Knowledge acquired on paper ultimately seems shallow still.
To thoroughly understand this, one must personally experience it all.
-- You Lu(Song Dynasty)

纸上得来终觉浅，
绝知此事要躬行
—— 陆游（南宋）

We are team Tsinghua-M 2024. As you can see from our logo, the curious cat symbolizes our spirit of exploration in Human Practice. Just as cats explore the world, our project is driven by curiosity, interest, and questions. Through education, collaboration, and interviews, we are dedicated to exploring the mysteries of the RNA world from the perspective of synthetic biology. We strive to combine cutting-edge scientific research with practical application scenarios, ensuring that our project not only has theoretical innovation but also practical value, bringing substantial contributions to society. Like that curious cat, we continuously leave our marks of exploration and change in the unknown RNA world.

我们是Tsinghua-M队伍，我们团队标志中的好奇猫象征着我们在人类实践中的探索精神。就像猫探索世界一样，我们的项目也是由好奇心、兴趣和问题驱动的。通过教育、合作和访谈，我们致力于从合成生物学的角度探索RNA世界的奥秘。我们努力将尖端科学研究与实际应用场景相结合，确保我们的项目不仅具有理论创新，而且具有实际价值，为社会带来实质性的贡献。就像那只好奇的猫一样，我们在未知的RNA世界中不断留下探索和改变的痕迹。

Our project this year is concentrated on the development of applications related to the ADAR system. Human practice has played a crucial role in the evolution and design of our project.

我们今年的项目集中在与ADAR系统相关的应用开发上，人类实践在项目的设计和调整中发挥了至关重要的作用。

Our integrated human practice commenced with an analysis of our stakeholders, focusing on two pivotal groups: businesses and academic research institutions. We structured our activities around the AREA cycle, synergizing it with the DBTL cycle to amplify the efficacy of our human practices. We pinpointed various communication targets across two primary sectors: advanced science and production application. This encompassed engagement with university professors, bioprocessing firms, brewing companies, and the general public. Furthermore, we engaged in profound discussions with our sponsors to comprehend the market potential for our project applications. These interactions have facilitated the optimization of our design and provided us with a clear grasp of the market for its practical application.

我们的integrated human practice始于对我们的利害关系人的分析，重点关注两个关键群体：企业和学术研究机构。我们围绕AREA循环构建我们的活动，并将其与DBTL循环相结合，以增强我们人类实践的有效性。我们确定了两个主要领域的多个沟通目标：先进科学和生产应用。这包括与大学教授、生物加工公司、酿酒公司和公众的接触。此外，我们与赞助商进行了深入讨论，以了解我们项目应用的市场潜力。这些互动有助于我们优化设计，并为我们提供了对其实际应用市场的清晰了解。

In education, we engaged in in-depth conversations with high school students, sowing the seeds of interest in synthetic biology. Through various conference exchanges, we learned from one another, absorbed suggestions from other teams, attempted to address issues for AIS-China, and engaged in profound cross-national communication with Munich 2024, who also utilized ADAR tools, experiencing diverse designs.

在education方面，我们与高中生进行了深入交谈，播下了对合成生物学的兴趣种子。通过各种会议交流，我们相互学习，吸收其他团队的建议，尝试解决AIS-China的问题，并与Munich 2024进行了深入的跨国交流，他们也使用ADAR工具，体验了不同的设计。

Additionally, we conducted an entrepreneurship analysis aimed at our project stakeholders and created accessible comics to enhance inclusivity.

此外，我们针对项目的stakeholders进行了entrepreneurship分析，并创建了可访问的漫画以增强inclusivity。

Throughout this journey of practice and exploration, many individuals have offered their assistance, contributing to the refinement of our project. We have witnessed the ripples our project and design have created in the market, observing a growing interest in RNAssay and synthetic biolog

在整个实践和探索的旅程中，许多人提供了帮助，促进了我们项目的完善。我们已经看到了我们的项目和设计在市场中产生的影响，观察到对RNAssay和合成生物学的兴趣日益增长。

In the early stages of the project, the team actively visited multiple enterprises for on-site tours and research, gaining in-depth insights into current market needs and technological application scenarios to ensure the design and implementation of the project have practical value.

在项目的早期阶段，团队积极走访多家企业进行实地考察和研究，深入了解当前市场需求和技术应用场景，确保项目的设计和实施具有实际价值。

In the later stages of the project, the team further engaged with more enterprises to conduct detailed needs analysis and application feedback, ensuring that the project outcomes genuinely meet the actual demands of the enterprises and have commercialization potential.

在项目的后期阶段，团队进一步与更多企业接触，进行详细的需求分析和应用反馈，确保项目成果真正满足企业的实际需求，并具有商业化潜力。

The team actively communicated with multiple university professors, inviting experts and scholars from various fields to provide professional review and guidance on the project. This included assistance with experimental design, data analysis, and project optimization, offering invaluable professional advice.

团队积极与多位大学教授沟通，邀请各领域专家学者为项目提供专业审查和指导。这包括在实验设计、数据分析和项目优化方面提供帮助，提供宝贵的专业建议。

Through collaboration with professors from different academic backgrounds, the team was able to integrate multidisciplinary knowledge and skills, thereby enhancing the comprehensiveness and innovation of the project.

通过与不同学术背景的教授合作，团队能够整合多学科知识和技能，从而提高项目的全面性和创新性。

Team members possess a strong passion for scientific research and exploration, striving to align personal interests with team goals to continuously drive the project forward.

团队成员对科学研究和探索充满热情，努力将个人兴趣与团队目标相结合，持续推动项目前进。

The team focuses on combining cutting-edge scientific research findings with practical application scenarios, ensuring that the project not only exhibits theoretical innovation but also has practical value, contributing substantively to society.

团队注重将前沿科研成果与实际应用场景相结合，确保项目不仅具有理论创新性，还具有实际价值，为社会做出实质性贡献。

Our tool project has three application directions, which are colony Strain Securitysystems, splice variants(spliced isoforms), and brewing. We delve into these three application directions, exploring the project's application prospects from various perspectives such as market, science, producers, and consumers. We constantly improve our plans and continuously enhance the feasibility of our project's applications, continuously expanding our project's influence and interactivity with the outside world.

我们的工具项目有三个应用方向，分别是菌株防盗门系统、剪接异构体和酿造。我们深入研究这三个应用方向，从市场、科学、生产者和消费者等多个角度探索项目的应用前景。我们不断完善计划，持续提高项目应用的可行性，不断扩大项目的影响力和与外界的互动性。

We first analyzed the stakeholders of our project, focusing on those with high power and high interest. We then selected the most important stakeholders to engage in human practice activities.

我们首先分析了我们项目的stakeholders，重点关注那些对我们的项目有着极大兴趣并能提供实质性帮助的人。然后，我们选择了最重要的stakeholders参与到人类实践活动中。

We use the AREA cycle to organize integrated human practices. Following the ASSESS-RESEARCH-EVALUATE-ACT cycle, we continuously improve the execution details of integrated human practices, enhancing their effectiveness in advancing the project. Additionally, our integrated human practices play a crucial role in the project's DBTL cycle, including validating certain concepts and guiding new designs.

我们使用AREA循环来组织整合的人类实践活动。遵循评估-研究-评价-行动（ASSESS-RESEARCH-EVALUATE-ACT）的循环，我们不断改进整合的人类实践活动的执行细节，提高它们在推进项目中的有效性。此外，我们的整合的人类实践活动在项目的DBTL循环（DESIGN-BUILD-TEST-LEARN）中扮演着关键角色，包括验证某些概念和指导新的设计。

EVOLYZER is also one of our sponsors. At the early stage of project design, we visited EVOLYZER company to explore the practical needs in several areas: RNA content detection, gene lock systems, and splice variant detection. From our discussions, we learned that there is a significant demand for gene lock systems in biological production, which plays an important role in patent protection and stimulating innovation.

北京衍微科技有限公司（下称衍微或EVOLYZER）也是我们的赞助商之一。在项目设计的早期阶段，我们访问了衍微公司，探讨了几个领域的实际需求：RNA含量检测、防盗门系统和剪接异构体检测。经过讨论，我们了解到生物生产中对防盗门系统有很大需求，这与专利保护和激发创新密切相关。

After completing the concept validation of the ADAR system in yeast, we plan to visit EVOLYZER again for further discussions.

在完成酵母ADAR系统的概念验证后，我们计划再次访问衍微进行进一步讨论。

Members of the iGEM Tsinghua-M team reported the project progress to Mr. Chen Bo, the general manager of Yanwei Technology, including an overview of the project, experimental progress, and optimization plans. During the Q&A session, the prevalence of strain theft, the duration of patent protection, strategies to avoid patent leakage, and methods of stealing strains and key genes were discussed.

我们的成员向研微科技总经理陈博先生汇报了项目进展，包括项目概况、实验进展和优化计划。在问答环节讨论了菌株盗窃的普遍性、专利保护期限、避免专利泄露的策略以及偷盗菌株和关键基因的方法。

Through the activity, we know that the market has a serious problem with strain theft, with a patent protection period of 20 years. Companies face challenges such as difficulty in providing evidence, some strains cannot be patented, the punishment for theft is light and limited to a single link, and the possible ignorance of upstream and downstream organizations. In addition, companies use methods such as adding inducers to avoid patent leakage, strain theft is usually carried out by insiders, and gene sequences may change in different environments. Mr. Chen Bo also suggested adding multiple mechanisms to achieve strategic integration, using yeast self-destruction, shearing, and transposition to amplify the effects of RNA variation, and considering environmental confidentiality measures such as light-controlled expression.

通过这次活动，我们了解到市场存在严重的菌株盗窃问题，专利保护期为20年。企业面临的挑战包括:取证困难、某些菌株无法申请专利、盗窃惩罚轻且仅限于单一环节、上下游组织可能存在不知情的情况。此外，企业使用添加诱导物等方法来避免专利泄露，菌株盗窃通常由内部人员进行，基因序列可能在不同环境中发生变化。陈博先生还建议添加多重机制以实现战略整合，利用酵母自毁、剪切和转座来放大RNA变异的效果，并考虑光控表达等环境保密措施。

In summary, through this exchange, we have become more familiar with the current market situation of security doors, and have also received specific suggestions for the direction of our project, from which we have greatly benefited.

总之，通过这次交流，我们对菌株防盗门的当前市场情况有了更深入的了解，也得到了针对我们项目方向的具体建议，从中获益良多。

To improve the design of the strain security system and to achieve more efficient multi-gene detection, we consulted with Professor Zhang Shuyi from the School of Pharmacy, Tsinghua University, who has in-depth research in the design of genetic circuits.

为了改进防盗门的设计并实现更高效的多基因检测，我们咨询了清华大学药学院的张数一教授，他在遗传电路设计方面有深入研究。

Professor Zhang suggested looking for a large yeast vector, determining a clear application scenario, and proposed considering promoters that can only initiate transcription after epigenetic methylation, to achieve reversible strain security system or the need to continuously add inducers to ensure the normal physiological state of the cells. He also suggested analyzing the relationship between gene expression and metabolic products, collecting data, and combining machine learning to establish a predictive model.

张教授建议寻找大型酵母载体，确定明确的应用场景，并提出考虑只有在表观遗传甲基化后才能启动转录的启动子，以实现可逆防盗或需要持续添加诱导物以确保细胞正常生理状态。他还建议分析基因表达与代谢产物的关系，收集数据，并结合机器学习建立预测模型。

The Strain Security system application team can update the project design, incorporating the idea of methylation promoters. At the same time, the team learned that the project should have practical significance and a clear research direction, and by in-depth analysis and data collection, it can add depth and breadth to the research.

防盗门应用团队可以更新项目设计，纳入甲基化启动子的想法。同时，团队了解到项目应具有实际意义和明确的研究方向，通过深入分析和数据收集，可以为研究增添深度和广度。

To evaluate the potential of the ADAR system in animal models of splicing-related diseases, we initially conducted a literature review. We found that progressive retinal atrophy (PRA) in cats is caused by a CEP290 gene mutation, IVS50 + 9T > G. This mutation creates a new splice site and causes a frameshift mutation, which ultimately disrupts the production of the CEP290 protein and leads to the development of PRA. Based on this finding, we proposed using plasmids containing sensors to detect the effects of this mutation in mammalian cells, given that mammalian cells possess endogenous ADAR activity.

为了评估ADAR系统在剪接相关疾病动物模型中的潜力，我们最初进行了文献综述。我们发现猫的进行性视网膜萎缩(PRA)是由CEP290基因突变IVS50 + 9T > G引起的。这种突变创造了一个新的剪接位点并导致移码突变，最终破坏CEP290蛋白的产生，导致PRA的发展。基于这一发现，我们提出使用含有传感器的质粒在哺乳动物细胞中检测这种突变的影响，因为哺乳动物细胞具有内源性ADAR活性。

To validate this approach, we reached out to a professor specializing in veterinary medicine via email. Through this discussion, we recognized that cultivating animal cells or tissues and introducing plasmids into these cells might present more challenges compared to our current yeast cell system. He suggested that we consider extracting RNA from cells and performing in vitro assays on the extracted RNA, which could be more practical at this stage. Additionally, he highlighted that PRA in cats serves as an excellent model for studying splicing-related diseases.

为了验证这种方法，我们通过电子邮件联系了一位专门研究兽医学的教授。通过这次讨论，我们认识到培养动物细胞或组织并将质粒引入这些细胞可能比我们目前的酵母细胞系统面临更多挑战。他建议我们考虑从细胞中提取RNA并对提取的RNA进行体外分析，这在目前阶段可能更加实用。此外，他强调猫的PRA是研究剪接相关疾病的优秀模型。

These advice inspired us to consider designing a cell-free system for in vitro RNA detection, which would mitigate the difficulties associated with animal cell or tissue culture and enhance the flexibility of our detection method.

这些建议启发我们考虑设计一个无细胞系统用于体外RNA检测，这将减轻与动物细胞或组织培养相关的困难，并增强我们检测方法的灵活性。

To investigate the academic value of a real-time detection system for the content of splicing isomers and explore its application potential in disease diagnosis and treatment, we interviewed Mr. Yan Chuangye from the School of Life Sciences at Tsinghua University.

为了研究剪接异构体含量实时检测系统的学术价值，并探索其在疾病诊断和治疗中的应用潜力，我们采访了清华大学生命科学学院的助理教授闫创业老师。

In the interview with Yan, we gained an in-depth understanding of the biological significance and molecular mechanism of splicing isomer phenomena. Yan elaborated on the diversity and complexity of splicing isomers in eukaryotic organisms and their key role in the regulation of genetic information expression. In addition, Yan discussed the association between splicing isomers and the occurrence of cancer, as well as the differences in splicing mechanisms between humans and yeast. These discussions provided valuable academic perspectives and research directions for our project.

在与闫创业教授的访谈中，我们深入了解了剪接异构体现象的生物学意义和分子机制。教授详细阐述了真核生物中剪接异构体的多样性和复杂性，以及它们在遗传信息表达调控中的关键作用。此外，教授还讨论了剪接异构体与癌症发生的关联，以及人类和酵母在剪接机制上的差异。这些讨论为我们的项目提供了宝贵的学术视角和研究方向。

Yan pointed out that abnormal expression of splicing isomers is related to a variety of diseases, including but not limited to cancer. Therefore, developing a system capable of real-time monitoring the content of splicing isomers is not only of significant academic value but also helps in early disease diagnosis and personalized medicine. Yan also emphasized the importance of in-depth research on splicing mechanisms, which may provide new strategies for treating diseases related to splicing abnormalities. In addition, Yan suggested that we consider the tissue-specific and stage-specific expression of splicing isomers in our project, as well as their mechanisms in disease occurrence.

教授指出，剪接异构体的异常表达与多种疾病有关，包括但不限于癌症。因此，开发一个能够实时监测剪接异构体含量的系统不仅具有重要的学术价值，还有助于早期疾病诊断和个性化医疗。教授还强调了深入研究剪接机制的重要性，这可能为治疗与剪接异常相关的疾病提供新的策略。此外，教授建议我们在项目中考虑剪接异构体的组织特异性和阶段特异性表达，以及它们在疾病发生中的机制。

We first communicated with Professor Xi Jingying from the School of Environmental Science. After reviewing our design, Professor Xi deemed it generally feasible but pointed out several potential issues in practical implementation:

我们首先与清华大学环境学院的席劲瑛教授进行了交流。在了解了我们的设计后，席教授认为总体上是可行的，但指出了实际实施中可能存在的几个问题：

1. Our system is primarily intended for detecting the relative levels of RNA encoding enzymes involved in flavor compound synthesis. However, this approach may have limited practical value. First, the synthesis of flavor compounds often involves many enzymes, so measuring the RNA levels of just one or a few enzymes in a pathway provides limited insight and may not offer significant theoretical support for brewing techniques or genetic modification. Second, compared to methods like spectroscopy or chromatography for measuring the concentration of flavor compounds directly, this approach is less straightforward. In response to the second point, we clarified that flavor compounds and RNA operate at different levels, and the latter is closer to the fundamental process of flavor compound production, thus having some potential value.

1. 我们的系统主要用于检测编码参与风味化合物合成酶的RNA的相对水平。然而，这种方法可能具有有限的实用价值。首先，风味化合物的合成通常涉及许多酶，因此仅测量一个或少数几个酶的RNA水平，这是较为有限的，可能不会为酿造技术或基因改造提供重要的理论支持。其次，与直接测量风味化合物浓度的光谱法或色谱法等方法相比，这种方法不够直接。针对第二点，我们指出，风味化合物和RNA在不同层面上起作用，后者更接近风味化合物生产的基本原理，因此具有一定的潜在价值。

2. The microorganisms used in Moutai production are largely natural and have undergone hundreds or even thousands of years of artificial selection. Therefore, the company may be reluctant to accept genetically engineered microorganisms.

2. 用于茅台酒生产的微生物大多是天然的，并且经历了数百甚至数千年的人工选择。因此，该公司可能不愿意接受基因工程微生物。

We followed up by asking Professor Xi to inquire with Moutai about two questions: 1. What is the company's acceptance level of using genetically engineered microorganisms in production? Are they completely opposed, or is there room for experimentation? 2. If our system can accurately measure the relative RNA levels of certain enzymes, would the company be willing to use this tool for data collection?

我们通过席劲瑛教授向茅台咨询了两个问题：1. 公司对在生产中使用基因工程微生物的接受程度如何，他们是完全反对，还是愿意尝试？2. 如果我们的系统能够准确测量某些酶的相对RNA水平，公司是否愿意使用这个工具进行数据收集？

A technical representative from Moutai responded from the perspective of the enterprise. For the first question, Moutai’s acceptance level of genetically engineered microorganisms in the brewing process is zero. For the second question, the representative stated that it would depend on whether our system offers advantages in measurement speed and accuracy compared to existing RNA detection devices, and whether it is more efficient than directly measuring enzyme activity or key metabolite levels, as our approach is relatively indirect. Based on our design principles, the RNAssay should offer better accuracy and convenience than current RNA detection technologies like qPCR. However, our system still faces challenges such as instability in accuracy and potential cytotoxicity of the proteins used, though it may outperform qPCR in the future.

茅台的技术代表从企业的角度回应了两个问题：首先，公司对于在酿造过程中使用基因工程微生物的接受度一般很低。其次，如果我们的系统在测量速度和准确性方面比现有的RNA检测设备有优势，并且比直接测量酶活性或关键代谢物水平更有效，那么公司可能会考虑使用这个工具进行数据收集，因为我们的方法相对间接。

The representative also pointed out that companies are generally less interested in basic research. They are more concerned with whether a technology can provide valuable applications, such as detecting or monitoring fermentation status. Therefore, if we aim to collaborate with companies to solve practical problems, it would be better to adopt a problem-oriented approach. Our detection method may not be suitable for Moutai's traditional solid-state fermentation process but could be more applicable in fields like biopharmaceuticals or biorefining, particularly for closed fermentation tanks. Another possible application could be anaerobic digestion scenarios in wastewater treatment, where our system could monitor methane production by measuring the RNA expression of methane-related enzymes in real time.

此外，代表还指出，公司通常对基础研究不太感兴趣。他们更关心的是，一项技术是否能提供有价值的应用，例如检测或监测发酵状态。因此，如果我们旨在与公司合作解决实际问题，最好采用以问题为导向的方法。我们的检测方法可能不适合茅台传统的固态发酵过程，但在生物制药或生物精炼领域，尤其是在封闭的发酵罐中，可能更加适用。另一个可能的应用是在废水处理中的厌氧消化场景，我们的系统可以通过实时测量与甲烷相关的酶的RNA表达来监测甲烷的产生。

Regarding our system itself, the representative offered practical advice, highlighting that our system's main advantage is time savings. In actual production, taking a small sample for testing and disrupting the cells is not unacceptable, so if our system can significantly reduce time, it could attract interest.

关于我们的系统本身，代表提供了实用的建议，强调我们的系统的主要优势是节省时间。在实际生产中，取出一小部分样本进行测试并破坏细胞是可以接受的，因此如果我们的系统能显著减少时间，可能会引起人们的兴趣。

During our visit to GenScript ProBio, their team inspired us with the idea that the ADAR system could potentially play a role in modifying Saccharomyces cerevisiae (brewer's yeast) to produce new flavors in wine. As a result, one of our project designs involves the genetic modification of brewer's yeast using synthetic biology techniques to create innovative alcoholic beverages with unique flavors. Our goal is to develop products that are both market-driven and innovative. In addition to literature reviews and corporate research, we designed and conducted a questionnaire survey aimed at gathering consumer opinions and expectations for such innovative products, to guide our product development and marketing strategy.

在我们访问GenScript ProBio时，他们的团队启发我们ADAR系统可能在改造Saccharomyces cerevisiae（酿酒酵母）以在葡萄酒中产生新风味方面发挥作用。因此，我们的一个项目设计涉及使用合成生物学技术改造酿酒酵母，以创造具有独特风味的创新酒精饮料。我们的目标是开发既符合市场需求又富有创新性的产品。除了文献综述和企业研究外，我们设计并进行了问卷调查，旨在收集消费者对这类创新产品的意见和期望，以指导我们的产品开发和营销策略。

1. Methodology and Sample

1. 方法与样本

The questionnaire was distributed both online and in person, ensuring the representativeness of the sample. The content covered demographic characteristics, drinking habits, knowledge of synthetic biology and genetic modification, as well as expectations and acceptance of new flavored wines.

问卷通过线上和线下方式发放，确保样本的代表性。内容涵盖人口统计特征、饮酒习惯、对合成生物学和基因改造的了解程度，以及对新风味葡萄酒的期望和接受度。

2. Demographic Analysis

2. 受众分析

The distribution of gender and age provides an initial profile of the target consumers, with females and those aged 31-40 representing a significant portion of the respondents.

性别和年龄分布为目标消费者提供了初步画像，其中女性和31-40岁年龄段占受访者的很大比例。

3. Analysis of Drinking Habits and Frequency

3. 饮酒习惯和频率分析

The data on drinking habits and frequency indicate that most respondents tend to drink moderately.

关于饮酒习惯和频率的数据表明，大多数受访者倾向于适度饮酒。

4. Awareness and Acceptance of Technology

4. 关于酿酒工艺中使用生物技术的了解和接受程度

Respondents’ knowledge of synthetic biology and genetic modification is relatively low, but their acceptance of new flavored wines is relatively high.

受访者对合成生物学和基因改造的了解程度相对较低，但对新风味葡萄酒的接受度相对较高。

5. Expectations for New Flavored Wines

5. 对于啤酒新风味的期待度

Consumers’ expectations for new flavored wines center on taste, flavor, and uniqueness. Desirable characteristics include a mellow, refreshing, sweet, and fruity flavor. Additionally, reducing alcohol content and enhancing unique flavors are key consumer concerns.

消费者对新风味葡萄酒的期望集中在口感、风味和独特性上。理想的特征包括醇厚、清爽、甜美和果香。此外，降低酒精含量和增强独特风味是消费者关注的关键问题。

1. Demographic Analysis

1. 受众分析

The demographic analysis revealed that respondents aged 31-40 constitute a large proportion. This finding suggests that this particular age group may have a higher interest and demand for innovative alcoholic products. Therefore, our product development and marketing strategies should focus specifically on this target audience to ensure our products appeal to and meet their needs.

人口统计分析显示，31-40岁的受访者占很大比例。这一发现表明，这个特定年龄段可能对创新酒精产品有更高的兴趣和需求。因此，我们的产品开发和营销策略应特别关注这个目标受众，以确保我们的产品吸引并满足他们的需求。

2. Analysis of Drinking Habits and Frequency

2. 饮酒习惯和频率分析

The survey results show that most respondents prefer moderate drinking. This trend indicates that, when designing products, we should consider offering options with lower alcohol content or at least provide products with varying alcohol levels to accommodate different consumer preferences. Furthermore, the emphasis on moderate drinking suggests that our product design should strike a balance between health and enjoyment, which could increase the market appeal of the products.

调查结果显示，大多数受访者倾向于适度饮酒。这一趋势表明，在设计产品时，我们应考虑提供酒精含量较低的选择，或至少提供不同酒精含量的产品，以适应不同消费者的偏好。此外，对适度饮酒的强调表明，我们的产品设计应在健康和享受之间取得平衡，这可能会增加产品的市场吸引力。

3. Awareness and Acceptance of Technology

3. 关于酿酒工艺中使用生物技术的了解和接受程度

Although respondents’ awareness of synthetic biology and genetic modification is generally low, their acceptance of new flavored wines is relatively high. This suggests that consumers are open to innovative products, even if they may not fully understand the underlying technology. This finding underscores the importance of educating and communicating with consumers during product promotion, raising awareness of how synthetic biology and genetic modification can contribute to the creation of unique and high-quality wines.

尽管受访者对合成生物学和基因改造的认知程度普遍较低，但他们对新风味葡萄酒的接受度相对较高。这表明消费者对创新产品持开放态度，即使他们可能不完全理解背后的技术。这一发现强调了在产品推广过程中教育和沟通消费者的重要性，提高他们对合成生物学和基因改造如何有助于创造独特和高质量葡萄酒的认识。

4. Expectations for New Flavored Wines

4. 对于啤酒新风味的期待度

Consumers' expectations for new flavored wines mainly focus on taste, flavor, and uniqueness. Desired characteristics include a mellow, refreshing, sweet, and fruity profile. Additionally, reducing alcohol content and enhancing unique flavors are important areas of focus for consumers. These expectations provide clear direction for our product development. We should prioritize these aspects in the formulation and production process to ensure the new products meet consumer expectations.

消费者对新风味葡萄酒的期望主要集中在口感、风味和独特性上。期望的特征包括醇厚、清爽、甜美和果香的口感。此外，降低酒精含量和增强独特风味是消费者关注的重要方面。这些期望为我们的产品开发提供了明确的方向。我们应在配方和生产过程中优先考虑这些方面，以确保新产品满足消费者的期望。

In conclusion, the survey results have provided valuable insights for our project, guiding both product development and marketing strategy. Our target consumer group tends to favor moderate drinking and is open to new flavored wines. In product development, we should focus on lower alcohol content, unique taste and flavor profiles, and increase consumer awareness of synthetic biology and genetic modification through education and communication. By adopting these strategies, we can create innovative alcoholic products that meet market demand and stand out as unique offerings.

总之，调查结果为我们的项目提供了宝贵的见解，指导产品开发和营销策略。我们的目标消费群体倾向于适度饮酒，并对新风味葡萄酒持开放态度。在产品开发中，我们应关注较低的酒精含量、独特的口感和风味，并通过教育和沟通提高消费者对合成生物学和基因改造的认识。通过采用这些策略，我们可以创造出满足市场需求并作为独特产品脱颖而出的创新酒精饮品。

Click here to know more about CABIO

点此进入嘉必优官网

CABIO is one of our sponsors. To gain more practical guidance for our application design, we visited CABIO to discuss the current progress of our project and several potential application directions. We also had the opportunity to tour their laboratory facilities.

CABIO是我们的赞助商之一。为了获得更多实际的应用设计指导，我们访问了CABIO，讨论了我们项目的当前进展和几个潜在的应用方向。我们还有机会参观了他们的实验室设施。

During our discussion with CABIO, we received comprehensive feedback and suggestions that spanned both foundational experiments and application design.

在与CABIO的讨论中，我们收到了涵盖基础实验和应用设计的全面反馈和建议。

System Design

系统设计

Considering the time delay between the introduction of ADAR plasmids and subsequent detection, it is advisable to introduce the ADAR plasmid in advance or replace the ADAR gene's promoter with an inducible promoter that can be activated prior to detection.

In terms of sensor design, given the limited preparation time before the competition, it may be more practical to develop sensors targeting the yeast genome or specific genes. The focus should be on establishing design principles and patterns for these sensors.

If we aim to develop a detection tool similar to qPCR, we may need to use it in conjunction with real-time monitoring instruments, such as plate readers or flow cytometers.

Integrating results from wet lab experiments can help validate and refine dry lab models, leading to a more robust experimental design.

考虑到引入ADAR质粒和随后检测之间的时间延迟，建议提前引入ADAR质粒或将ADAR基因的启动子替换为可在检测前激活的诱导型启动子。

在传感器设计方面，考虑到比赛前的有限准备时间，开发针对酵母基因组或特定基因的传感器可能更加实用。重点应放在建立这些传感器的设计原则和模式上。

如果我们的目标是开发类似于qPCR的检测工具，可能需要将其与实时监测仪器(如酶标仪或流式细胞仪)结合使用。

整合湿实验结果可以帮助验证和完善干实验模型，从而导致更稳健的实验设计。

Wet Lab Experiment Design and Results

湿实验设计和结果

When analyzing confocal microscopy imaging results, it is important to exclude the effects of background fluorescence from the cells. Furthermore, compared to fluorescent proteins, using luciferase can provide a more stable and reliable signal under confocal microscopy, thereby improving the accuracy and reproducibility of the results.

When using flow cytometry to detect cell fluorescence, it is beneficial to simultaneously assess cell health and status to determine the impact of ADAR on the cells.

在分析共聚焦显微镜成像结果时，重要的是排除细胞背景荧光的影响。此外，与荧光蛋白相比，使用荧光素酶可以在共聚焦显微镜下提供更稳定和可靠的信号，从而提高结果的准确性和可重复性。

使用流式细胞仪检测细胞荧光时，同时评估细胞的健康状况和状态是有益的，以确定ADAR对细胞的影响。

Application Design

实际应用设计

The suggestions we received provided significant insights that could influence our application approach:

The demand for multi-gene detection is primarily in the fields of biopharmaceuticals and upstream R&D in industrial production, with lower demand in actual production processes. This suggests a need to adjust our focus in application design to better align with market demands.

Regarding gene lock technologies, existing designs are relatively easy to bypass, especially with advances in whole-genome sequencing technologies, where genetic information is rarely secure. However, there is a significant market potential for developing more robust gene lock solutions.

For splice variant detection, our current design does not cover all possible splicing forms. A more comprehensive approach is required, especially to address abnormal splicing events related to branch sites.

Mr. Liu from CABIO also proposed a novel application direction: using this system to manipulate yeast metabolic pathways during fermentation to produce wines with different flavors.

Inspired by the insights from CABIO, we are now investigating the potential applications of the ADAR system in brewing and the detection of diseases associated with abnormal splicing in animals.

我们收到的建议为应用方向提供了重要建议，可能影响我们的应用方法:

多基因检测的需求主要集中在生物制药和工业生产的上游研发领域，在实际生产过程中需求较低。这表明我们需要调整应用设计的重点，以更好地符合市场需求。

关于防盗门技术，现有设计相对容易被绕过，尤其是随着全基因组测序技术的进步，遗传信息很少是安全的。然而，开发更强大的基因锁解决方案具有重要的市场潜力。

对于剪接异构体检测，我们当前的设计并未涵盖所有可能的剪接形式。需要一个更全面的方法，特别是针对与分支位点相关的异常剪接事件。

CABIO的刘先生还提出了一个新的应用方向：利用这个系统在发酵过程中操纵酵母的代谢途径，以生产具有不同风味的葡萄酒。

受CABIO建议的启发，我们现在正在研究ADAR系统在酿造和检测动物异常剪接相关疾病方面的潜在应用。

Click here to know more about EVOLYZER

点此进入衍微官网

EVOLYZER is one of our sponsors of us. To promote the development and industrial application of the project tool—a tool based on ADAR enzyme for RNA editing, the iGEM Tsinghua-M team, in order to explore corporate needs and seek expert guidance, sent some members to visit, learn, and exchange at EVOLYZER.

1. Communication Process

1. 交流过程

SessionA: Project Progress Report

第一部分：项目进展报告

Members of the iGEM Tsinghua-M team introduced the project progress to Mr. Chen Bo, the general manager of Yanwei Technology, focusing on three aspects: overall overview, experimental progress, and optimization plan. In the first part, the team members highlighted the mechanism and specific applications of the team's RNA editing project, and elaborated in detail on its application ideas in the "Strain Security system design." In the second part, experimental progress, the team members demonstrated the feasibility of the RNA editing system at the levels of transcription, translation, and expression interaction through experimental results of qPCR, Western Blot, and fluorescence characterization. In the third part, the optimization plan, the team members expressed their expectations for the design of system optimization, including replacing the promoter to increase the expression of ADAR, and changing the number of MS2 on gRNA to improve selectivity.

SessionB: Q&A

第二部分：问答环节

Q1: Are there many cases of market strain theft?
A: The number of patents protecting the strains themselves is not large, and the concept of "strain theft prevention" emerged relatively late. However, the theft of strains is indeed a significant issue. It is alarming that a complete theft industry chain has already been formed internally, with an even open market for pricing targeting specific university laboratories. Moreover, companies also encounter other problems, such as the difficulty for the plaintiff company to provide evidence, and some strains cannot be included in patents. In addition, the scale of punishment for stealing strains is very small and is often limited to a single link. The upstream and downstream cooperative organizations are presumed to be unaware. In summary, both external supervision and internal mutual supervision are weak, and there has been no good solution so far.

Q1：市场上菌种盗窃的情况多吗？
A：保护菌种本身的专利数量并不多，“菌种防盗”的概念出现得相对较晚。但是，菌种的盗窃确实是一个严重的问题。令人担忧的是，内部已经形成了完整的盗窃产业链条，甚至针对特定大学实验室的菌种有公开的市场定价。此外，公司还会遇到其他问题，如原告公司难以提供证据，有些菌种无法纳入专利保护。此外，盗窃菌种的惩罚力度非常小，通常只限于单一环节。上下游的协作组织被推定为不知情。总之，外部监管和内部相互监管都较弱，目前还没有好的解决办法。

Q2: How long is the patent protection period for microbial strains?
A: The patent term is generally twenty years.

Q2：微生物菌种的专利保护期是多久？
A：专利保护期通常为二十年。

Q3: How do companies avoid patent leaks caused by the theft of strains?
A: Generally, companies can accept the method of continuously adding inducers like IPTG to the reaction system to regulate the expression of key proteins, and the cost is not very high; or they can add other substances that have restrictive or regulatory effects, to some extent avoiding being deciphered through sequencing.

Q3：公司如何避免因菌种盗窃导致的专利泄露？
A：通常，公司可以接受向反应系统中不断添加IPTG等诱导剂的方法来调节关键蛋白的表达，成本不是很高；或者可以添加其他具有限制性或调节作用的物质，在一定程度上避免通过测序被破译。

Q4: How are strains stolen? How are key genes stolen?
A: The process of stealing strains is actually not complicated. It might be as simple as walking around the factory area, and the bacteria could be carried away on one's body. Stealing strains from the refrigerator is relatively rare, and most of the people who steal strains are insiders. It is highly likely that key genes are stolen through sequencing for gene sequencing. However, the gene sequence is not unchanging. For example, the S9114 Bacillus subtilis strain will naturally change its gene sequence under different environments and process treatments in different fermentation factories, and the yield will naturally increase. This means that there may be cases where the marked detection is a certain strain, but there are also differences in actual use, which is more complex.

Q4：菌种是如何被盗的？关键基因是如何被盗的？
A：盗窃菌种的过程实际上并不复杂。可能就像在工厂区域走动一样简单，细菌就可能附着在身上被带走。从冰箱中盗窃菌种相对较少，大多数盗窃菌种的人是内部人士。关键基因很可能是通过基因测序被盗窃的。然而，基因序列并非一成不变。例如，S9114枯草杆菌在不同环境下和不同发酵工厂的不同工艺处理下，其基因序列会自然变化，产量自然会提高。这意味着有时标记检测是某种菌种，但实际使用中也存在差异，情况更加复杂。

Q5: Do you have any suggestions for improvement on our project?
A: Consider adding multiple mechanisms to achieve strategy integration. You could utilize yeast's self-destruction, shearing, and transposition to amplify the effects of RNA variation. Additionally, you might consider photocontrol, such as allowing colonies to express specific substances only under a certain sequence of light exposure, and not being able to do so outside of this environment. To enhance environmental secrecy, you could also place the light source inside the fermentation tank, so that those who steal the yeast cannot deduce the cultivation conditions through the factory environment. Furthermore, the yield of compounds by yeast is not high. If not limited by laboratory constraints, you might consider other microbial strains.

Q5：对我们的项目有什么改进建议吗？
A：考虑增加多种机制来实现策略整合。可以利用酵母的自我毁灭、剪切和转座来放大RNA变异的效果。此外，可以考虑光控，例如只允许菌落在一定顺序的光照下表达特定物质，而在这种环境之外无法做到。为了增强环境的保密性，也可以将光源放置在发酵罐内部，这样盗窃酵母的人就无法通过工厂环境推断培养条件。此外，酵母产生的化合物产量不高。如果不受到实验室条件的限制，可以考虑使用其他微生物菌种。

2. Laboratory Visit

2. 实验室参观

After the interview concluded, the team members, proceeded to visit the company's laboratory and the internal facilities of the factory for observation and learning. This gives us a better understanding of the actual production.

采访结束后，团队成员接着参观了公司的实验室和工厂内部设施，进行观察和学习。这有助于我们更好地了解实际生产情况。

In our interactions with CCiC as well as the Beijing iGEM team, and online exchanges with AIS-China, we discussed the progress of our respective projects, shared the issues we encountered and the final solutions we found, Even shared different perspectives on the same article.. We had a collision of ideas, and each group also absorbed the experiences of each other.

在与CCiC以及北京iGEM团队的互动中，以及与AIS-China的在线交流中，我们讨论了各自项目的进展，分享了我们遇到的问题和最终找到的解决方案，甚至分享了对同一篇文章的不同观点。我们产生了思想的碰撞，每个团队也吸收了彼此的经验。

We visited the Bayi School in Beijing and found that high school students lack a systematic understanding of synthetic biology. However, in our enthusiastic exchanges, we felt their thirst for knowledge and curiosity. We introduced our project progress in a more accessible way, received many direct but crucial questions and shortcomings, and also allowed us to constantly reflect on and improve our project.

我们访问了北京八一学校,发现高中生对合成生物学缺乏系统的了解。然而,在我们热情的交流中,我们感受到了他们对知识的渴望和好奇心。我们以更易于理解的方式介绍了我们的项目进展,收到了许多直接但关键的问题和指出的不足,这也让我们不断反思和改进我们的项目。

[1] Narfström, K., Holland Deckman, K. & Menotti-Raymond, M. The domestic cat as a large animal model for characterization of disease and therapeutic intervention in hereditary retinal blindness. J. Ophthalmol. 2011, 906943 (2011).
[2] Travor, M.D. Preclinical models of retinitis pigmentosa. Methods Mol. Biol. 2023, 181–215 (2023).

[1] Narfström, K., Holland Deckman, K. & Menotti-Raymond, M. The domestic cat as a large animal model for characterization of disease and therapeutic intervention in hereditary retinal blindness. J. Ophthalmol. 2011, 906943 (2011).
[2] Travor, M.D. Preclinical models of retinitis pigmentosa. Methods Mol. Biol. 2023, 181–215 (2023).

1. Academic Research Institutions

1. 学术研究机构

Specific Needs: There is a requirement for professional-grade microbial strain protection solutions to ensure the security of sensitive biological materials and research outcomes.

具体需求：需要专业级别的微生物菌株保护解决方案，以确保敏感生物材料和研究成果的安全。

Existing Solutions: Standard biosafety equipment and protocols may not be suitable for all types of research applications or laboratories with highly specialized needs.

现有方案：标准的生物安全设备和协议可能不适用于所有类型的研究应用或具有高度专业化需求的实验室。

Disadvantages: Current solutions may not provide a sufficient level of security, especially in preventing the illegal transfer and use of strains.

缺点短板：当前解决方案可能无法提供足够的安全级别，尤其是在防止菌株的非法转移和使用方面。

Ultimate Needs: There is a need for a solution specifically designed for research environments that can comprehensively safeguard the security of biological materials and research findings.

最终需求：需要一种专为研究环境设计的解决方案，能够全面保障生物材料和研究成果的安全。

2. Large-Scale Biotech Companies

2. 大型生物技术公司

Specific Needs: Customized and scalable strain theft prevention for complex R&D and production.

具体需求：为复杂的研发和生产定制和可扩展的菌株防盗解决方案。

Existing Solutions: Generic biosafety products requiring custom development for specific needs.

现有方案：通用生物安全产品需要针对特定需求进行定制开发。

Disadvantages: High custom development costs and lack of flexibility for new R&D requirements.

缺点短板：高昂的定制开发成本，缺乏针对新研发需求的灵活性。

Ultimate Needs: Customizable, scalable, cost-effective strain theft prevention solutions.

最终需求：Customizable, scalable, cost-effective strain theft prevention solutions.

3. Small to Medium-Scale Biotech Companies

3. 中小规模生物技术公司

Specific Needs: Comprehensive protection of microbial strains from unauthorized use or theft, safeguarding intellectual property.

具体需求：全面保护微生物菌株免受未经授权的使用或盗窃，保护知识产权。

Existing Solutions: Adding substances to strains for a certain level of protection against sequencing.

现有方案：向菌株添加物质，以在一定程度上防止测序。

Disadvantages: High costs and complex logistics for substance addition.

缺点短板：添加物质的高成本和复杂物流。

Ultimate Needs: Economical, reliable solutions with intrinsic anti-theft mechanisms like self-destruction under specific conditions.

最终需求：经济实惠、可靠的解决方案，具有内在的防盗机制，如在特定条件下自我销毁。

1. SWOT Analysis

1. SWOT 分析

S - Strengths

S - 优势

High Confidentiality: Post-theft inutility due to colony death or gene absence.

高保密性：被盗后由于菌落死亡或基因缺失而无法使用。

Technological Advancement: Innovative, hard-to-imitate biotechnological methods.

技术先进性：创新的、难以模仿的生物技术方法。

Legal Protection: Patent protection for related technologies or mechanisms.

法律保护：相关技术或机制的专利保护。

W - Weaknesses

W - 劣势

R&D Challenges: Technical hurdles requiring interdisciplinary expertise.

研发挑战：需要跨学科专业知识的技术障碍。

Operational Complexity: Complex biological operations and precise control needed.

操作复杂性：需要复杂的生物操作和精确控制。

Maintenance Costs: Continuous investment in funds, equipment, and personnel.

维护成本：需要持续投入资金、设备和人员。

O - Opportunities

O - 机会

Market Demand: Growing need for high-confidentiality strains in various biotech sectors.

市场需求：各种生物技术领域对高保密性菌株的需求日益增长。

Technical Cooperation: Partnerships for joint development of confidentiality tech.

技术合作：合作伙伴关系以共同开发保密技术。

Policy Support: Government support for biosafety and IP protection.

政策支持：政府对生物安全和知识产权保护的支持。

T - Threats

T - 威胁

Technological Cracking Risk: Potential for advanced tech to be compromised.

技术破解风险：先进技术可能被破解的潜在风险。

Ethical and Legal Issues: Controversies and varying legal restrictions on techniques.

伦理和法律问题：围绕技术的争议和不同的法律限制。

Market Competition: Other companies developing alternative confidentiality technologies.

市场竞争：其他公司开发替代保密技术。

2. PEST Analysis

2. PEST 分析

P - Political

P - 政策

Scientific Research Support Policies: Funding and incentives for high-tech fields.

科研支持政策：对高科技领域的资金支持和激励措施。

Regulatory Protection: Legal safeguards for intellectual property.

监管保护：对知识产权的法律保护。

E - Economic

E - 经济

Adequate Funding: Financial support for research and commercialization.

充足资金：对研究和商业化的财政支持。

Market Potential: Significant opportunities in the growing biotech industry.

市场潜力：在不断增长的生物技术行业中的重大机遇。

S - Social

S - 社会

Demand from Research Institutions and Enterprises: Drive for IP protection in biotech.

研究机构和企业的需求：生物技术领域对知识产权保护的驱动力。

Intellectual Property Awareness: Positive social attitudes towards IP protection.

知识产权意识：社会对知识产权保护的积极态度。

T - Technological

T - 技术

Innovation Drive: Advances in biotech and IT for enhanced confidentiality measures.

创新驱动：生物技术和信息技术的进步，增强保密措施。

Technological Integration: Improved efficiency and security with new tech integration.

技术整合：新技术整合提高效率和安全性。

1. Marketing Channels Analysis

1. 营销渠道分析

Exhibitions and Trade Shows: Showcase products, engage with potential customers, and gather feedback.

展览和贸易展：展示产品，与潜在客户互动，收集反馈。

Presentations and Seminars: Demonstrate expertise and promote product features through educational content.

演讲和研讨会：通过教育性内容展示专业知识并推广产品特点。

Direct Mail and Email Marketing: Send personalized product info and promotions to target customers.

直邮和电子邮件营销：向目标客户发送个性化的产品信息和促销。

Digital Marketing: Increase online visibility through SEO, online ads, and social media.

数字营销：通过搜索引擎优化、在线广告和社交媒体增加在线曝光率。

Social Media Marketing: Post product info and interact with the audience on platforms like LinkedIn, Twitter, and Facebook.

社交媒体营销：在LinkedIn、Twitter和Facebook等平台上发布产品信息并与受众互动。

For the "inclusivity" section, members of our team designed a short story based on the basic principles of RNAssay called "The Adventures of the Cat", which was caricatured and translated into different languages. In this way, we have made it easier for people of different countries and ages to understand the principles of the project in a more accessible way.

对于"包容性"部分，我们团队的成员基于RNAssay的基本原理设计了一个简短的故事，名为"猫咪的冒险"。这个故事被制作成漫画并翻译成不同的语言。通过这种方式，我们让来自不同国家和年龄段的人能够以更易理解的方式掌握项目的原理。

世界の果てには、終止子の扉があると言われています。扉が開くと、中に山積みにされていた宝物たちが、まばゆい光を放ちながら虹をつくり、空を横切っていきました。 無数のネコ王国の民がこの門を開け、この無尽蔵の財宝を手に入れようと山を越えてやってきました。何百年もの間成功した猫はいませんでしたなぜなら──あの奇妙な錠です。通常の猫の爪には3つの「隙間」がありますACCロックの隙間はUAGで、両者は一致しません。 ある日、猫の戦士RNAssayも世界の果てに来ました。そっと爪をかけてみましたが、残念ながら、鍵はびくともしません。しかし、Rさんはあきらめませんでした。彼はひらめいて、ADARという猫の爪切りを取り出しました。ADARを使って錠の形状を丁寧に修正し、真ん中のAをIに修正しました。それが終わると、Rさんはまた爪をかけました。すると不思議なことが起こりました。かちり、と鍵がひらいて、まぶしい光が、ドアの中からさし出しました。

世界の果てには、終止子の扉があると言われています。扉が開くと、中に山積みにされていた宝物たちが、まばゆい光を放ちながら虹をつくり、空を横切っていきました。 無数のネコ王国の民がこの門を開け、この無尽蔵の財宝を手に入れようと山を越えてやってきました。何百年もの間成功した猫はいませんでしたなぜなら──あの奇妙な錠です。通常の猫の爪には3つの「隙間」がありますACCロックの隙間はUAGで、両者は一致しません。 ある日、猫の戦士RNAssayも世界の果てに来ました。そっと爪をかけてみましたが、残念ながら、鍵はびくともしません。しかし、Rさんはあきらめませんでした。彼はひらめいて、ADARという猫の爪切りを取り出しました。ADARを使って錠の形状を丁寧に修正し、真ん中のAをIに修正しました。それが終わると、Rさんはまた爪をかけました。すると不思議なことが起こりました。かちり、と鍵がひらいて、まぶしい光が、ドアの中からさし出しました。

Die legende besagt, dass am ende der welt ein beendetes tor liegt. Wenn die türen geöffnet werden, wachsen da berge Von schätze, die bunt leuchtenden lichtern emporragen, regenbogen bilden sich und schweben quer über den himmel. Tausende Von elvis-unterbewohnern Kamen her und versuchten, die tür für den unerschöpflichen schatz zu öffnen. Aber in tausenden Von jahren schaffte es keine katze. Denn das seltsame schloss. Es gibt drei "leerräume" : ACC; Und die lücke, die geschlossen war, war eine UAG, die nicht zu einer ag gehörte. Mit der katze und dem helden RNAssay ging die welt zu ende. Er hat ganz leicht die klauen zu einem schloss gehalten, leider flattert das. Aber kleiner R gibt nicht auf. Der film drehte sich und legte eine katze mit krallen raus ADAR. Er hat durch ADAR das schloss gründlich bearbeitet und aus der mitte ein großes A gemacht. Nachdem die kleine R fertig ist, legt sie die krallen wieder an. Jetzt geschieht etwas ganz wundervolles! Das schloß schloß zusammen, ein donnerschlag, und der grelle lichtblitz schoß durch die tür.

Die legende besagt, dass am ende der welt ein beendetes tor liegt. Wenn die türen geöffnet werden, wachsen da berge Von schätze, die bunt leuchtenden lichtern emporragen, regenbogen bilden sich und schweben quer über den himmel. Tausende Von elvis-unterbewohnern Kamen her und versuchten, die tür für den unerschöpflichen schatz zu öffnen. Aber in tausenden Von jahren schaffte es keine katze. Denn das seltsame schloss. Es gibt drei "leerräume" : ACC; Und die lücke, die geschlossen war, war eine UAG, die nicht zu einer ag gehörte. Mit der katze und dem helden RNAssay ging die welt zu ende. Er hat ganz leicht die klauen zu einem schloss gehalten, leider flattert das. Aber kleiner R gibt nicht auf. Der film drehte sich und legte eine katze mit krallen raus ADAR. Er hat durch ADAR das schloss gründlich bearbeitet und aus der mitte ein großes A gemacht. Nachdem die kleine R fertig ist, legt sie die krallen wieder an. Jetzt geschieht etwas ganz wundervolles! Das schloß schloß zusammen, ein donnerschlag, und der grelle lichtblitz schoß durch die tür.

La légende raconte qu’au bout du monde, il y a une porte de la fin des fils. Quand la porte fut ouverte, la montagne de trésors à l’intérieur brillait d’un éclat multicolore, formant un arc-en-ciel qui traversait tout le ciel. De nombreux habitants du royaume des miaouls ont traversé les montagnes pour tenter d’ouvrir cette porte et acquérir ce trésor inépuisable. Mais depuis des milliers d’années, aucun chat n’a réussi. Parce que - la serrure étrange. Il y a trois «vides» dans les pattes d’un chat normal: ACC; Et l’espace dans la serrure est UAG, les deux ne correspondent pas. Un jour, le guerrier chat RNAssay arrive aussi au bout du monde. Il pose doucement ses pattes sur la serrure, qui, malheureusement, ne bouge pas. Mais le petit R n’a pas abandonné. Il a eu un coup d’âme et a sorti une pelle à pattes de chat - ADAR. Il A sérieusement modifié la forme de la serrure avec ADAR, en changeant le A central en I. Quand cela a été fait, le petit R a reposé ses pattes. C’est alors que quelque chose d’étonnant s’est produit! La serrure «claquait» et une lumière éblouissante s’échappait de la porte.

La légende raconte qu’au bout du monde, il y a une porte de la fin des fils. Quand la porte fut ouverte, la montagne de trésors à l’intérieur brillait d’un éclat multicolore, formant un arc-en-ciel qui traversait tout le ciel. De nombreux habitants du royaume des miaouls ont traversé les montagnes pour tenter d’ouvrir cette porte et acquérir ce trésor inépuisable. Mais depuis des milliers d’années, aucun chat n’a réussi. Parce que - la serrure étrange. Il y a trois «vides» dans les pattes d’un chat normal: ACC; Et l’espace dans la serrure est UAG, les deux ne correspondent pas. Un jour, le guerrier chat RNAssay arrive aussi au bout du monde. Il pose doucement ses pattes sur la serrure, qui, malheureusement, ne bouge pas. Mais le petit R n’a pas abandonné. Il a eu un coup d’âme et a sorti une pelle à pattes de chat - ADAR. Il A sérieusement modifié la forme de la serrure avec ADAR, en changeant le A central en I. Quand cela a été fait, le petit R a reposé ses pattes. C’est alors que quelque chose d’étonnant s’est produit! La serrure «claquait» et une lumière éblouissante s’échappait de la porte.

Легенда гласит, что в конце мира есть врата, которые закрывают сыны.Когда дверь была открыта, горы сокровищ сияли ярким светом, создавая радугу, охватывающую все небо. Дети бесчисленных мяу-элвис прошли через горы, чтобы попасть сюда, чтобы попытаться открыть эту дверь и получить это безграничное сокровище.Но за тысячи лет ни одна кошка не преуспела. Потому что... этот странный замок.Лапа нормальной кошки имеет три "пробела" : ACC;И брешь в замке для UAG, оба не совпадают. Однажды, воин котов, эрнасэй, также прибыл на край света.Он мягко кладет когти на замок, который, к сожалению, не открывается.Но малышка р не сдалась.У него возникла идея и он вытащил лопату для кошачьих когтей-адар.Он серьезно изменил форму замка с помощью ADAR, чтобы превратить среднюю A в I.После того, как малыш р закончил, он снова запустил когти.И тогда случилось что-то чудесное!Замок "щелк-чао" захлопнулся и ослепительный свет вышел из двери.

Легенда гласит, что в конце мира есть врата, которые закрывают сыны.Когда дверь была открыта, горы сокровищ сияли ярким светом, создавая радугу, охватывающую все небо. Дети бесчисленных мяу-элвис прошли через горы, чтобы попасть сюда, чтобы попытаться открыть эту дверь и получить это безграничное сокровище.Но за тысячи лет ни одна кошка не преуспела. Потому что... этот странный замок.Лапа нормальной кошки имеет три "пробела" : ACC;И брешь в замке для UAG, оба не совпадают. Однажды, воин котов, эрнасэй, также прибыл на край света.Он мягко кладет когти на замок, который, к сожалению, не открывается.Но малышка р не сдалась.У него возникла идея и он вытащил лопату для кошачьих когтей-адар.Он серьезно изменил форму замка с помощью ADAR, чтобы превратить среднюю A в I.После того, как малыш р закончил, он снова запустил когти.И тогда случилось что-то чудесное!Замок "щелк-чао" захлопнулся и ослепительный свет вышел из двери.

据说在世界的尽头有一扇终止子之门。当门打开时，堆积在里面的宝物散发出耀眼的光芒，形成彩虹横跨天空。 无数的猫王国子民翻山越岭来到这里，试图打开这扇门，获取这无穷无尽的财宝。数百年来，没有一只猫成功过，原因在于——那个奇怪的锁。普通猫爪有三个"间隙"ACC，而锁的间隙是UAG，两者不匹配。 有一天，一位名叫RNAssay的猫战士也来到了世界尽头。他轻轻地用爪子试了试，但遗憾的是，锁纹丝不动。然而，R先生并没有放弃。他灵光一闪，拿出了一个叫做ADAR的修剪器。他用ADAR小心翼翼地修改了锁的形状，将中间的A改成了I。完成后，R再次用爪子试了试。于是不可思议的事情发生了。咔嗒一声，锁开了，耀眼的光芒从门内透了出来。

据说在世界的尽头有一扇终止子之门。当门打开时，堆积在里面的宝物散发出耀眼的光芒，形成彩虹横跨天空。 无数的猫王国子民翻山越岭来到这里，试图打开这扇门，获取这无穷无尽的财宝。数百年来，没有一只猫成功过，原因在于——那个奇怪的锁。普通猫爪有三个"间隙"ACC，而锁的间隙是UAG，两者不匹配。 有一天，一位名叫RNAssay的猫战士也来到了世界尽头。他轻轻地用爪子试了试，但遗憾的是，锁纹丝不动。然而，R先生并没有放弃。他灵光一闪，拿出了一个叫做ADAR的修剪器。他用ADAR小心翼翼地修改了锁的形状，将中间的A改成了I。完成后，R再次用爪子试了试。于是不可思议的事情发生了。咔嗒一声，锁开了，耀眼的光芒从门内透了出来。