Contribution

old: BBa_K4818026

dCas9 is a modified version of Cas9 enzyme, originally designed to cut DNA. dCas9 has been changed in a way that makes it incapable of performing this process of cutting DNA. As a result, it is pretty useful in regulating gene expression. Cas9 is prone to produce unwanted DNA cutting, while dCas9 can be utilized to induce or suppress the activity of any certain genes without disturbing the coding sequence.

However, one of the biggest challenges was the regulation of dCas9 activity and to serve this purpose, we integrated CRISPR-dCas9 technology into a novel synthetic receptor based-system called dCas9-TF Syn-VEGFR-1\2. So that we could improve ( BBa_K4818026) which was designed by iGEM23_INSAENSLyon1 .

As a result, dCas9 activity will be linked and regulated through the receptor state in response to the VEGF concentration within the microenvironment of our engineered cells expressing the system.

Furthermore, to limit the basal activity of the CRISPR-dCas9 that is usually associated with gene off targeting effects. According to that, we optimized the safety of dCas9 based gene regulation strategies through splitting dCas9 protein into two non-functional domains including: one part containing the C-terminal domain (c) dCas9-TF and another part, the N-terminal domain (N) dCas9. Each domain is attached to a different chain of the receptor forming dCas9 (C/N)-TF Syn-VEGFR-1\2 system. VEGF dependent dimerization of the two chains of the receptor will mediate release of the two non-functional domains into the cytoplasm and spontaneous assembly of the two fragments into an effector complex.

Moreover, the translocation of the dCas9-TF to target genes is mediated through nuclear translocation signal (NLS) and guide RNA which constantly guides dCas9 coupled with other transcription activators to target genes. In our design dCas9-TF activity will be recruited to Nanog enhancer gene locus which lies within chromosome 11 upstream to YAP-1 gene, regulating cell differentiation and proliferation.

Figure(3). This figure illustrates VEGF dependent dimerization of the two domains of dCas9

TEV protease, a crucial enzyme in molecular biology, has been widely used for protein purification and analysis. Also, it has high specificity and efficiency in recognizing and cleaving peptide bonds between specific amino acids sequence known as TEV cleavage site (TCS).

We tried to improve this part (BBa_K3139012) which was designed by iGEM19_NAU-CHINA. However, controlling TEV activity has been a longstanding challenge.To overcome this limitation, we've Integrated a CRISPR-dCas9 technology into a novel synthetic receptor based-system which ensures that TEV protease is activated only following the dimerization of the two receptor chain specific conditions. The dimerization of the receptor is conditioned upon the presence of VEGF, a substance elevated in wound injuries, particularly burns. This conditional activation mechanism significantly enhances the safety and precision of TEV protease-mediated protein release.

For more strict control over TEV protease, we've divided TEV enzyme into two non-functional domains: C-terminal domain (C-TEV) and N-terminal domain (N-TEV). Each domain is linked to a different chain of the synthetic receptor. The assembly of the two domains of TEV to form a catalytically active version of TEV protease is conditioned by the binding of VEGF to the receptor that mediates the dimerization of the two chains of the receptor.

By applying this design we could ensure the specificity of TEV protease, preventing unintended receptor activity to reduce VEGF-independent basal activity

Figure(4). This figure illustrates VEGF dependent dimerization of the two domains of TEV and its proteolytic activity for releasing of dCas9

This MS2 coat protein (MCP) exhibits a strong affinity for a specific stem-loop structure (MS2). This binding ability makes it a valuable tool for studying RNA localization, purification, and interactions. It can be used for RNA Visualization By fusing it to fluorescent protein which in turn can be used to study RNA-protein interactions.

In our model, we have added a new feature to MCP (BBa_K2923011) ,which was designed by iGEM19_Strasbourg to act as a protein sensor that could recruit different effector proteins to its specific stem loop (MS2) .

While it originally relied solely on its high binding affinity to MS2 RNA, we have implemented MCP in our metalloproteinase-9 (MMP-9) based mRNA switch by incorporating a specific sensor (nanobody) that recognizes MMP9, a structural protein that increases significantly within cells during tissue injury like burns. Nanobodies are a unique type of antibody which is used to recognize specific types of proteins making them ideal for applications in diagnostics and therapeutics This modification allows our system to switch from an inactive to an active state in response to tissue damage. Moreover, MCP could act as an intracellular protein sensor by combining MCP to a nanobody specific to the protein of interest.

Figure(5). This figure illustrates our improvement on MCP by combining it to nanobody to act as an intracellular protein sensor

Our proposal for the new SDG 18 on "Ensuring Fire Safety and Minimizing Global Burn Injuries" is a huge step toward harnessing the power of iGEM teams to drive real-world change. We introduce this proposal to the public and the UN with the hope that it will enable future iGEM teams to work outside the box of synthetic biology on projects that will have long-term, tangible societal benefits.

This effort will serve as an example for iGEM teams on how science projects with innovation can be embedded within larger global policy frameworks such as the SDGs. It encourages and opens up new dimensions of public health, safety, and environmental Sustainability that teams may push beyond in synthetic biology.

Beyond the scope of iGEM, the global impact of our proposal lies in its potential to save lives, reduce economic losses, and protect vulnerable populations from preventable injuries. By representing fire safety measures through equal healthcare access to burn injuries.

This proposal not only shows the abilities of iGEM teams to have an effect on policy, it also highlights the critical role of science-based advocacy in shaping a better future. We hope that this contribution will be an inspiration to iGEM teams worldwide into making action on pressing global challenges and further continue to make meaningful, far-reaching impacts beyond the competition. We can create together a legacy of innovation and responsibility that transcends borders and disciplines in the pursuit of the greater good for all of humanity.

We believe that the music is a powerful tool for reaching children. By creating a catchy song with clear and simple lyrics, we can make fire safety information not only understandable but also memorable, so we made a song called Be safe from fire and launched it on YouTube, Tiktok, Facebook. Suno AI enables us to make the song without any music experience, making fire safety messages accessible and enjoyable. With AI, we can generate various musical styles appealing to children and their caregivers, holding their attention in a way that is impossible with traditional methods alone.

We are proud to pave the way for a new approach to raising awareness that future iGEM teams can adopt and customize for their own styles .

During our podcast series, we went in-depth into the vital connections between the UN's Sustainable Development Goals (SDGs) and pressing global issues.

The first episode conveys the urgent message that all these SDGs address interconnected challenges—such as poverty, inequality, and climate change—emphasizing the need for collective action. The episode on SDG 18 "Fire Safety and Burn Prevention" emphasized how SDG 18 will advance equitable fire safety measures and international cooperation.

In the SDG 11 episode , we discussed the inclusive and resilient urban environments, focusing on fire safety because of urban planning and community involvement.

SDG Podcast Episode 1

SDG Podcast Episode 2

SDG Podcast Episode 3

In our journey this year, our team members have contributed to many human practice events that raise awareness about burn injuries. Our team has established a burn guidebook. This guidebook includes basic safety measures for fire prevention, first aid procedures in case of burn injuries, and pharmacological and natural treatments for burn injuries.

We used social media as a tool to reach many people, we have made three educational podcasts about SDGs and their significant impact on our world, how to reduce fire accidents in cities and develop a sustainable city, and the last one was about our new SDG (Ensuring fire safety and minimal burn injuries). Additionally, our team designed a brochure for women and shared it on social media. This brochure includes safe cooking tips and procedures to deal with minor burn injuries.

Figure(6). Cooking safety , child safety, and first aid procedures brochure.

We created a guidebook that provides a comprehensive overview of the AFCM-Egypt iGEM team's approach to organize human practices events. It was designed to help us planning and outlining the steps we follow to plan our events. Also, it serves as a reference for other iGEM teams to ensure that any event is organized successfully and reaches the desired goal.

We created a new story named Maya and the Kitchen dragon. It aims to teach children about burn prevention and appropriate first aid in case of burns. It also delivers important instructions that children must follow to protect themselves from the risk of burns in a simple way . We designed this story in two languages : Arabic , English.In addition, We collaborated with iGEM IOANNINA team to translate it into Greek.