose peak in the enzymatic hydrolysate of the BZ-11 strain's polysaccharide fermentation supernatant indicates that, in addition to β-glucan, the extracellular polysaccharides also contain pullulan.

## Blocking the biosynthetic pathways of melanin and pullulan

#### Identification of key genes

In order to ensure the purity of β-glucan and to reduce the difficulty of extraction, we attempted to knock out the genes encoding key proteins in the melanin and pullulan synthesis pathways of the BZ-11 strain. Based on the genomic information of the BZ-11 strain and the modeling analysis of the dry laboratory group, the polyketide synthase gene *PKS*, which is crucial for melanin synthesis, and the pullulan synthesis key genes *AGS2-1* and *AGS2-2*, were ultimately identified for knockout.

#### Construction of knockout vectors and Cre recombinase expression vectors

We designed a gene knockout strategy using homologous recombination to disrupt the synthesis pathways of impurities. Consequently, we amplified the FL4A-NAT-loxp plasmid along with the 3' and 5' homologous arms of the target gene to construct the gene knockout vector. The primer sequences designed for amplifying the homologous arms are presented in the table below.

Eventually, three knockout vectors were constructed: FL4A-NAT-loxp-*PKS*, FL4A-NAT-loxp-*AGS2-1*, and FL4A-NAT-loxp-*AGS2-1*, as shown in Figure 4.

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| ![](https://static.igem.wiki/teams/5314/result/4-new.png) |
| Figure 4: Gene Knockout Vectors Containing loxp Sites |

Concurrently, a Cre enzyme expression plasmid was designed, as depicted in Figure 5, for the removal of the NAT gene that was integrated into the genome following homologous recombination.

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| ![](https://static.igem.wiki/teams/5314/result/5.png) |
| Figure 5: Cre Enzyme Expression Vector for Eliminating Antibiotic Resistance |

#### Knockout and validation experiments

Firstly, the FL4A-NAT-loxp-*PKS* knockout vector was introduced into BZ-11 via protoplast transformation. At this juncture, if the knockout vector was successfully transformed and homologous recombination occurred, a strain lacking the *PKS* gene and incapable of producing melanin should be obtained, which we designate as BZW. Subsequently, genetic knockout validation for BZW was carried out. Primers were designed to amplify the upstream and downstream fragments of the *PKS* gene, using the genomic DNA of BZ-11 and BZW as templates. PCR amplification was performed on both the BZ-11 and the knockout strain BZW, with the results shown in Figure 6 (A).

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| ![](https://static.igem.wiki/teams/5314/result/6-new.png) |
| Figure 6: PCR validation experiments for triple gene knockout. (A) PCR validation for *PKS* gene knockout; (B) PCR validation for *AGS2-1* gene knockout; (C) PCR validation for *AGS2-1* gene knockout. (Adjacent to BZ-11 in each group is the 1 kb DNA ladder: from top to bottom 10 kb, 8 kb, 7 kb, 6 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb) |

The amplified band for the BZ-11 strain is 4715 bp, which corresponds closely with the length of the *PKS* gene; the amplified band for the BZW strain is 1914 bp, which corresponds closely with the length of the NAT gene. This indicates that the *PKS* gene in the BZW strain has been completely knocked out.

Thereafter, the Cre-loxP system was utilized to excise the introduced NAT gene from the genome. Subsequently, the FL4A-NAT-loxp-*AGS2-1* knockout vector was introduced. Following the same validation procedure as for the *PKS* gene, repeat PCR validation experiments were conducted, with the results shown in Figure 6 (B). The validation results for *AGS2-1* are depicted in Figure 6 (C).

Based on the results of the PCR validation experiments, it can be concluded that the *PKS*, *AGS2-1*, and *AGS2-1* genes have been completely knocked out.

## Validation of the properties of strain BZWΔags2-1/2

#### Melanin

In PDA medium, streak cultivation was performed for both the BZ-11 and BZW strains, with the results illustrated in the figure below.

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| ![](https://static.igem.wiki/teams/5314/result/7-new.png) |
| Figure 7: Colony morphology of BZ-11 and BZW strains. (a) Colony characteristics of the BZWΔags2-1/2 strain after cultivation; (b) Extracellular polysaccharides produced by the collected BZWΔags2-1/2 strain; (c) Colony characteristics of the BZ-11 strain after cultivation; (d) Extracellular polysaccharides produced by the collected BZ-11 strain. |

After the *PKS* gene knockout, melanin synthesis was completely blocked, resulting in a significant change in the color of the fermentation liquid and extracellular polysaccharides. The original strain A. melanogenum BZ-11 cultivated on PDA medium exhibited black colonies (Figure 7c and Figure 7d), whereas the A. melanogenum BZW strain showed white colony morphology without melanin production (Figure 7a and Figure 7b).

#### Pullulan

High-performance liquid chromatography (HPLC) analysis was conducted on the enzymatic hydrolysates of the exopolysaccharides from the fermentation supernatant of BZWΔags2-1/2, and the results are depicted in Figure 8.

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| ![](https://static.igem.wiki/teams/5314/result/3d-new.png) |
| Figure 8: HPLC profiles of pullulanase hydrolysates of pullulan standard, glucan standard, and exopolysaccharides produced by BZ-11 and BZWΔags2-1/2, as well as the maltotriose standard without pullulanase hydrolysates. |

Using the fermentation supernatant of the original strain BZ-11, pullulan standard, β-glucan standard, and maltotriose standard as controls for comparative analysis, the results are as shown in the figure. The enzymatic hydrolysate of the exopolysaccharides from the fermentation supernatant of BZWΔags2-1/2 lacks a maltotriose peak, whereas the enzymatic hydrolysate from BZ-11 exhibits a maltotriose peak. Since pullulanase can hydrolyze pullulan to produce maltotriose but cannot hydrolyze β-glucan, this indicates that the BZWΔags2-1/2 strain is no longer capable of synthesizing pullulan.

In addition to the aforementioned analyses, Fourier-transform infrared spectroscopy (FTIR) was also conducted on the extracellular β-glucan, cell wall β-glucan of BZWΔags2-1/2, and extracellular β-glucan of BZ-11. The detection was performed using a Nicolet Nexus 470 FTIR spectrometer with a scanning range of 4000-400 cm⁻¹, a resolution of 2 cm⁻¹, and 16 scans, and the results are depicted in Figure 9.

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| ![](https://static.igem.wiki/teams/5314/result/9-new.png) |
| Figure 9: FTIR spectra of the exopolysaccharides β-glucan, cell wall β-glucan produced by BZWΔags2-1/2, and cell wall β-glucan produced by BZ-11. |

According to the FTIR analysis, all three types of polysaccharides exhibited a broad and intense O-H stretching vibration absorption peak near 3321 cm⁻¹, indicating their typical saccharide characteristics. Additionally, the C-H stretching vibration absorption peak was observed at 2919 cm⁻¹. It is also noteworthy that the extracellular polysaccharides of the original BZ-11 strain showed characteristic absorption peaks for α-configuration at 850 cm⁻¹ and β-configuration at 890 cm⁻¹, whereas the extracellular β-glucan and cell wall β-glucan of the engineered BZWΔags2-1/2 strain only exhibited a β-configuration absorption peak at 890 cm⁻¹. This suggests that the extracellular and cell wall β-glucan samples of the BZWΔags2-1/2 strain are free from contamination with α-configuration glycosidic bond polysaccharides, while the extracellular polysaccharides of the original BZ-11 strain contain both β-glucan and α-configuration glycosidic bond polysaccharides. As pullulan is an α-configuration glycosidic bond polysaccharide, this result is consistent with the high-performance liquid chromatography (HPLC) analysis, further confirming that the engineered strain no longer synthesizes pullulan.

To ensure the authenticity and reliability of the results, we also conducted 1H-13C HSQC (heteronuclear single quantum coherence) analysis using a JEOL JNM-ECP 600M HZ NMR spectrometer, and the results are shown in Figure 10.

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| ![](https://static.igem.wiki/teams/5314/result/10-new.png) |
| Figure 10: 1H-13C HSQC spectrum of exopolysaccharides β-glucan produced by BZWΔags2-1/2. |

As can be seen from the figure, the terminal carbon signal appears at 103 ppm, and the terminal hydrogen signal appears at a chemical shift of 4.5 ppm, which allows us to identify the extracellular polysaccharide as β-glucan. Additionally, the peaks at 86.84 ppm, 77.38 ppm, 73.34 ppm, 70.84 ppm, and 61.44 ppm correspond to the positions of carbons C3, C5, C2, C4, and C6, respectively. This further confirms that the extracellular polysaccharide produced by the engineered BZWΔags2-1/2 strain is β-glucan.

The consistency of the results from HPLC, infrared spectroscopy, and NMR spectroscopy indicates that after the knockout of the *AGS2-1* and *AGS2-1* genes, the extracellular polysaccharide product of BZWΔags2-1/2 no longer contains pullulan, and is a pure β-glucan.

## Optimization of the growth medium for BZWΔags2-1/2

To further increase the β-glucan yield of BZWΔags2-1/2, the medium was optimised.

#### Nitrogen source

Through optimization of the nitrogen source, the results are shown in Figure 11.

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| ![](https://static.igem.wiki/teams/5314/result/11-new.png) |
| Figure 11: Optimization of the nitrogen source in the culture medium. |

When NaNO₃ was used as the nitrogen source, the extracellular β-glucan yield of the engineered BZWΔags2-1/2 strain was significantly higher than that in the medium with (NH₄)₂SO₄. Further optimization of NaNO₃ concentration indicated that at NaNO₃ concentrations of 0.1-0.2 g/L, the β-glucan yield was the highest, reaching 25.48-26.97 g/L. At 0.2 g/L NaNO₃, the total β-glucan yield was the highest, reaching 26.97 g/L, with cell wall β-glucan yield at 10.92 g/L and extracellular β-glucan yield at 16.05 g/L.

#### Carbon source

The concentration of the carbon source (glucose) was optimized in a gradient, and the results are shown in Figure 12.

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| ![](https://static.igem.wiki/teams/5314/result/12-new.png) |
| Figure 12: Optimization of the carbon source in the culture medium. |

When the glucose concentration was within the range of 90-140 g/L, the β-glucan yield was 22.11-29.64 g/L. At a glucose concentration of 130 g/L, the β-glucan yield of the engineered BZWΔags2-1/2 strain was the highest, reaching 29.64 g/L, with cell wall β-glucan yield at 11.92 g/L and extracellular β-glucan yield at 17.81 g/L.

#### Composition of the culture medium

The β-glucan yield of the engineered BZWΔags2-1/2 strain after optimization in this experiment has significantly increased, reaching a maximum of 29.64 g/L, which is much higher than the 9.9 g/L in existing technologies. In addition, the engineered strain completely eliminated the production of the byproduct pullulan, demonstrating significant technological progress.
The final optimized culture medium composition is: 90-140 g/L glucose, 0.2 g/L yeast extract, 0.1-0.2 g/L NaNO₃, 7 g/L potassium dihydrogen phosphate, 2.5 g/L sodium phosphate dodecahydrate, 2.5 g/L magnesium sulfate heptahydrate, 0.15 g/L ferric chloride, 0.15 g/L calcium chloride, 0.02 g/L manganese sulfate, and 0.02 g/L zinc sulfate.

## The scale-up cultivation and extraction of β-glucan

#### Scale-up cultivation

Inoculating the engineered BZWΔags2-1/2 strain into a fermenter containing fermentation medium for scaled-up culture and fermentation, continuous monitoring of extracellular glucan, cell wall glucan, cell dry weight, and residual glucose in the fermenter was conducted. The data recorded up to 120 hours were plotted into a line graph, as shown in Figure 13.

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| ![](https://static.igem.wiki/teams/5314/result/13-new.png) |
| Figure 13: Monitoring of extracellular glucan, cell wall glucan, cell dry weight, and residual glucose in the fermenter. |

Upon calculation, it was found that by the 120th hour, the glucose in the medium was essentially completely consumed. At this point, the total content of β-glucan, as well as the contents in the cell wall and extracellularly, all reached their peak values, with slow growth in cell dry weight.

#### Extraction of β-glucan

The engineered BZWΔags2-1/2 strain was inoculated into a fermenter containing fermentation medium and fermented at 28℃-30℃. After fermentation, centrifugation was performed, and the results are shown in Figure 14.

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| ![](https://static.igem.wiki/teams/5314/result/14-new.png) |
| Figure 14: Separation and extraction of extracellular and cell wall β-glucan. |

Extracellular β-glucan was separated from the supernatant, and cell wall β-glucan was separated from the biomass. The method for separating extracellular β-glucan from the fermentation liquid was precipitation with alcohol, and the method for separating cell wall β-glucan from the microbial cells was alkali extraction. After freeze-drying, high-quality β-glucan products were obtained.

## Anchor Protein Efficiency Comparison

#### Result

After reviewing the literature and the work of the dry laboratory group, we ultimately identified three candidate anchor proteins, AM.CWP, AM.SED, and AM.TIP, to assist in the binding of VHb to the cell wall. To compare these three options, we fused them with GFP and constructed the verification plasmids pNATX13-loxp-AM.CWP-EGFP, pNATX13-loxp-AM.SED-EGFP, and pNATX13-loxp-AM.TIP-EGFP, as shown in the figure below.

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| ![](https://static.igem.wiki/teams/5314/result/15.png) |
| Figure 15: pNATX13-loxp-AM.CWP-EGFP |

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| ![](https://static.igem.wiki/teams/5314/result/16.png) |
| Figure 16: pNATX13-loxp-AM.TIP-EGFP |

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| ![](https://static.igem.wiki/teams/5314/result/17.png) |
| Figure 17: pNATX13-loxp-AM.SED-EGFP |

After transformation, fluorescence observation was conducted, with results as shown in Figure 18.

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| ![](https://static.igem.wiki/teams/5314/result/18-new.png) |
| Figure 18: Fluorescence observation of AM.SED, AM.CWP, and AM.Tip as anchor proteins at 48h and 72h |

The fluorescence intensity of all three groups of experiments was enhanced along with the extension of incubation time, but the overall fluorescence intensity was not very high. It was observed that the fluorescence intensity and distribution of fluorescence with AM.CWP was significantly better than that with AM.SED and AM.Tip, and the difference in fluorescence intensity was seen at the cell wall. No significant difference in fluorescence intensity was seen at the cell wall with AM.SED and AM.Tip. Therefore, we also conducted further tests on AM.CWP and the results are shown in Figure 19.

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| ![](https://static.igem.wiki/teams/5314/result/19.png) |
| Figure 19: Fluorescence observation of CWP-EGFP. (A) Bright-field observation at 24h; (B) Observation under 488nm excitation light at 24h; (C) Bright-field observation at 48h; (D) Observation under 488nm excitation light at 48h; (E) Bright-field observation at 72h; (F) Observation under 488nm excitation light at 72h |

The transformed strain 48H exhibited a green fluorescent halo around the cells, which expanded with the extension of cultivation time. By the 72-hour mark, the cells were uniformly green, and it was observable that the fluorescence intensity was not high. Moreover, the difference in fluorescence intensity between inside the cells and on the cell wall was not significant. This observation suggests that the fusion protein constructed could potentially assist VHb in binding to the cell wall of BZWΔags2-1/2, thereby providing the cells with a more oxygen-rich microenvironment and enhancing the production of glucan. But our work still needs to be further optimised.

#### Discussion

A region rich in Ser/Thr structural domain is located near the C-terminal anchoring sequence, and this area contains multiple O-glycosylation sites. The serine or threonine at these sites may covalently bind to glucans on the cell wall. These sites could be crucial for the stable attachment of the anchor protein to the cell wall. In the initial experiments, we did not include this sequence in the design of the fusion protein. This might be one of the reasons for the low fluorescence intensity and the lack of significant difference in fluorescence intensity between the cell wall and the inside of the cell. The next experimental approach is to incorporate the aforementioned sequence into the design and optimize the length of the sequence included.

## Future Work

Firstly, adjust the C-terminal domain of the anchor protein until satisfactory results are achieved, characterized by significantly higher fluorescence intensity on the cell wall compared to the interior of the cell, and a high overall fluorescence intensity. Then, construct an expression vector for VHb using the optimal anchor protein sequence. After transforming the plasmid into BZWΔags2-1/2, perform PCR validation to confirm the insertion of the VHb gene into the genome. Subsequently, use Cre recombinase to eliminate the resistance gene. Following this, conduct CO-difference spectrum and oxygen uptake rate (OUR) analysis to verify whether there is an enhancement in the oxygen uptake capacity of the strain. In addition, we can employ real-time quantitative PCR technology to measure the transcription levels of genes involved in the β-glucan synthesis pathway, to assess the impact of VHb on β-glucan synthesis. Finally, we can also construct a fusion protein of VHb-EGFP to directly observe the expression and distribution of VHb [5].

## References

[1] Wang, M., Danesi, P., James, T. Y., Al‐Hatmi, A. M. S., Najafzadeh, M. J., Dolatabadi, S., Ming, C., Liou, G., Kang, Y., & De Hoog, S. (2019). Comparative pathogenicity of opportunistic black yeasts in *Aureobasidium*. *Mycoses*, 62(9), 803–811. https://doi.org/10.1111/myc.12931
[2] Zhou, R., Ma, L., Qin, X., Zhu, H., Chen, G., Liang, Z., & Zeng, W. (2023). Efficient Production of Melanin by *Aureobasidium melanogenum* Using a Simplified Medium and pH-Controlled Fermentation Strategy with the Cell Morphology Analysis. *Applied Biochemistry and Biotechnology*, 196(2), 1122–1141. https://doi.org/10.1007/s12010-023-04594-8
[3] Jiang, H., Chen, T., Chi, Z., Hu, Z., Liu, G., Sun, Y., Zhang, S., & Chi, Z. (2019). Macromolecular pullulan produced by *Aureobasidium melanogenum* 13-2 isolated from the Taklimakan desert and its crucial roles in resistance to the stress treatments. *International Journal of Biological Macromolecules*, 135, 429–436. https://doi.org/10.1016/j.ijbiomac.2019.05.190
[4] Zhang, L., Li, Y., Wang, Z., Xia, Y., Chen, W., & Tang, K. (2006). Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering. *Biotechnology Advances*, 25(2), 123–136. https://doi.org/10.1016/j.biotechadv.2006.11.001
[5] Xue, S., Jiang, H., Chen, L., Ge, N., Liu, G., Hu, Z., Chi, Z., & Chi, Z. (2019). Over-expression of *Vitreoscilla* hemoglobin (VHb) and flavohemoglobin (FHb) genes greatly enhances pullulan production. *International Journal of Biological Macromolecules*, 132, 701–709. https://doi.org/10.1016/j.ijbiomac.2019.04.007

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