After six month of effort, team members of Tsinghua-M 2024 not only proved the feasibility of RNAssay but also demonstrated its functionality in various application fields including in situ detection of multigene expression, Strain Security System and in vivo dynamic monitoring of splice variants. We provide iGEMers with biological information of RNAssay, useful possible application strategies and other useful tools to utilize RNAssay into their own project, including:

经过六个月的努力，我们不仅证明了RNAssay的可行性，还证明了其在各种应用领域的功能，包括多基因表达的原位检测、菌株安全系统和剪接变体的胞内动态监测。我们为iGEMers提供RNAssay的生物信息、有用的可能应用策略和其他有用的工具，以将RNAssay应用于他们自己的项目中，包括：

1.Basic biological information of ADAR1_p150 and ADAR2(DD)E488Q–MCP in yeast

1.酵母中ADAR1_p150和ADAR2（DD）E488Q-MCP的基本生物学信息

2.Well characterized parts used in RNAssay

2.基于RNAssay的Parts

3.Work flow of in situ detection of multigene expression

3.多基因表达原位检测工作流

4.Work flow of engineering strain security system

4.菌株防盗门工作流

5.Work flow of in vivo dynamic monitoring of splice variant to help the next generation organ transplantation

5.助力下一代器官移植技术的剪接异构体动态传感工作流

6.Prediction model to guide ogRNA design

6.指导ogRNA设计的预测模型

7.Bioinformatics data of the capacity for splice variants detection

7.有助于剪接异构体检测的生物信息学数据

8.ADAR-Cat game for hands-on practicing of the basic principle of RNAssay

8.基于项目的推广游戏

9.Children's book to better explain the mechanism of RNAssay to the public

9.基于项目的漫画书

We have carried out a series of experiment and have well-characterized the two ADAR enzyme we used. Many of the characterization were first down in yeast and we provide valuable information including:

我们进行了一系列实验，并对我们使用的两种ADAR酶进行了充分的表征。许多表征工作最初是在酵母中完成的。基于此，我们获得了大量关于ADAR的宝贵的信息，包括：

1.Successful expression of ADAR1_p150 and ADAR2(DD)E488Q-MCP, characterized by qPCR and western blot. We also proved that utilizing stronger promoter of yeast(TEF1) can greatly boost the expression of the two enzymes.

1.通过qPCR和蛋白质印迹（western blot）进行表征，我们验证了ADAR1_p150和ADAR2(DD)E488Q-MCP的成功表达。此外，我们还证明使用酵母的更强启动子（TEF1）可以大大提高这两种酶的表达水平。

2.Proved cytotoxicity of ADAR1_p150 and ADAR2(DD)E488Q-MCP for yeast, characterized by the growth curve of the yeast transformed with ADAR. Our experiment shows that both ADARs can suppress the growth of yeast while ADAR2 display stronger cytotoxicity. This information will guide further optimization of RNAssay.

2.我们证实了ADAR1_p150和ADAR2(DD)E488Q-MCP对酵母的细胞毒性，这是通过转化了ADAR的酵母的生长曲线来表征的。我们的实验表明，这两种ADAR都能抑制酵母的生长，而ADAR2表现出更强的细胞毒性。这将指导RNAssay的进一步优化。

3.Nuclear-cytoplasmic localization of ADAR1_p150 and ADAR2(DD)E488Q-MCP, characterized by fluorescence confocal microscopy.

3.我们通过荧光共聚焦显微镜，确定了ADAR1_p150和ADAR2(DD)E488Q-MCP的核质定位情况，

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We have constructed dozens of plasmids with a lot of parts, which have been successfully characterized by qPCR, western blot, fluorescence confocal microscopy and flow cytometry. Some parts are first introduced into iGEM parts registry, including:

我们已经构建了数十个含有大量parts的质粒，并已通过qPCR、Western blot、荧光共聚焦显微镜和流式细胞术成功进行了表征。其中一些部件首次被引入iGEM parts registry，包括：

1.HTA1 promoter: useful promoter in Saccharomyces cerevisiae.

1.HTA1 promoter: useful promoter in Saccharomyces cerevisiae.

2.ECM10 terminator: useful terminator in Saccharomyces cerevisiae.

2.ECM10 terminator: useful terminator in Saccharomyces cerevisiae.

3.ADAR1_p150: codon optimized ADAR1_p150 for Saccharomyces cerevisiae with proved RNA editing activity.

3.ADAR1_p150: codon optimized ADAR1_p150 for Saccharomyces cerevisiae with proved RNA editing activity.

4.ADAR2(DD)E488Q–MCP: codon optimized ADAR2(DD)E488Q–MCP for Saccharomyces cerevisiae with proved RNA editing activity.

4.ADAR2(DD)E488Q–MCP: codon optimized ADAR2(DD)E488Q–MCP for Saccharomyces cerevisiae with proved RNA editing activity.

5.ADAR1_p150-mScarlet: fusion protein of ADAR1_p150 and mScarlet to observe the location of ADAR1_p150.

5.ADAR1_p150-mScarlet: fusion protein of ADAR1_p150 and mScarlet to observe the location of ADAR1_p150.

6.ADAR2(DD)E488Q–MCP-mScarlet: fusion protein of ADAR2(DD)E488Q–MCP and mScarlet to observe the location of ADAR2(DD)E488Q–MCP.

6.ADAR2(DD)E488Q–MCP-mScarlet: fusion protein of ADAR2(DD)E488Q–MCP and mScarlet to observe the location of ADAR2(DD)E488Q–MCP.

7.eGFP-eBFP sensor: sensor RNA with eGFP as basic gene and eBFP as reporter gene.

7.eGFP-eBFP sensor: sensor RNA with eGFP as basic gene and eBFP as reporter gene.

8.mScarlet-eGFP sensor: sensor RNA with mScarlet as basic gene and eGFP as reporter gene, performing better than eGFP-eBFP sensor.

8.mScarlet-eGFP sensor: sensor RNA with mScarlet as basic gene and eGFP as reporter gene, performing better than eGFP-eBFP sensor.

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Through experiment, we have fished out a feasible work flow for in situ detection of multigene expression. With this, iGEMers of the future are able to:

通过实验，我们筛选出了一种可行的工作流程，用于原位检测多基因表达。有了这个流程，未来的iGEM参赛者能够：

1.Design their own RNA sensor

1.设计他们自己的RNA sensor

2.Achieve in situ detection of target gene expression easily

2.轻松实现目标基因表达的原位检测

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Through experiment, we have fished out a feasible work flow for engineering strain security system. With this, iGEMers of the future are able to:

通过实验，我们筛选出了一种可行的工作流程，用于原位检测多基因表达。有了这个流程，未来的iGEM参赛者能够：

1.Design their own RNA sensor that corresponding with different stress responsing gene

1.设计自己的RNA传感器，与不同的应激反应基因相对应

2.Construct and select the microorganism with the security system

2.构建和选择具有安全系统的微生物

3.Utilizing different environmental stress as the key to the lock

3.利用不同的环境应力作为锁的钥匙

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Through experiment, we have fished out a feasible work flow for in vivo dynamic monitoring of splice variant to help next generation organ transplantation. With this, iGEMers of the future are able to:

通过实验，我们找到了一种可行的剪接异构体胞内动态监测工作流程，以帮助新一代器官移植。在这套流程的基础上，未来的iGEMers能够：

1.Design their own RNA sensor that can sense different cancerization signals

1.设计自己的RNA传感器，可以感知某些特定的癌变信号

2.Utilize RNAssay to kill cells with carcinomatous features, thus guaranteeing the safety of next generation organ transplantation.

2.利用RNAssay杀死具有癌变特征的细胞，从而保证新一代器官移植的安全

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Using experiment data, we developed our own prediction model using molecular dynamics simulation, molecular docking and fitting algorithm, which will help future iGEMers to:

利用实验数据，我们使用分子动力学模拟、分子对接和拟合算法开发了自己的预测模型，这将有助于未来的iGEMers：

1.Better design the ogRNA candidates.

1.更好地设计 ogRNA 候选序列

2.Select one ogRNA from candidates with possibly best performance.

2.从候选序列中选择一个可能具有最佳性能的 ogRNA

3.Observe the docking condition of dsRNA and ADAR.

3.观察 dsRNA 和 ADAR 的接合情况

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Applying bioinformatics methods, we successfully build our model to evaluated the capacity of RNAssay for splice variants detection in human. With the code we provided, future iGEMers will be able to:

通过某些生物信息学手段，我们构建了自己的模型，以评估RNAssay在人类剪接异构体检测中的能力。在我们提供的代码的基础上，未来的iGEM参与者将能够：

1.Figure out whether their splice variants of interest can be monitored by RNAssay

1.确定他们感兴趣的剪接变体是否可以通过 RNAssay 进行监测

2.Evaluate the capacity of RNAssay for splice variants detection in other organisms

2.评估 RNAssay 在其他生物中检测剪接变体的能力

3.Optimize the algorithm with their own experiment data

3.使用他们自己的实验数据优化算法

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We wrote a game that is integrated with the basic mechanism of RNAssay, which is easy to pick up. We developed this game with the aim to:

我们编写了一个与RNAssay基本机制相结合的游戏，并且我们让这个游戏很容易上手。我们开发这个游戏的目的是为了：

1.Serve as an engaging, hands-on way to help the public understand our design principles

1.作为一种引人入胜的、实践性的方式，帮助大家理解我们的设计原则。

2.Integrate the game into their science curriculum to make learning more dynamic

2.将游戏融入科学课程，使学习更加生动活泼。

3.Be used at public science events, museums, and exhibitions to engage the broader community with synthetic biology. Interactive experiences like games can capture the attention of people of all ages, encouraging them to explore scientific ideas in a fun, approachable format.

3.在公共科学活动、博物馆和展览中使用，以吸引更广泛的社区参与合成生物学。像游戏这样的互动体验可以吸引各个年龄段人群的注意力，鼓励他们以一种有趣、易于接受的方式探索科学理念。

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We created delicacy Children's book to help young kids and public better grasp our design principles. We believe the children's book can be used:

我们创作了精美的儿童漫画书籍，以帮助公众更好地理解我们的设计理念。这本儿童书可以用于：

1.To introduce young audiences to the principles of synthetic biology in a simplified and engaging way, by breaking down complex concepts into fun, age-appropriate narratives and illustrations

1.通过将复杂概念分解成有趣且适合儿童年龄的叙述和插图，以简洁而吸引人的方式向年轻受众介绍合成生物学的原理。

2.In public events, museums, or libraries to educate the general public, including parents and children, about synthetic biology and RNAssay

2.在公共活动、博物馆或图书馆中，向包括父母和儿童在内的普通大众教育合成生物学和RNAssay的知识。

3.As a tool to integrate synthetic biology into the curriculum at the elementary or middle school level.

3.作为一种工具，将合成生物学整合到小学或初中阶段的课程中。

4.To promote inclusivity by targeting diverse audiences, helping bridge the gap in science education between different demographic groups.

4.通过针对不同群体，促进包容性，帮助缩小不同人口群体之间在科学教育方面的差距。

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