| Name | Number | Type | Length | Description |
| PsppA | BBa_K5238012 | Other | 137bp | The PsppA promoter is a strong inducible promoter that regulates gene expression in Lactobacillus plantarum. |
| PsppA+LysKB317 | BBa_K5238013 | Composite | 1031bp | The PsppA promoter controls the expression of the endosome LysKB317, leading to the programmed cell death of Lactobacillus plantarum. |
In this composite part, we have selected the PsppA promoter and the lysin LysKB317 to achieve the self-destruction of Lactobacillus plantarum.
The PsppA promoter, derived from the sakacin P gene cluster, plays a role in regulating gene expression in Lactobacillus plantarum, particularly in the production of bacteriocins, which are typically regulated through a secretion-based peptide quorum-sensing mechanism. In the expression vector we constructed, the PsppA promoter works in concert with other regulatory elements to precisely control the expression of target genes, such as LysKB317. This mechanism is of significant importance for the development of novel microbial control strategies.
In relevant literature[ Sørvig, Elisabeth et al. “High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors.” Microbiology (Reading, England) vol. 151,Pt 7 (2005): 2439-2449. doi:10.1099/mic.0.28084-0], researchers have found that swapping different promoters, such as the PsppA and PorfX promoters, affects the production of GusA and PepN. In experimental studies, it was discovered that the PsppA promoter can enhance the production of GusA in two host strains. Therefore, we chose the PsppA promoter from Lactobacillus plantarum to participate in the expression of target genes in the vector, thereby promoting the expression of the downstream target gene LysKB317 and achieving the function of a self-destruction switch.
The core of our self-destruction switch is the lysin gene LysKB317, which originates from the phage genome. LysKB317 is an enzyme that degrades bacterial cell walls, leading to cell lysis. In our design, the expression of LysKB317 is strictly regulated by the inducible promoter PsppA, ensuring the safety of the engineered Lactobacillus plantarum.
In this component, for the first time, we students have utilized the PsppA promoter to regulate the expression of the self-destruction gene LysKB317. In our experiments, we designed a control group (without inducer), an experimental group (with inducer), and a blank group (vector without the self-destruction switch to exclude the possible impact of the inducer itself on bacteria). We verified the effectiveness of the self-destruction switch by plotting growth curves.
In the experiment, initially, we constructed a control strain (L168-pSIP403-PsppA-empty, abbreviated as EP) and a strain expressing a suicide switch (L168-pSIP403-PsppA-LysKB317, abbreviated as LysKB317). Subsequently, we established control groups (EP, EP+sakacin P) and experimental groups (LysKB317, LysKB317+ sakacin P), with the inducer concentration of sakacin P set at 25 ng/ml. We activated the corresponding bacteria, cultured them overnight, and prepared the culture medium under the respective conditions in a 96-well plate. Finally, we inoculated the activated EP and LysKB317 into the corresponding wells at a ratio of 1:50. After completion, we placed the 96-well plate into a microplate reader and adjusted the automatic timing measurement parameters of the reader: 37°C, 10 s of shaking before measurement, medium shaking intensity, wavelength at 600 nm, and a full-plate detection every hour for a continuous monitoring period of 24 hours.
Below are the growth curves we detected. As shown in the figure, EP (blue circles) represents the empty vector control, EP+ (purple squares) represents the empty vector control with inducer, LysKB317 (black triangles) represents the strain with the suicide switch plasmid but without inducer, and LysKB317+ (red inverted triangles) represents the strain with the suicide switch plasmid and with inducer. We continuously monitored the growth of Lactobacillus plantarum over a 24-hour period, reflecting the growth of bacteria in different wells of the 96-well plate through the OD values detected by the microplate reader. The x-axis represents time in hours, with each small division representing an automatic OD value detection by the microplate reader every hour. The y-axis represents the detected OD values. (n=4)
First, we compared the EP (blue circles), which represents the empty vector control, with the EP+ (purple squares), which represents the control with inducer, to eliminate the influence of the inducer on bacterial growth. As observed from the graph, the purple line is slightly lower than the blue line, indicating that the addition of the inducer indeed has a subtle effect on bacterial growth.
Next, we focused on comparing LysKB317 (black triangles), which represents the strain with the suicide switch plasmid but without inducer, with LysKB317+ (red inverted triangles), which represents the strain with the suicide switch plasmid and with inducer. As observed from the graph, the red line is indeed lower than the black line, and the inhibitory effect on the growth of Lactobacillus plantarum L168 by the group with the suicide switch and inducer is greater than the subtle influence caused by the inducer alone. Therefore, this experiment demonstrates that the PsppA+LysKB317 module can suppress the growth of Lactobacillus plantarum L168.
Additionally, from the graph, we can observe that after the addition of the inducer, around the 10th hour, the growth curve of Lactobacillus plantarum L168 begins to gradually slow down, indicating a decrease in growth rate. After the 12th to 13th hour, the red line with the suicide switch and inducer starts to flatten, indicating that Lactobacillus plantarum L168 has entered the stationary phase of growth.. This indicates that the PsppA+LysKB317 suicide switch can inhibit the growth of Lactobacillus plantarum within 12 to 13 hours. Assuming that our Lactobacillus plantarum L168 is administered as a medication to patients with sakacin P inducer, it can control the growth of Lactobacillus plantarum in the gut within about 12 hours, thereby controlling the amount of small molecules expressed and released by Lactobacillus plantarum. This effectively prevents the accumulation of excessive amounts of small molecules in the body, achieving controllable dosage.
The sakacin P-induced promoter PsppA is being used for the first time in iGEM, and LysKB317 is a lysin targeting the cell wall of Lactobacillus plantarum. Theoretically, this design should effectively regulate the growth of Lactobacillus plantarum; this ensures the safety of Lactobacillus plantarum when applied for therapeutic purposes and prevents biological leakage.
For the treatment of Alzheimer's disease, we identified tetrahydrofolate as an effective LilrB3 binder and produce it using the key enzyme sequence folKE. Tetrahydrofolate binds to LilrB3 receptor, preventing APOE4 interaction and inhibiting Aβ deposition. Accordingly, we genetically engineered Lactobacillus plantarum L168 to biosynthesize theanine and niacin, aiming to develop therapy for Alzheimer's disease, respectively.
We successfully identified a small molecule, tetrahydrofolate, capable of binding to the LilrB3 receptor through SPR biacore experiments. By overexpressing folKE, we successfully constructed a plasmid, pSIP403-P9-folKE, in Lactobacillus plantarum L168, which significantly enhances the production of tetrahydrofolate. The qPCR results demonstrate that the relative expression levels of two genes, folK and folE, were markedly upregulated in the L168 strain containing the pSIP403-P9-folKE plasmid compared to the control group (p＜0.0001).
We also used Liquid Chromatograph Mass Spectrometer (LC-MS) to examine the production of THF in the supernatant of L168.
It’s easy to see the level of THF increased dramatically in L168 containing the pSIP403-P9-folKE plasmid compared to the control group, demonstrating that as expected, the folKE gene was successfully expressed in L168 and produced THF.
Accordingly, we genetically engineered Lactobacillus plantarum L168 to biosynthesize theanine and niacin, aiming to develop therapy for Alzheimer's disease, respectively.
We identified the sequences of the key genes glnA and pncA, and designed both upstream and downstream primers for them. The selected vector for this purpose was pSIP403. Subsequently, we designed and constructed the two plasmids in the DH5α prior to introducing it into the sensory Lactobacillus plantarum L168 to produce theanine and niacin.
As shown in the figure 3, these are our qPCR results. The left group represents the transcription level of glnA, and the right one represents pncA. The qPCR results indicate that our designed plasmid effectively and significantly upregulated the key gene expression of both theanine and niacin in L168 compared to the control group respectively.
For depression, utilizing molecular docking and SPR, we identified inosine as a molecule capable of decoupling SERT-nNOS. We engineered Lactobacillus plantarum L168 to produce inosine by incorporating the key enzyme gsk. The engineered probiotics release inosine in the gut, which then travels through the bloodstream to the brain, disconnecting SERT from nNOS to alleviate depressive symptoms.
By overexpressing gsk, we successfully constructed a plasmid, pSIP403-P9-gsk, in Lactobacillus plantarum L168, which significantly enhances the production of inosine. The qPCR results demonstrate that the relative expression levels of gsk, were markedly upregulated in the L168 strain containing the pSIP403-P9- gsk plasmid compared to the control group （p＜0.0001）
We also used Liquid Chromatograph Mass Spectrometer (LC-MS) to examine the production of inosine in the supernatant of L168.
It’s easy to see the level of inosine increased dramatically in L168 containing the pSIP403-P9-gsk plasmid compared to the control group, demonstrating that as expected, the gsk gene was successfully expressed in L168 and produced inosine.
Accordingly, we genetically engineered Lactobacillus plantarum L168 to biosynthesize theanine and niacin, aiming to develop therapy for Alzheimer's disease, respectively.
We identified the sequences of the key genes glnA and pncA, and designed both upstream and downstream primers for them. The selected vector for this purpose was pSIP403. Subsequently, we designed and constructed the two plasmids in the DH5α prior to introducing it into the sensory Lactobacillus plantarum L168 to produce theanine and niacin.
As shown in the figure 3, these are our qPCR results. The left group represents the transcription level of glnA, and the right one represents pncA. The qPCR results indicate that our designed plasmid effectively and significantly upregulated the key gene expression of both theanine and niacin in L168 compared to the control group respectively.
During the 11th China iGEMer Conference (CCiC), the NJMU-CHINA team had the privilege of attending a lecture by iGEM Liaison Officer Ms. Bao Yuhan on "How to Improve Your Human Practices." Ms. Bao provided an in-depth explanation of the Human Practices feedback model and the importance of the maturity evaluation system from a professional perspective, which gave us a deeper understanding of reflection and improvement within our team activities. Inspired by this, the NJMU-CHINA team decided to design a maturity model tailored to our characteristics, aiming to better guide and evaluate our Human Practices activities in our synthetic biology project, thus further promoting project improvement and development.
While designing the maturity model, we integrated the six dimensions recommended by the iGEM website and, after multiple discussions and reflections, optimized and consolidated these dimensions to create the "Holistic Maturity Model" (HMM of NJMU-CHINA). This model encompasses five core dimensions: Reflection, Responsibility, Responsiveness, Innovativeness, and Principle-Centered. We believe these five dimensions comprehensively cover every aspect of team activities, from thought processes to actions, serving as key indicators for evaluating the maturity of Human Practices activities.
1. Five Core Dimensions of the Model
①Reflection: Reflection is the foundation of continuous team improvement, implying an in-depth review and enhancement of activities. Our team conducts systematic reflection after every activity, evaluating the outcomes and summarizing lessons learned. After applying the maturity model, we place greater emphasis on using reflective results to guide and improve future activities, ensuring substantial progress with each endeavor. We have developed from unconscious reflection to making it an integral part of our team culture, establishing a comprehensive reflection mechanism.
②Responsibility: Conducting a synthetic biology project requires a sense of responsibility toward society, the environment, and all stakeholders. Our model emphasizes the need for the team to consider the impact of their actions on society at every stage, fully accounting for all interests involved. After implementing the model, the team's sense of responsibility has significantly improved, particularly during activities in collaboration with communities and schools. The team demonstrated a high degree of responsibility for the outcomes, actively taking measures to mitigate any potential negative impacts.
③Responsiveness: Responsiveness refers to the team's ability to adapt to external changes and feedback. We recognize the importance of being flexible in adjusting activity plans and promptly addressing societal concerns in synthetic biology practices. By applying the HMM model, we have established an effective response mechanism during activity planning and execution, allowing us to quickly adapt to environmental changes and adjust based on feedback. This high level of responsiveness has made our activities more aligned with actual needs, garnering more social recognition.
④Innovativeness: Innovation is the core driving force behind continuous project development. The team constantly explores new forms and methods when designing activities to ensure they inspire participants' interest and enthusiasm. With guidance from the HMM model, we have progressed from tentative to systematic innovation, continuously introducing creative and impactful initiatives in our activities. For example, we combined modern social media to disseminate knowledge about synthetic biology, presenting it in vivid and engaging ways to the public, greatly expanding our impact.
⑤Principle-Centered: Adhering to principles and ethical standards is crucial throughout the activities. The HMM model helped us recognize this and integrate principle-centeredness into our daily practices. From planning to execution, we consistently adhere to ethical and moral standards, ensuring every action reflects the team's integrity. This has not only enhanced our social credibility but also laid a solid foundation for the project’s long-term development.
2. Application and Effects of the Maturity Model
In the past few months, we have successfully applied the "Holistic Maturity Model" (HMM) to various team activities, achieving remarkable results. The model’s application not only helped us evaluate the effectiveness of our activities but also provided clear directions for improvement. Specifically, in terms of reflection, we established a systematic reflection process through the HMM model, ensuring that experiences were effectively summarized after each activity and applied to subsequent work. Regarding responsibility, the model guided us to prioritize social responsibility, proactively respond to feedback, and adjust activity plans as needed. The model also helped us build an efficient feedback mechanism, ensuring a prompt response to challenges. In terms of innovation, our activities were enriched by the constant emergence of creative ideas, such as using new media for knowledge dissemination and organizing interactive online and offline activities. Lastly, the HMM model ensured that we maintained our adherence to ethical and moral standards, reflecting the team’s core values in every action.
By integrating the HMM model into our practices, we effectively evaluated activity outcomes and significantly improved our professionalism and execution capabilities. Many participants have expressed high regard for our model, believing it helps clarify team goals and promotes continuous improvement.
3. Promotion and Recommendations for the HMM Model
Given the successful application of the HMM of NJMU-CHINA in our team’s practice, we hope to recommend this model to more teams. The HMM model can help teams gain a comprehensive understanding of their strengths and weaknesses in Human Practices activities, set clear development goals, and provide directions for improvement. We believe this model will assist other teams in achieving higher levels of practice in synthetic biology projects, creating a greater positive impact on society. To promote this model effectively, we plan to create documentation that introduces the HMM model’s design philosophy, application methods, and practical effects. By sharing case studies and experiences, we aim to help other teams understand how to apply this model to their activities. We hope this initiative encourages more teams to pursue excellence in Human Practices activities and collectively contribute to the sustainable development of synthetic biology.
In conclusion, the "Holistic Maturity Model" (HMM of NJMU-CHINA) is a result of our team’s continuous exploration, refinement, and innovation. It has successfully guided our activities and played a significant role in enhancing the quality and impact of our endeavors. We sincerely hope the HMM model can serve as a reference tool for other teams, supporting more synthetic biology projects in achieving outstanding results and contributing to a profound understanding and positive impact on Human Practices.
If you are interested in our HMM model, feel free to click to download the Python code for our model for more details. If your team is interested in applying our model in future activities, please contact us. （上传代码pdf）
The NJMU-CHINA team has always been dedicated to lowering the learning barriers for synthetic biology and medical knowledge, aiming to popularize basic biological and medical concepts to a broader audience, thereby contributing to public health and the advancement of medicine. After conducting a series of offline activities, the team realized that while these efforts achieved some degree of success, their reach was still limited. To achieve wider dissemination of knowledge, the team conducted extensive social research and internal brainstorming sessions, ultimately deciding to leverage multiple online platforms to establish a comprehensive and multi-layered synthetic biology and medical knowledge outreach system that could cover a larger audience.
Part 1: Major Events in Synthetic Biology- Knowledge Dissemination via QQ and Little Red Book
In response to the general public’s unfamiliarity and misconceptions about synthetic biology, NJMU-CHINA team members utilized social media platforms like QQ and Little Red Book (Xiaohongshu) to transform complex scientific knowledge into easy-to-understand content through images, text, and short videos. This approach effectively dismantled stereotypes about synthetic biology. Firstly, within the university, we conducted synthetic biology popularization activities via the NJMU-CHINA official QQ account, regularly publishing informative posts that vividly demonstrated the principles and applications of synthetic biology to teachers and students. In addition, we distributed meticulously designed science outreach brochures offline, guiding more people to participate in our online social media activities.
To further reach a younger demographic, we chose the highly popular Little Red Book platform as our knowledge dissemination channel. On this platform, we regularly update content and have organized an interactive "Knowledge Challenge" activity, where participants randomly draw a synthetic biology-related question and then submit their answers either via private messages or by tagging them on the social platform. This innovative approach has garnered widespread attention and participation, injecting new vitality into our efforts to popularize synthetic biology knowledge. The NJMU-CHINA team will continue to expand its presence on these platforms, striving to establish our social media accounts as a representative figure in synthetic biology and biomedical knowledge dissemination, guiding more people to engage in learning and discussions on synthetic biology.
Part 2: Activities for All - Open Sharing through WeChat
During the team recruitment in 2024, we received enthusiastic applications from many students at Nanjing Medical University. However, due to the limitations on team members, many students were unfortunately unable to participate directly in our human practice activities. To ensure more people could engage in our outreach activities, the NJMU-CHINA team launched the "Activities for All" initiative.
We made full use of WeChat as a platform for information dissemination and activity showcasing, regularly sharing the team's daily human practice activities through diverse formats such as text, images, and videos, capturing and displaying our experiences and insights. Additionally, we posted recruitment information via this platform to attract more students to join our activities. Moreover, the team collaborated with other iGEM teams like NAU-CHINA and LZU-CHINA, sharing project exchanges on each other’s WeChat public accounts, spreading various teams’ research achievements and project knowledge to a broader community.
To date, the NJMU-CHINA team’s official WeChat account has published over 50 articles, amassing nearly 10,000 views. We hope to leverage this platform to create an open and inclusive activity system, enabling more students and members of the public interested in synthetic biology to participate and learn together.
Part 3: Together with Light and Dust - Joining the Short Video Trend on Bilibili and TikTok
In recent years, short video platforms have become a primary source of knowledge and entertainment for young people. Recognizing this trend, the NJMU-CHINA team seized the opportunity by establishing official accounts on Bilibili and TikTok, actively participating in the short video outreach wave. By sharing snippets of our activities, educational talks, and team dynamics, NJMU-CHINA showcased the vibrant daily life and scientific exploration journey of iGEMers.
As our video content is regularly updated, we have gradually received positive social feedback, with increasing levels of attention and interaction on these platforms. We acknowledge that while the communication power of short video platforms is immense, consistently improving content quality is key to maintaining long-term influence. Therefore, the NJMU-CHINA team will continue to refine its presence on these platforms, leveraging diverse content formats to attract more people to learn about synthetic biology and medical knowledge, thereby promoting the outreach work in this field to reach new heights.
By utilizing multiple platforms such as QQ, Little Red Book, WeChat, Bilibili, and TikTok, the NJMU-CHINA team has built a comprehensive synthetic biology knowledge dissemination system that covers the entire society. In the future, we will remain committed to creating high-quality outreach content, expanding our dissemination channels, and inviting more people to explore the world of synthetic biology, thereby advancing the process of scientific outreach together.
Introduction:
The NJMU-CHINA team has meticulously designed a series of synthetic biology popular science lesson plans that use accessible teaching methods and engaging storytelling to introduce children aged 6-10 to the basic concepts, development, and practical applications of synthetic biology in fields such as medicine and agriculture. This lesson plan series aims to stimulate children's interest in life sciences, cultivate their scientific thinking and inquiry skills, and encourage them to reflect on the impact of technology on human life from ethical and societal perspectives.
Lesson Plan 1: "Wonderful Life Engineering: Exploring the World of Synthetic Biology"
Starting with a story about "Life Engineers" creating new life, this lesson plan introduces the basic principles and development of synthetic biology, helping students understand the core concepts of cells, DNA, and genes. It emphasizes the fundamentals of gene editing and biological components while encouraging students to discuss ethical issues related to life sciences, thereby nurturing their scientific thinking and critical analysis skills.
Lesson Plan 2: "Life DIY: The Wonderful World of Synthetic Biology"
This lesson plan introduces synthetic biology through a DIY pet shop story, explaining the concepts of cells, DNA, genes, chromosomes, and plasmids in detail, with an emphasis on the principles and applications of gene editing technology. By guiding students to discuss the ethical issues surrounding synthetic biology, the plan fosters a sense of awe and reflection toward science, sparking interest in life sciences and providing an excellent foundation for students to explore synthetic biology.
Lesson Plan 3: "Life Wizards: The Magical Journey of Gene Editing"
This lesson plan uses the story of "Life Wizards" to vividly introduce the CRISPR-Cas9 gene-editing technology and its applications in synthetic biology, with a particular focus on its potential role in treating Alzheimer's disease and depression. Through detailed explanations and ethical discussions, it cultivates students' curiosity about life sciences and their sense of social responsibility regarding technological advancements.
Highlights:
1.Engaging Story Introductions:
Each lesson begins with an imaginative story featuring roles like "Life Engineers" and "Life Wizards," combining scientific knowledge with fantasy elements to stimulate students' curiosity and interest in synthetic biology. This approach effectively transforms complex scientific concepts into content that children enjoy, achieving an educational yet entertaining effect.
2.Comprehensive Knowledge Delivery:
he lesson plans are well-structured, starting with basic concepts and progressing to more complex ideas, covering essential topics such as gene editing, chromosomes, plasmids, cells, DNA, and genes. By incorporating interactive questioning, blackboard teaching, and video aids, students build a complete understanding of synthetic biology.
3.Focus on Ethical and Social Responsibility:
In addition to scientific knowledge, the lesson plans guide students to consider ethical issues related to synthetic biology, such as animal ethics, genetic risks, life patents, and ecological risks. This element not only broadens their thinking but also fosters respect and responsibility toward life and science.
4.Flexible Interactive Teaching:
The lesson plans emphasize interaction between teachers and students, utilizing questions, discussions, and storytelling to actively engage students, encouraging independent learning. Post-class discussions and reflections guide students to delve deeper into synthetic biology.
Significance of the
Activity:Applicability of the Lesson Series
1.Wide Applicability: Although designed for children aged 6-10, the content and teaching methods are highly adaptable, making them suitable for science education across different age groups. Other teams can adjust the content and difficulty level based on the actual situation to meet the learning needs of different students.
2.Practicality and Ease of Use: The lesson plans have a clear structure and detailed teaching process, including course objectives, key teaching points, introduction methods, knowledge explanations, discussion sessions, and concluding assessments. Teachers can carry out the lessons with minimal preparation, making the teaching process easy to execute, ideal for use in schools, communities, and science museums.
3.Emphasis on Comprehensive Skill Development: Beyond imparting scientific knowledge, the series integrates scientific thinking, ethics, and social responsibility into the curriculum, helping to cultivate students' overall abilities and qualities.
4.Aligned with Modern Educational Ideals: The lesson plans reflect modern education's emphasis on "quality education" and "inspirational teaching," stimulating students' interest, guiding them to explore actively, and integrating scientific knowledge into real-life situations, helping students understand and apply it better.
If you are interested in our series of lesson plans, feel free to download them for more details. We warmly welcome iGEM teams to use our lesson plans in their future activities.
