Human Practices

Our journey begins with a compelling source of inspiration: iGEM LINKS China 2021, who astutely emphasized that "The core of Human Practices is to show that your project is good and responsible to the world, so the first thing iGEM team should do is to clarify their own values." Guided by this profound wisdom, our initial step upon identifying our central predicament was to undertake a thorough value-sensitive analysis. This was a crucial endeavor aimed at ensuring the righteousness of our project's trajectory while steadfastly preserving our core values as the cornerstones of our project's design.

Furthermore, our commitment to excellence led us to apply for and secure the Impact Grant, an achievement that encouraged us to delve even deeper into the essence of our project. Through extensive interactions with external stakeholders, we engaged in profound reflection, scrutinizing the compatibility of our project and research methodologies with the overarching goal of promoting global welfare and responsibility, enabled us to make Reflection & Conclusion. This journey of introspection ultimately culminated in a profound realization of the responsibility we bear in shaping the world for the better, prompting us to draw valuable insights and conclusions that we share with you today.

Make sure to read the Integrated human practice page to learn how we implemented the feedback of stakeholders in our project.

After many engagements with professors outside and inside of our team, we also did a background investigation about our project to better understand and explain the urgency of the problem and the meaning of tackling this problem.

In United States, ethanol yields have risen from 8.3 L of ethanol per bushel of corn to more than 10.2 L per bushel, capital costs have decreased from $0.95 per liter to approximately $0.26 per liter. Improving fermentation temperature every 2℃ can reduce the energy consumption of distillation by 1%. The tolerance modify of the industry strains also can saving energy consumption. Operation at high temperatures and using thermotolerant yeast can significantly reduce the cooling costs and help preventing contamination. High-temperature cultivation will not only benefit a simultaneous saccharification and fermentation process, given that the current compromise between the optimal fermentation temperature (30-35℃) and saccharification temperature (50℃) ¹, but also mitigate the cooling costs, avoid or minimize microbial contamination.

Take million tons of chemicals enterprise for example, according to the calculations, the cooling water and cooling power consumption will be reduced 15% and 10% respectively every year if the fermentation temperature is increased 5℃. Moreover, the optimal growth temperature of S. cerevisiae is also dismatch with the optimum enzymatic temperature (35-50℃) leading to the poor enzymatic efficiency. ²

There are typically three strategies are widely used, namely irrational, rational and semi-rational strategy. The irrational strategy follows an directed evolution approach, generating a large number of mutants and screening strains with the desired phenotype by high-throughput techniques. The rational strategy is built upon a fundamental understanding of cellular processes, aiming to confer stress tolerance to cell factories by directly modifying its genome. And semi-rational strategy, which is the preferred method for the majority of researchers, emphasizes the integration of partial understanding of cellular processes with evolutionary techniques, involving preparing mutants libraries more accurately. Here, we discuss the exploration of stress-tolerant parts and different strategies in constructing stress-tolerant S. cerevisiae cell factories. This background investigation gave us many resources to develop our project and made us have more deeply understand about the advance.

Irrational strategy

Certain important traits in production, such as high temperature and acid-alkaline tolerance, are often regulated by multiple genes. Obtaining specific target and elucidating the associated signaling pathways in detail are crucial steps in obtaining cell factories with corresponding stress tolerance capabilities, which can be resource-intensive. However, the irrational strategy involves the iterative process of random mutagenesis and directed selection to obtain cell factories with improved stress tolerance, meaning that this strategy can help reduce the complexity of the overall research.

Specifically, applying external pressure on cell factories through continuous cultivation, followed by further isolation and purification, is a common approach to directly obtain populations with desired traits. For example, continuous cultivation and passaging of S. cerevisiae under conditions such as high temperature and low pH, followed by detection and isolation of corresponding advantageous mutants, have been employed to obtain heat- and acid-tolerant strains, respectively. However, this method consumes a mount of efforts and resources, and it relies on the instability of endogenous cellular systems(such as denaturation of protein and formation of reactive oxygen species (ROS)) to induce mutations, making it sensitive to factors such as genome accessibility, thereby making it difficult to generate high-quality and homogeneous mutant libraries. Applying random mutated techniques directly targeting genome, which includes physical/chemical methods and biological methods, can rapidly generate a large mutant library for subsequent stress screening. In addition to traditional methods such as radiation and alkylating agents, atmospheric and room temperature plasma (ARTP) represents a novel physical and chemical mutagenesis method, which can generate libraries with a large number of random mutations without relying on toxic or hazardous reagents. For example, ARTP mutagenesis was used to obtain S. cerevisiae cell factories with low pH tolerance³. On the other hand, gene editing techniques represented by the CRISPR/Cas9 are the main biological mutagenesis methods, which offer easier control over mutation rates but come with higher costs. By combining the principles of homology-directed repair(HDR)⁴ or cDNA library preparation technology⁵, researchers have also obtained S. cerevisiae cell factories with tolerance to furfural, acetic acid and others.

Above discussed strategies are almost based on the principle of genetic mutation, however, chromosomal aberration, including changes in chromosome structure and chromosome number, is equally important for irrational strategies. Large-scale chromosomal structural variations rely on efficient protoplast fusion, which suffers from instability now. S. cerevisiae containing artificially synthesized chromosomes, with non-directional recombination sites (loxPsym) inserted on both sides of a large number of genes, serve as excellent materials for constructing mutant libraries through gene recombination. However, direct utilization of the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system may result in high lethality. To overcome this difficulty, two groups used wild-type S. cerevisiae chromosome in combination with an artificially synthesized chromosome to create a partial heterozygous diploid, which provides a repair template for lethal rearrangements and effectively reduced the rate of lethality, and conferred heat/caffeine and alkali tolerance to S. cerevisiae cell factories, respectively⁶⁷. Despite these efforts, imprecise control of SCRaMbLE system over gene recombination still poses challenges in generating high-quality mutant libraries. However, when combined with the reporter of SCRaMbLEd cells using efficient selection (ReSCuES) system⁸, the positive rate of reprogrammed yeast can be improved, enabling the rapid screening and selection of strains tolerant to ethanol, acetic acid and heat. According to above works, to further expand the range and quantity of mutants, integrating SCRaMbLE-mediated gene recombination and CRISPR/Cas9-mediated gene mutation may be a great choice. Chromosome number variation has been shown to be of significant importance for the adaptation of biological systems to the environment. Researchers have found that changes in genome ploidy could enhance vanillin tolerance and ethanol fermentation capability in S. cerevisiae cell factories ⁹. This suggests that studying chromosome number variation through traditional chemical methods that prevent mitosis or physical methods that introducing synthetic genomes by transformation may provide new avenues for enhancing stress tolerance in cell factories.

The application of irrational strategies allows cell factories to autonomously and maximally explore the fitness landscape of stress resistance. However, the potential of this strategy can’t be fully harnessed due to the large size of the mutants and inability of screening throughput. Therefore, enhancing screening throughput and reducing screening costs are crucial scientific questions. Moreover, although irrational strategies currently contribute to some extent in improving stress tolerance of S. cerevisiae cell factories, explaining the origin of this tolerance is not a straightforward task. That is because of the insufficient understanding of the parts responsible for stress tolerance. Therefore, further exploration and characterization of these parts can partially address this issue.

Data Mining

Life systems constantly face stimuli in complex and ever-changing environments. It is not an exaggeration to say that biological processes have developed through interactions with environmental stimuli. Throughout billions of years of evolution, most life systems have developed the ability to cope with various adversities, which is functioning by stress-responsive parts that encompass gene expression products that directly or indirectly confer various defense mechanisms against environmental stress. Exploring stress-responsive elements not only helps us understand the origins of the complexity within life systems, but also provides fundamental parts for engineering stress-tolerant traits.

The complete sequencing and chemical synthesis of the S. cerevisiae genome have provided a solid foundation for exploring endogenous stress-responsive parts within the yeast genome. Researchers have constructed various mutant libraries, including deletion and overexpression mutants, resulting in abundant genome information. For instance, a mutant strain with tolerance to isobutanol was identified by screening a yeast deletion library, specifically targeting GLN3¹⁰. Furthermore, extensive omics studies have revealed endogenous stress-related elements in S. cerevisiae. Two groups employed transcriptome analysis under high-temperature[11] and acetic acid¹² stress conditions, respectively, and identified endogenous elements associated with heat and acid tolerance. Similarly, transcriptome data was utilized to construct a CRISPRa screening library and discovered the heat resistance-related gene OLE1¹³.

In addition S. cerevisiae itself, another promising approach is to mine and characterize stress-responsive parts from the genomes of different species. Particularly, plants that can’t easily adapt environment through broad migration and microorganisms living in extreme environments, are worth special attention. Performing multi-omics analyses on organisms(such as plants) with multiple systems or growth stages can rapidly identify differentially expressed genes among various systems under stress conditions. C. Jia et al. have elucidated the crucial role of the phenylpropanoid biosynthesis pathway under salt stress by metabolomics and transcriptomics, providing insights into enhancing the salt tolerance of S. cerevisiae cell factories¹⁴. However, these methods may not be fully applicable to extreme microorganisms due to challenges associated with culturing them in the laboratory. A viable strategy is to perform metagenomic analysis directly from environmental samples. Kaushal et al. has discovered a highly active β-glucosidase, BglM, capable of maintaining high activity in high temperature, high glucose concentration and ethanol, from a hyperthermophilic aquatic habitat metagenomic data resource¹⁵. This enzyme has the potential to improve the overall performance of cell factories. However, this approach only reveals the most typical stress-responsive parts and does not provide a systematic understanding of extreme microorganisms’ metabolic network patterns. Therefore, there is still considerable room for improvement in methods for genome resource mining specifically targeting extreme microorganisms.

The exploration of stress-responsive parts requires both the development of new technologies to improve efficiency and the meticulous annotation and mining of existing information by numerous research teams. Only with a sufficient foundation of information on stress-responsive elements can rational design strategies be fully applied to construct cell factories with various stress-tolerant capabilities.

Rational/semi-rational

It is true that irrational strategies rely on the preparation of mutant libraries and the development of high-throughput screening techniques. However, there are still challenges associated with the efficiency and quality of mutant library preparation, as well as the high cost and limited applicability of high-throughput screening, which prevent these strategies from being widely used in everyday laboratory settings. Actually, from the understanding of the biological processes of tolerance, researchers often employ rational design approaches to modify the metabolism and regulatory networks of S. cerevisiae or construct synthetic gene circuits using stress-responsive parts from different sources to enhance the stress tolerance of S. cerevisiae cell factories.

To address stress from factors such as acidity, high osmolarity, and toxic compounds that present in fermentation broth, researchers often employ various efficient transport proteins to directly export the stressors out of the cell. For example, overexpress the dicarboxylic acid transporter SpMae1 and its homolog AcDct gene¹⁶, or a human fatty alcohol transporter protein FATP1¹⁷, can improve the tolerance of S. cerevisiae cell factories to dicarboxylic acids and fatty alcohols, respectively. However, factors beneficial to cell factory production, such as the K(+)/H(+) transmembrane concentration gradient, can be harnessed by transporting them into the cell. For example, overexpress the potassium ion importer gene TRK1 enhances the tolerance of S. cerevisiae cell factories to fatty alcohols. Regarding external stressors like high temperature, where the number of directly associated targets is limited, it is often more advantageous to improve the overall performance of the cell factory or enhance the strength of certain stress response systems within the cell factory. For example, constructing an artificial protein quality control system to improve protein stability can enhance the heat resistance of S. cerevisiae cell factories.

In addition, several stress response systems developed in other cell factories are worth expanding for application in S. cerevisiae. For example, the IMHeRE system¹⁸ has been constructed in E. coli, utilizing a riboswitch to respond to different temperatures and producing different heat shock proteins (HSPs) to cope with high-temperature stress. On the other hand, the genetic pH shooting (GPS) system¹⁹, uses acid/alkali-inducible promoters for intelligent pH regulation, thereby improving the fitness of the cell factory in acidic or alkaline environments. Combining with quorum sensing and suicide systems, these basic designs can achieve intelligent control of cell density and enhance production performance. And by leveraging riboswitches and various stress-responsive parts, it is possible to construct automated regulatory circuits for different and sole stressors. Designs incorporating promoters that respond to different stimuli can enable intelligent responses to multiple stressors, such as implementing AND gates or OR gates, and can also be used to explore the interplay between various stress factors.

Compared to non-rational strategies, employing rational strategy in the construction of stress-tolerant yeast cell factories offers enhanced design precision, significantly reduces screening challenges, and allows for more refined target selection. Moreover, this approach enables researchers to conduct in-depth analyses of selected genes, encompassing both phenotypic and molecular mechanisms, leading to comprehensive characterizations. However, the success of rational design relies on the prior identification of stress-responsive parts and the availability of tools for precise genomic manipulation. The exploration of stress-responsive parts often generates substantial data, making it challenging to achieve fully characterization of all parts within the S. cerevisiae cell factories.

Therefore, based on a partial understanding of biological systems, integrating both non-rational and rational strategies, a new approach has emerged for constructing stress-tolerant yeast cell factories by narrowing the focus and generating locally dense mutant libraries. This approach combines high-throughput screening and limited directed engineering and is known as a semi-rational strategy. For instance, the semi-rational strategy involves the use of error-prone PCR to construct mutant libraries targeting the global transcription factor SPT15, which participates in numerous regulatory processes in S. cerevisiae, leading to the rapid generation of highly ethanol-tolerant strains²⁰. Additionally, constructing random libraries using stress-responsive parts and different strength promoters and terminators from various species enables the screening of cell factories with improved stress tolerance and production capacity²¹. Furthermore, in vivo continuous evolution techniques can facilitate autonomous strain iteration, greatly enhancing the efficiency of laboratory-based evolutionary simulations. Crook, N et al.²² have employed retrotransposon-based in vivo continuous evolution (ICE) systems to enhance 1-butanol tolerance in S. cerevisiae, demonstrating the potential of these methods for broader applications. These approaches warrant further development to enhance their universality and applicability.

After decades of development, the construction of stress-tolerant S. cerevisiae cell factories has yielded significant achievements. The flexible application of non-rational, rational, and semi-rational strategies has enabled researchers to transform S. cerevisiae cell factories in multiple dimensions to enhance stress tolerance. However, improving stress tolerance often comes at the cost of decreased production performance, and the desired outcomes are not always achieved. This is because, on one hand, the generation of stress tolerance features and the expression levels of metabolites required for production may exhibit antagonistic effects, on the other hand, quantifying the impact of stress-related elements on the metabolic network itself is challenging. Therefore, achieving a balance between production performance and stress tolerance is a crucial consideration when constructing S. cerevisiae cell factories that can be effectively used for production.

In conclusion, previous research has provided numerous methods and tools for the construction of S. cerevisiae cell factories. We try to imitate the rational design method to develop our project due to our inspiration source.

During the investigation, we noticed in one paper pointed out that rational design by overexpressing multiple genes to improve cell tolerance and robustness is challenging.

Through brainstorming we analyzed the stakeholders of our project involves identifying individuals, groups that are directly or indirectly affected by or have an interest in the outcomes and activities of our biomanufacturing project focused on improving Saccharomyces cerevisiae cell factories' heat resistance. Here are our key stakeholders:

Understanding these stakeholders and their interests can help us navigate the project effectively, communicate its importance, and foster collaborations where necessary. It's important to engage with and consider the needs and concerns of these stakeholders throughout our research and development process.

In the spirit of iGEM, our journey commenced with a deliberate and meticulous approach. Upon confirming the paramount nature of our central predicament, our foremost endeavor was to embark on a comprehensive voyage of value-sensitive analysis. Our primary objective in this endeavor was to ensure that the trajectory of our efforts remained steadfast in alignment with our fundamental principles. As we embarked on this journey, we held fast to the bedrock of our initial values, guiding every facet of our project design.

In pursuit of this endeavor, we undertook a series of distinct surveys and analyses that encompassed a multifaceted spectrum. These analytical pursuits were meticulously structured to delve into the intricate domains of the economy, environment, social considerations, ethical implications, regulatory frameworks, scientific foundations, safety protocols, and the intricate realm of data collection. The forthcoming discourse will delineate the specifics of each of these analyses, providing a comprehensive view of our rigorous investigative process.

Through extensive interactions with external stakeholders, we engaged in profound reflection, scrutinizing the compatibility of our project and research methodologies with the overarching goal of promoting global welfare and responsibility, enabled us to make Reflection & Conclusion.

Build a diverse Team

Our story begins with life, and after a journey, it will return to life. Synthetic biology has added vibrant colors to this experience.

Synthetic biology is an emerging discipline that encompasses wide-ranging intersections between life sciences and various other fields. We firmly believe that confining synthetic biology solely within the realm of biological sciences does not facilitate the true realization of interdisciplinary collaboration. Our team leader had previously participated as a team member in the 2022 iGEM competition with a team primarily led by Sichuan University's College of Life Sciences. In an effort to propagate synthetic biology to every corner of this university, renowned for its engineering disciplines, he decided to step out of his comfort zone and take the first step toward this goal. In the College of Light Industry Science and Engineering, he found a like-minded teammate, and together, they set out to form a team. Before long, they successfully gathered students with diverse academic backgrounds, forging a united team to embark on the iGEM journey together.

Explore Content——Get Inspired

Increasing the stress resistance of engineered microorganisms → Enhancing the stress resistance of saccharomyces cerevisiae → Enhancing the heat resistance of saccharomyces cerevisiae.

Get inspired initially

Our journey, born from the intersection of chemical engineering and economics, initially sparked our inspiration. Dr. Kaifeng Du's lecture elucidated the multifaceted prospects inherent in establishing a robust green chemical industry. Within these insights, one thread piqued our curiosity — the use of microbial cell factories for production.

As microorganisms engage in fermentation, complex interactions of metabolic reactions unfold, accompanied by the inevitable rise of thermal stress, impacting the normal growth and expression of microorganisms. To ensure the continuity of optimal production conditions, the introduction of cooling water is crucial. However, as we observed the operation of this cooling mechanism, we uncovered a fact — excessive consumption of energy and capital.

This observation further solidified our determination, making the necessity for innovation abundantly clear. Beyond the domains of traditional cooling practices, an opportunity emerged to harmonize energy efficiency and economic prudence with microbial capabilities. Our focus shifted from mere cooling mechanisms to encompass a broader vision: the seamless integration of sustainable production with microbial prowess. As we navigate this uncharted territory — the enhancement of engineering microbial stress resistance — we are summoned forward.

Narrow “microorganisms” into “sacchromice cerevisia”

Purpose: We decided to engage in a discussion with our inspiring teacher, Dr. Kaifeng Du, with the aim of validating the feasibility of our ideas.

Contribution: Dr. Du confirmed the urgency and feasibility of enhancing the stress resistance of engineering microorganisms. He suggested narrowing the scope of engineering microorganisms to a specific type, such as saccharomyces cerevisiae, Escherichia coli, or lipolytic yeasts, among others.

Implementation: Through our dialogue with Dr. Du, we garnered a wealth of feedback regarding this idea. We conducted thorough research and analysis of several engineering microorganisms proposed by Dr. Du. Among these, saccharomyces cerevisiae emerged as the most suitable eukaryotic organism for expressing exogenous genes. It possesses a well-understood mechanism for gene expression regulation, relatively straightforward genetic manipulation, and a eukaryotic protein translation post-processing system not found in prokaryotes. It has a specific safety gene engineering receptor system, is devoid of specific viruses, can secrete the products of exogenous gene expression into the culture medium, and offers a straightforward large-scale fermentation process with low costs. Furthermore, the initial chassis organism that initially captivated our interest for our ethanol cell factory was also saccharomyces cerevisiae. Therefore, we decided to narrow our research focus from engineering microorganisms to saccharomyces cerevisiae.

Narrow “stress resistance” into “heat resistance”

Purpose:After literature research, we learned that Saccharomyces cerevisiae is sensitive to the environment, and to obtain an efficient cell factory, improving stress resistance is the key, but for different stresses, the strategies adopted are different, perhaps we need to choose a kind of coercion for tackle, so we asked Professor Faqing Huang with questions

Contribution:Professor Faqing Huang affirmed our idea of focusing on one type of stress for tackle, and suggested that starting from our original inspirational background, the biological production of ethanol, focus on thermal stress among many stresses, and improve the resistance of Saccharomyces cerevisiae to thermal stress.

Implementation:After talking with Professor Faqing Huang and reviewing relevant literature, we finally decided to focus on heat stress, with the goal of improving the heat resistance of Saccharomyces cerevisiae.

Brainstorm Broadly——Find Possible Solutions——Construct Our Initial Design of the solution——Furthermore Verify The Possibility Of The Solution——Conduct and improve our project

Focus on polyamines as our strategy

Purpose: To explore the feasibility of achieving this goal and gain insights from teachers in different fields.

Contribution:

1. Professors Chun Li and Wenyi Zhang affirmed our idea and introduced us to strategies such as the exploration of heat-resistant elements like HSPs (heat shock proteins) and the selection of strains with improved heat resistance through directed evolution mutations.
2. Professor Jiufu Qin introduced us to the compound polyamines, providing a new possibility for our project.
3. Learning about RNA switches provided us with ideas for pathway design.

Implementation:

After discussions with these professors and extensive literature review, we received valuable feedback on this issue. After comparing various strategies and previous work in the field, we developed a strong interest in polyamines. We learned that polyamines, as positively charged compounds, can bind to various macromolecules such as phospholipid membranes, nucleic acids, and proteins, stabilizing molecular structures and regulating gene expression. Several studies have reported the important role of polyamines in stress resistance in plants, and there are also relevant reports in microorganisms. These findings suggest that it may be possible to construct a gene device in the form of polyamines and their derivatives to build a stress-resistant yeast cell factory. As a result, we decided to focus our project design on polyamines, hoping to provide a new strategy for stress resistance.

Discuss the initial design of our project

Purpose:

1. Inviting Professor Jiufu Qin to serve as the Principal Investigator (PI) for our competition project.
2. Further discussing the strategy design for using polyamines to enhance yeast cell heat resistance with Professor Qin.

Contribution:

Professor Jiufu Qin agreed to become our PI and provided further insights into the use of polyamines, particularly thermospermine, which is found in higher levels in thermophilic extremophilic microorganisms. He also mentioned that literature reports suggest that polyamines can bind to nucleic acids, stabilizing their structures and reducing the risk of double-strand breaks (DSB). This may be one of the reasons why polyamines can enhance the heat resistance of plants and certain microorganisms.

Implementation:

We are very grateful to Professor Qin for agreeing to be our PI. After brainstorming with him, we further solidified the possibility of constructing a stress-resistant saccharomyces cerevisiae cell factory using polyamines and their derivatives. We outlined a basic solution design approach: constructing saccharomyces cerevisiae strains that express different polyamines, subjecting them to high-temperature stress characterization, and conducting product detection. We aim to select strains with improved heat resistance, further regulating the types and levels of polyamines, ultimately obtaining a heat-resistant saccharomyces cerevisiae cell factory.

Visited INGIA-BIO getting design insights and funding support

Purpose:

1. Obtain insights from frontline researchers in the enterprise on the issue.
2. Explore the feasibility of the initial project concept and brainstorm further.
3. Seek support for our team's participation in the 2023 iGEM competition.

Contribution:

1. Senior Scientist Wei Li suggested that we consider the advantages of using polyamines to build a stress-resistant yeast cell factory compared to other strategies. This forms the foundation of our project, and he recommended trying external addition tests as an initial step.
2. President Jian Hua suggested that we consider a specific production scenario for subsequent validation of our ideas, as a successful cell factory needs to be tested in practical production.
3. Director of Strategic Development Zhenghong Li recommended that while determining the production scenario, we should also think about the impact of the polyamine metabolism pathway on other metabolic pathways. Building a stress-resistant yeast cell factory with polyamines involves improvements to certain core metabolic pathways, and this might affect the metabolic pathways needed for the production of specific additional products.
4. Director of Strategic Development Zhenghong Li proposed that we consider using existing and upcoming polyamine resources to build a robust database. This database could be analyzed and explored in-depth using artificial intelligence methods to make precise predictions about the stress resistance properties of polyamines.

Implementation:

After discussions with the teachers from Yingjia Hesheng Company, we received valuable feedback on the topic. We believe that considering the advantages of polyamines is crucial for our project, including its feasibility and innovation, and external addition experiments can provide an initial assessment of polyamines' heat resistance. We will consider conducting this experiment. Additionally, we agree that adding a practical production scenario for validation is important; as an iGEM project, this could be a simplified version of a production problem. We also fully support the idea of considering the robustness of polyamines with respect to other metabolisms. While we should aim to avoid this as much as possible in subsequent validation, we also need to conduct more tests after proving system usability. Finally, we greatly appreciate the brainstorming session with the teachers from Yingjia Hesheng Company, and we are seriously considering the idea of using artificial intelligence for mathematical modeling to achieve a higher-level understanding and creation of life systems.

After this exchange, we have carefully considered our project and gained a basic direction for its next steps. First, we plan to review more reference literature, focusing on the relationship and mechanisms between polyamines and stress resistance, as well as the robustness of polyamine metabolism with other metabolisms. Second, we intend to communicate with more industry experts to explore practical application scenarios and demands for the stress-resistant yeast cell factory. Of course, this will involve extensive literature collection and organization. Lastly, we are considering interviewing experts or teachers with backgrounds in bioinformatics or artificial intelligence to discuss whether our project can truly benefit from the power of artificial intelligence to increase dimensions.

Held "Forum on Biomass Science and Engineering - Microbiology and Biosynthesis - Exploring Life Health and Carbon Neutrality" for diverse communication

Purpose:

1. Facilitate knowledge exchange and showcase in the fields of microbiology and synthetic biology, promoting the development of these disciplines.
2. Learn about research achievements, advancements, and technological innovations related to microbiology and biosynthesis, providing inspiration and reference for our team's project.

Contribution:

1. The Dong Li team focused on developing carbon-neutral and carbon-negative technologies, emphasizing the significance of carbon peak and carbon neutrality for China's high-quality development. This reaffirmed our belief that our project, which aims to reduce energy consumption and contribute to carbon neutrality, is on the right track.
2. Researcher Shengxiong Huang's report on "Analysis, Evolution, and Application of Secondary Metabolic Pathways in Plant Natural Medicine" sparked our thoughts on the possibility of using deep learning, machine learning, or meta-learning methods to predict how yeast cell factories with stress resistance respond to various environmental and nutritional changes, allowing for better control of the production process.
3. The Qun Sun team made many explorations into the functional interactions of protein-polyphenol complexes and their impact on the gut microbiota. This provided significant insights into our research on the interaction between polyamines and DNA under high-temperature conditions and suggested that we could explore the interaction between polyamines and proteins through molecular docking methods.
4. The Hai Zhao team conducted active research on functional microorganisms and enzyme systems in the Daqu fermentation system and contributed to the fine detection and classification in this field. Their systematic approach provided us with new perspectives.

Implementation:

Through the "Forum on Biomass Science and Engineering - Microbiology and Biosynthesis - Exploring Life Health and Carbon Neutrality," we learned about research achievements, advancements, and technological innovations related to microbiology and biosynthesis, providing inspiration and reference for our team's project. In particular, we believe it is essential to consider and explore the societal impact of our project, such as "contributing to carbon neutrality." Furthermore, we consider the concept verification of using deep learning methods to predict how yeast cell factories with stress resistance respond to environmental and nutritional changes, especially in real production scenarios like ethanol production. Additionally, we support the idea of exploring the interactions of polyamines not only with DNA but also with other macromolecules such as proteins. Moreover, we learned to approach problems from a systemic perspective.

Lastly, we express our sincere gratitude to Professor Jiufu Qin for giving us the opportunity to attend the conference as volunteers. This allowed us not only to learn about the cutting-edge knowledge in microbiology and biosynthesis but also provided us with inspiration and experience in preparing academic conferences.

After this forum exchange, we have carefully considered our project's next steps based on this feedback. First, we plan to review more reference literature, focusing on the relationship and mechanisms between polyamines and stress resistance, as well as the interaction of polyamines with biological macromolecules. Second, we intend to communicate with more industry experts to explore practical application scenarios and demands for the stress-resistant yeast cell factory, as well as to gauge public attitudes through surveys or similar methods. Lastly, we are still considering interviewing experts or teachers with backgrounds in bioinformatics or artificial intelligence to discuss whether our project can truly benefit from the power of artificial intelligence to increase dimensions.

Chose CRISPR/cas9 as genome editing system

Purpose:

1. Understand the basic strategies of molecular cloning to find suitable molecular cloning-related approaches for our experiments.
2. Learn about the application of gene editing technology in saccharomyces cerevisiae.
3. Explore other approaches to enhance the heat resistance of saccharomyces cerevisiae cell factories.

Contribution:

Faqing Huang suggested considering homologous recombination-based molecular cloning methods for plasmid construction. For genome editing, he recommended considering the use of Recombineering-related systems and CRISPR/cas9-related systems. Regarding our project, Professor Huang advised us to consider other heat tolerance-related systems, such as HSPs from different species, in addition to polyamines. He mentioned that heterologous expression might face issues of expression system incompatibility, requiring extensive screening. Furthermore, he advised us that once we determine the final expression product, to maximize intracellular resource utilization, we should design dynamic regulation strategies to ensure that the expression product is appropriate in terms of time and space. This can be achieved through the design of secondary structures at the 5' end of the expression frame sequence or the design of miRNAs complementary to mRNA. Lastly, Professor Huang believed that using single-point regulation strategies like polyamines or HSPs alone might not achieve a significant improvement in heat resistance. Therefore, another approach is to enhance the overall performance of the saccharomyces cerevisiae cell factory through irrational techniques.

Implementation:

After our discussion with Professor Faqing Huang, we have progressed further in the actual implementation of our project. We have adopted his suggestions for molecular cloning and gene editing. Additionally, we consider his proposal of using HSPs as a supplementary approach to our project. However, we find the concepts of dynamic regulation and irrational techniques to be somewhat distant for our current stage, but we will reconsider these suggestions as the project progresses.

Following this exchange, we have integrated and reflected on the information received, which provides us with specific plans for further developing our project. First, we will gather more information and learn about the two gene integration methods proposed by Professor Faqing Huang. Based on this, we will assess their feasibility for our project and select a suitable gene editing approach. Second, we plan to review literature related to HSPs to understand the feasibility of using HSPs to construct a heat-resistant saccharomyces cerevisiae cell factory. Lastly, we plan to have preliminary knowledge about dynamic regulation strategies and irrational techniques, with the intention to discuss them further with Professor Huang as the project matures.

Attended CCiC promoting our project to audience from different teams

Purpose:

1. Participate in CCiC and share our project with the other teams in the China region to gain their insights.
2. Understand the projects of other teams in the China region and seek teams working on resilience-related topics for discussions.

Contribution:

We shared our own project during CCiC through preparation and presentation. This allowed us to gain a deeper understanding of our own project while also learning about several other teams working on resilience-related topics. We look forward to engaging in discussions with these teams.

Got advice from experienced professor Yao Nie

Purpose: To obtain advice from experienced Professor Nie Yao.

Contribution:

1. Professor Nie emphasized that our project should start from real-world issues, investigate the significance of this problem both domestically and internationally, and he hopes that our team can contribute to the world.
2. Professor Nie provided guidance on our experimental plan, affirming the logicality of our approach and the planned workload.
3. Professor Nie suggested that we practice multiple times before the defense, ensuring a well-structured presentation of our experiments, integrated human practice, and other work, anticipating potential questions and preparing thoughtful answers in advance.

Implementation:

In implementing Professor Nie's valuable advice, our project took a proactive approach. We conducted thorough research to address real-world issues, determining their importance both nationally and internationally. This involved extensive literature review and engaging with relevant stakeholders to gain a comprehensive understanding of the broader context surrounding these issues. Furthermore, under Professor Nie's expert guidance, we diligently refined our experimental protocols, ensuring a logical approach and carefully planned workload.

Additionally, we heeded Professor Nie's advice, preparing extensively for our project defense. We conducted multiple practice sessions to effectively present and explain our experiments, integrated human practice, and other project elements. We also anticipated potential questions and prepared well-thought-out responses in advance. These practical implementations underscore our commitment to addressing real-world challenges and making significant contributions to the global scientific community.

Discussions drove our experiments

During the project's progress, we timely communicated with Professor Jiufu Qin (PI), pushing the project forward.

Purpose: To communicate project progress with Professor Jiufu Qin and establish a plan.

Contribution: We discussed and confirmed the schedule for wet experiments and human practice arrangements.

1. Wet experiments: The main experiments (as follows) were reconfirmed, with work primarily scheduled from August 15th to September 10th, concluding wet experiments on September 10th. (Experiment 1 (completed): Knocking out the OAZ1 gene; Experiment 2 (completed): Knocking in the SPE1 gene; Experiment 3: Knocking in SPE1 and AtACL5 genes (at the same locus); Experiment 4: Transforming engineered strains from Experiment 2 with the AtACL5 plasmid; Experiment 5: Synthesizing and characterizing the blast gene).
2. Dry experiments: Exploring SPE gene and AtACL gene components, constructing a part library.
3. human practice: We have confirmed communication with relevant industry experts in stress resistance and component exploration. Currently, we have scheduled a meeting with Professor Li Chun during the Beijing academic conference period, and we plan to communicate with a biorefinery enterprise at the end of August. We also plan to conduct a synthetic biology lecture for first-year students as part of the education section and plan to communicate with the XJU-China and SCU-China teams in August.

Implementation: Through communication with Professor Qin, we have determined the future plans for wet and dry experiments as well as integrated human practice, laying the foundation for the orderly progress and success of the project.

Attend “the National Chemical Engineering and Green Chemical Engineering Annual Conference” for communication

To further discuss our research topic, we participated in the National Chemical Engineering and Green Chemical Engineering Annual Conference.

Promoted our project to audience from different research background

Realized the bigger problem is to enhance thermal resistance while increasing the yield of the target product

Purpose:

During the conference, we became very interested in Dr. Zhang Jinwei's research, which was highly inspiring to us. We would like to engage in further discussions with him and gain his insights into our current research project.

Contribution:

1. Dr. Zhang advised us that improving the thermal resistance of the strain individually is not a challenge. The real challenge is to enhance thermal resistance while increasing the yield of the target product simultaneously without extending the fermentation cycle. He emphasized that this is the most crucial aspect.
2. Dr. Zhang suggested coupling production with thermal resistance enhancement and affirmed our choice of ethanol as the target product.

Implementation:

We greatly appreciate and resonate with Dr. Zhang Jinwei's perspective. Firstly, we plan to focus on optimizing the genetic background and growth conditions of our strain to enhance its thermal resistance, while ensuring that the fermentation cycle does not significantly lengthen. We have applied gene editing techniques to alter the expression of key genes, enhancing the cell's adaptability to high-temperature environments. Throughout this process, we pay special attention to ethanol yield, maximizing production efficiency through carefully designed fermentation conditions, such as temperature, pH, and nutrient supply. Additionally, we intend to strengthen the monitoring and data analysis aspects to ensure the simultaneous improvement of thermal resistance and ethanol production. We employ advanced bioinformatics tools to track changes in gene expression and metabolic pathways, better understanding the correlation between these two objectives. Meanwhile, we maintain close communication and consultation with Dr. Zhang to ensure our experiments and analyses receive professional guidance and support.

In summary, our implementation plan aims to achieve high ethanol production while enhancing thermal resistance and ensuring a reasonable fermentation cycle. This comprehensive strategy will help address the most critical challenge emphasized by Dr. Zhang and lay a solid foundation for the successful execution of the project.

Got the idea of quantity and effect

Purpose:

We hope to gain insights and recommendations on our research project's technical pathway from the perspective of renowned stress resistance expert Professor Chun Li.

Contribution:

1. Professor Chun Li suggested investigating the role of polyamines in yeast cell pH balance to understand how polyamines may affect the comprehensive mechanisms of stress resistance. He encouraged conducting a more comprehensive study to uncover various aspects of stress resistance mediated by polyamines.
2. Professor Chun Li recommended that we design experiments to study the impact of polyamines on pH regulation and temperature within yeast cells. This may involve altering polyamine levels and monitoring pH changes. To understand the mechanisms involved, we can examine gene expression, protein interactions, and metabolic pathways influenced by polyamines. As Li Chun suggested, comparing the effects of polyamines with other stress-enhancing compounds requires conducting control experiments under defined conditions to determine potential synergistic effects.
3. Professor Li Chun also emphasized the issue of quantity, reminding us that both small amounts and excessive amounts can affect our effectiveness.

Implementation:

In summary, Professor Chun Li's suggestions provide a purposeful approach to understanding the role of polyamines in yeast stress resistance, making a substantial contribution to the depth and breadth of the research. His insights provide a clear path for analysis through carefully designed experiments and molecular biology. These insights enhance our understanding of yeast stress resistance and its applications across various industries, inspired us and made one of our final future goals is to regulate the dose-response relationship.

Tried to solve spore concern

Determined the content and style of the wiki

Purpose: We conducted discussions regarding the wiki with the PI Jiufu Qin.

Contribution:

1. We have decided that the style of this wiki should be concise and clear, with a focus on enhancing the overall design of the iGEM wiki to ensure visual appeal and user-friendliness. This includes optimizing layout, formatting, and mobile responsiveness.
2. Professor Qin suggested that our team should significantly enrich and pay attention to the content of the wiki.
3. Deadline Management: According to the advice of the PI, the team's top priority should be diligently adhering to all project milestones and iGEM competition deadlines. This entails creating a well-structured timeline, assigning clear responsibilities, and conducting regular checks to track and meet the established deadlines.
4. Team Collaboration: The PI emphasized the importance of fostering effective collaboration among team members for wiki development. The team should implement collaboration tools and establish communication channels to facilitate real-time cooperation and information sharing.

Implementation:

We will focus on improving the design of the iGEM wiki to ensure it has an visually appealing and user-friendly interface. Meeting deadlines will be accomplished through the creation of a structured timeline, progress tracking, and regular check-ins. To promote team collaboration, collaboration tools and communication channels will be adopted. Finally, a feedback loop will be established, actively seeking and incorporating feedback from all stakeholders to continually refine and enhance the wiki.

Solve contamination and transformation problems in experiments

Purpose:

To discuss the current status of experiments with Professor Qin and address the challenges of contamination and transformation failures in our ongoing experiments.

Contribution:

Professor Qin suggested that we should completely replace all equipment and reagents and conduct the experiments anew. Additionally, he recommended that three team members simultaneously conduct parallel experiments to ensure the experiments progress correctly and to identify the causes of contamination and transformation failures.

Implementation:

We will heed Professor Qin's advice by replacing all reagents and equipment and conducting parallel experiments with multiple team members simultaneously. This approach ensures the experiments progress correctly and helps us pinpoint the reasons for contamination and transformation failures.

Invite Yajie Wang for teaching us bio-informatics

Purpose:

We invited graduate student Yajie Wang to provide us with insights into bioinformatics methods and to gather his opinions on the bioinformatics aspect of our project.

Contribution:

During this interaction, we gained knowledge about deeply analyzing the sequences of specific genes and uncovering their homologous genes and potential relationships.

Implementation:

We will follow a structured approach to collect and organize sequences of the target genes of interest. Using bioinformatics tools and software, we will conduct BLAST analysis, carefully scrutinizing the results to identify sequence similarities, differences, and potential evolutionary connections. Simultaneously, we plan to construct expression systems for existing genes, such as replacing promoters, to conduct heat resistance and yield tests. This comprehensive approach will provide valuable insights into the genetic aspects of our project.

After a lot of engagement with stakeholders outside and inside of our team, and many times group discussions, we improved our project to our best.

November——iGEM the Grand Jamboree, here we come!