This year, we are addressing the induction of healing in non-regenerative organs. by leveraging tissues' regenerative capabilities, which can be achieved through altering their native functions which depends on modulation of the internal pathways. We decided to exploit MSCs’ regenerative features, besides their potential to act as a modular platform for cell-based gene therapy in addition to , inducing the expression of a novel protein called yes-associated protein (YAP-1) that is involved in regulating cellular growth, proliferation, and differentiation.
Our engineered MSCs possess a receptor dCas9(N/C) Syn VEGFR-1/2 that is specifically designed to recognize VEGF , a protein whose levels rise significantly in response to tissue damage. When this receptor binds to VEGF, it triggers a signaling pathway that leads to an increase in the intracellular levels of YAP-1 within the MSCs. This elevated YAP-1 level motivates the MSCs to proliferate and differentiate into various cell types found in the local wound environment, thereby promoting tissue regeneration.
Our receptor is formed of two separate longitudinal chains. Each chain consists of components from the three domains of the receptor( external , transmembrane, and internal). Thus, we divided TEV protease and dCas9 into two separate components, each in a separate chain.
Our receptor is formed of two separate longitudinal chains. Each chain consists of components from the three domains of the receptor( external , transmembrane, and internal). Thus, we divided TEV protease and dCas9 into two separate components, each in a separate chain.
Our first chain contains VEGFR1 as an external domain while the internal domain contains C- TEV ,its cleavage site(TCS(Q,G)) , nuclear localization signal (NLS) and C-dCas9 which is attached to our transcription activators: VP64, GAL4 and UAS transCMV enhancer
| Part Name | Description | ID | Designer |
|---|---|---|---|
| VEGFR-1 | It is vascular endothelial growth factor receptor which contain 3 members contain similar structures and their external parts are made up entirely of repeating segments that resemble parts of antibodies (immunoglobulin homology repeats) and they have a critical role in the formation of blood vessels and lymphatic vessels. | BBa_K5036000 | AFCM Egypt 2024 |
| Part Name | Description | ID | Designer |
|---|---|---|---|
| CMV promoter | CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues. Due to its high transcription levels, it is a potent promoter that is employed to induce gene expression. | BBa_K4586016 | AFCM Egypt 2023 |
| C-TEV | This is known as the tobacco etch virus which is very selective for cleaving proteins at particular amino acid sequences and has been modified to have better qualities like greater heat stability, decreased self-cleavage and also allows researchers to precisely cleave the tag off, leaving behind the receptor in its unmodified form. In our model, TEV was divided into N-terminal and C-terminal fragments. So the C-terminal fragment was grafted onto the first chain of our dCas9(C)-TF- synVEGFR1 receptor. | BBa_K5036001 | AFCM Egypt 2024 |
| NLS | It is a nuclear localization signal which is a short sequence of amino acids found in certain proteins that acts as a zip code, directing those proteins to the nucleus of a cell. In our model it is attached to dCas9(C) in the first chain of dCas9(C)-TF- synVEGFR1 receptor. | BBa_K5036002 | AFCM Egypt 2024 |
| TCS(Q,G) | This is the TEV cleavage site, a particular amino acid sequence detected by TEV to start cleavage. | BBa_K5036003 | AFCM Egypt 2024 |
| dCas9(C) | It is a modified version of the CRISPR-Cas9 gene editing tool which cannot cut DNA. Instead, it can bind to a specific DNA sequence guided by an RNA molecule so it can be fused to transcriptional activators or repressors. In our model dcas9 was divided into N-terminal and C-terminal fragments. our dCas9 (c) is attached to our dCas9(C)-TF-synVEGFR1 receptor's first chain and it is linked to 64, GAL4 and UAS Trans CMV enhancer. | BBa_K5036004 | AFCM Egypt 2024 |
| VP-64 | It is a strong transcriptional activator composed of four tandem copies of VP16 (Herpes Simplex Viral Protein 16) connected with glycine-serine (GS) linkers. When fused to another protein domain, it binds near the gene promoter. | BBa_K5036005 | AFCM Egypt 2024 |
| TCS (Q, G)-E1D | This is mutant form of TEV Cleavage Site which is a particular amino acid sequence that the TEV protease detects and cleaves | BBa_K5036052 | AFCM Egypt 2024 |
| UAS Trans CMV enhancer | It is a Powerful Tool for Gene Expression which is derived from the cytomegalovirus (CMV). Moreover, it can significantly boost gene expression in various cell types. Furthermore, it works by binding to specific transcription factors, which then recruit other proteins to the gene promoter region. This complex interaction helps to initiate and sustain gene transcription. | BBa_K5036021 | AFCM Egypt 2024 |
| GAL-4 | GAL4 is a kind of protein that can be found and combined with UAS DNA structure domain as it can recognize and combine with UAS. Because GAL4 originated from yeast cells, the activation domain can not work in mammalian cells. Therefore, we only used its combining domain and used it together with VP64 . | BBa_K4585001 | CSU-CHINA 2023 |
| Part Name | Description | ID | Designer |
|---|---|---|---|
| VEGFR-2 | It is vascular endothelial growth factor receptor which contain 3 members contain similar structures and their external parts are made up entirely of repeating segments that resemble parts of antibodies (immunoglobulin homology repeats) and they have a critical role in the formation of blood vessels and lymphatic vessels. | BBa_K5036006 | AFCM Egypt 2024 |
| Part Name | Description | ID | Designer |
|---|---|---|---|
| N-TEV | This is known as the tobacco etch virus which is very selective for cleaving proteins at particular amino acid sequences and has been modified to have better qualities like greater heat stability, decreased self-cleavage and also allows researchers to precisely cleave the tag off, leaving behind the receptor in its unmodified form. In our model, TEV was divided into N-terminal and C-terminal fragments. So the N-terminal fragment was grafted onto the second chain of our dCas9(N)-synVEGFR2 receptor. | BBa_K5036007 | AFCM Egypt 2024 |
| NES | It is a Nuclear Export Signal which is a short sequence of amino acids found in certain proteins that direct those proteins out of the nucleus of a cell. . In our model it is engineered in the second chain of dCas9(N)-TF- synVEGFR2 receptor. | BBa_K5036008 | AFCM Egypt 2024 |
| TCS(Q,L) | This is the TEV cleavage site, a particular amino acid sequence detected by TEV to start cleavage. | BBa_K5036022 | AFCM Egypt 2024 |
| TCS(Q.L)-G6Q | This is mutant form of TEV Cleavage Site which is a particular amino acid sequence that the TEV protease detects and cleaves. | BBa_K5036051 | AFCM Egypt 2024 |
| d-Cas9 (N) | It is a modified version of the CRISPR-Cas9 gene editing tool which cannot cut DNA. Instead, it can bind to a specific DNA sequence guided by an RNA molecule so it can be fused to transcriptional activators or repressors. In our model dcas9 was divided into N-terminal and C-terminal fragments. our dCas9 (N) is attached to our dCas9(N)-synVEGFR2 receptor's second chain. | BBa_K5036009 | AFCM Egypt 2024 |
| Part Name | Description | ID | Designer |
|---|---|---|---|
| VEGF-R1, C-TEV, NLS, TCS(Q,G),HA, dCas9(C), VP64, GFP | In our first receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R1, is attached to an internal domain composed of C terminal domain of TEV protease, a nuclear localization signal (NLS), a TEV cleavage site(TCS(Q,G)), and dCas9(C) which is linked to transcription activator VP64 | BBa_K5036026 | AFCM Egypt 2024 |
| VEGF-R1, C-TEV, NLS, TCS(Q,G),HA, dCas9(C),VP64, GAL4, GFP | In our first receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R1, is attached to an internal domain composed of C terminal domain of TEV protease, a nuclear localization signal (NLS), a TEV cleavage site(TCS(Q,G)), and dCas9(C) which is linked to transcription activators: VP64 and GAL4. | BBa_K5036027 | AFCM Egypt 2024 |
| VEGF-R2, N-TEV, NES, TCS (Q, L), HA, dCas9(N), mCherry | In our second receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R2, is attached to an internal domain composed of the N terminal domain of TEV protease, a nuclear export signal (NES), a TEV cleavage site(TCS(Q,L)), and dCas9(N). | BBa_K5036028 | AFCM Egypt 2024 |
| VEGF-R1, C-TEV, NLS, TCS(Q,L), HA, dCas9(C), VP64, GFP | In our first receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R1, is attached to an internal domain composed of C terminal domain of TEV protease, a nuclear localization signal (NLS), a TEV cleavage site(TCS(Q,L)), and dCas9(C) which is linked to transcription activator VP64. | BBa_K5036029 | AFCM Egypt 2024 |
| VEGF-R2, N-TEV, NES, TCS (Q, G), HA, dCas9(N),mCherry | In our second receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R2, is attached to an internal domain composed of N terminal domain of TEV protease, a nuclear export signal (NES), a TEV cleavage site(TCS(Q,G)), and dCas9(N). | BBa_K5036030 | AFCM Egypt 2024 |
| VEGF-R1, C-TEV, NLS, TCS(Q,G),HA, dCas9(N), VP64, GFP | In our first receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R1, is attached to an internal domain composed of C terminal domain of TEV protease, a nuclear localization signal (NLS), a TEV cleavage site(TCS(Q,G)), and dCas9(N) which is linked to transcription activator VP64. | BBa_K5036031 | AFCM Egypt 2024 |
| VEGF-R2, N-TEV, NES, TCS (Q, L), HA, dCas9(C),mCherry | In our second receptor's chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R2, is attached to an internal domain composed of N terminal domain of TEV protease, a nuclear export signal (NES), a TEV cleavage site(TCS(Q,L)), and dCas9(C). | BBa_K5036032 | AFCM Egypt 2024 |
| VEGF-R1, N-TEV, NLS, TCS(Q,G), HA, dCas9(C), VP64, GFP | In our first receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R1, is attached to an internal domain composed of N terminal domain of TEV protease, a nuclear localization signal (NLS), a TEV cleavage site(TCS(Q,G)), and dCas9(C) which is linked to transcription activator VP64. | BBa_K5036033 | AFCM Egypt 2024 |
| VEGF-R2, C-TEV, NES, TCS (Q, L), HA, dCas9(N), mCherry | In our second receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R2, is attached to an internal domain composed of C terminal domain of TEV protease, a nuclear export signal (NES), a TEV cleavage site(TCS(Q,L)), and dCas9(N). | BBa_K5036034 | AFCM Egypt 2024 |
To sum up, our receptor is confined to two separate chains. When an injury occurs, extracellular VEGF levels increase and bind to our receptor causing the dimerization of TEV protease. This leads to the cleavage of the receptor chains at the TCS site, resulting in dimerization and release of dCas9.
According to our design, Our dCas9(N/C) Syn VEGFR-½ is engineered to target two different locations:one is a natural gene where promoting transcription activity of YAP-1 to increase YAP-1 expression and increase our MSCs proliferation and differentiation, and the other is our TID switch that has been added to cells to induce transcription of our genetic circuit, so we have used a new CRISPR technology that allows us to express multiple guide RNAs from a single transcript of RNA separated by hammerhead ribozyme (HHR). This HHR allows each part to be separated and function independently.
So after VEGF dependent release of dCas9 complexed with VP64,GAL4 and UAS Trans CMV enhancer , the complex will be taken by gRNA and NLS to target Nanog gene which is located upstream of YAP-1 encoding region to induce transcription and production of YAP-1, respectively.
| Part Name | Description | ID | Designer |
|---|---|---|---|
| Human U6 promoter | It is a type III RNA polymerase III promoter that controls the expression of short RNAs. Distance sequence element (DSE), proximal sequence element (PSE), and TATA box are the three primary components of the U6 promoter. The nucleotide G indicates the U6 promoter's transcriptional beginning location. | BBa_K4586017 | AFCM Egypt 2023 |
| Nanog gRNA1 | Guide RNA is a crucial component of the CRISPR-Cas9 gene editing system as it acts as a dCas9 guide to its target. It consists of two main parts. Firstly, Spacer Sequence: which is a short, user-defined sequence that complements (matches) the target DNA sequence. Secondly, Scaffold Sequence which is pre-designed RNA structure allows gRNA to bind to the Cas9 protein and facilitate its interaction with the target DNA. | BBa_K5036010 | AFCM Egypt 2024 |
| Nanog gRNA2 | Guide RNA is a crucial component of the CRISPR-Cas9 gene editing system as it acts as a dCas9 guide to its target. It consists of two main parts. Firstly, Spacer Sequence: which is a short, user-defined sequence that complements (matches) the target DNA sequence. Secondly, Scaffold Sequence which is pre-designed RNA structure allows gRNA to bind to the Cas9 protein and facilitate its interaction with the target DNA. | BBa_K5036011 | AFCM Egypt 2024 |
| Nanog gRNA3 | Guide RNA is a crucial component of the CRISPR-Cas9 gene editing system as it acts as a dCas9 guide to its target. It consists of two main parts. Firstly, Spacer Sequence: which is a short, user-defined sequence that complements (matches) the target DNA sequence. Secondly, Scaffold Sequence which is pre-designed RNA structure allows gRNA to bind to the Cas9 protein and facilitate its interaction with the target DNA. | BBa_K5036012 | AFCM Egypt 2024 |
| HHR | HHRis a type of self-catalytic RNA molecule that has been engineered to cleavespecific RNA targets which is essential for various biological processes,such as gene regulation. | BBa_K5036039 | AFCM Egypt 2024 |
| gRNA scaffold for Cas9 | It is pre-designed RNA structure allows gRNA to bind to the Cas9 protein and facilitate its interaction with the target DNA. | BBa_K2217004 | AFCM Egypt 2017 |
| gRNA operator | It is guide operator sequences composed of a 20 base pair target sequence and a PAM sequence and it has high optimization score ( > 98%) and high expression in an initial screen containing near-infrared fluorescent protein (iRFP). | BBa_K1875004 | BostonU 2016 |
| Part Name | Description | ID | Designer |
|---|---|---|---|
| U6 promoter-Nanog gRNA1 (scaffold- spacer RNA ) | This part consists of the U6 promoter, which constantly activates the creation of our Nanog guide RNA, and The Nanog guide RNA comprises a spacer sequence that matches the Nanog DNA sequence, enabling gene editing. Additionally, the guide RNA includes a scaffold sequence that binds to the dCas9 protein, allowing it to interact with the target DNA. | BBa_K5036035 | AFCM Egypt 2024 |
| U6 promoter- Nanog gRNA2 (scaffold- spacer RNA ) | This part consists of the U6 promoter, which constantly activates the creation of our Nanog guide RNA, and The Nanog guide RNA comprises a spacer sequence that matches the Nanog DNA sequence, enabling gene editing. Additionally, the guide RNA includes a scaffold sequence that binds to the dCas9 protein, allowing it to interact with the target DNA | BBa_K5036036 | AFCM Egypt 2024 |
| U6 promoter- Nanog gRNA3 (scaffold- spacer RNA ) | This part consists of the U6 promoter, which constantly activates the creation of our Nanog guide RNA, and The Nanog guide RNA comprises a spacer sequence that matches the Nanog DNA sequence, enabling gene editing. Additionally, the guide RNA includes a scaffold sequence that binds to the dCas9 protein, | BBa_K5036037 | AFCM Egypt 2024 |
| CRISPR multiplexing system (Nanog gRNA-HHR-gRNA operator) | CRISPR multiplexing system is a versatile genetic engineering tool, that empowers scientists to simultaneously target and modify multiple genes within an organism. By designing multiple guide RNAs that precisely bind to specific DNA sequences of interest, researchers can efficiently and accurately introduce desired genetic changes. This capability enhances efficiency and precision, making CRISPR a valuable asset for a wide range of biological applications. | BBa_K5036040 | AFCM Egypt 2024 |
To mitigate the potential adverse effects associated with YAP-1 overproduction, we developed a switch that controls its translation. This switch comprises several key components: a cap with NSP3A bound to an MMP-9 Nanobody; a coding region encoding the YAP-1 protein; an MS2 element linked to MCP and another MMP-9 Nanobody; and an HHR element upstream to the poly A tail.
| Part Name | Description | ID | Designer |
|---|---|---|---|
| Ptet promoter | This part is inducible promoter Ptet which is induced by tetracycline. This is the basic principle of Tet induced expression system, it is to regulate the conformation of proteins by inducer such as Tet, thus controlling the expression of target proteins. When there is no tetracycline, TetR will bind to TetO to block downstream expression of resistant genes. When tetracycline is added, it will bind to TetR and change its conformation, then TetR is separated from TetO, thereby relieving the inhibition of resistance genes. | BBa_K2800027 | HUBU-Wuhan 2018 |
| MMP-9 Nano body1 | Nanobodies are a unique type of antibody derived from camelids. These single-domain antibodies are significantly smaller and more stable, making them ideal for applications in diagnostics and therapeutics. Nanobodies can be used to develop highly sensitive and specific tests for a variety of diseases, including cancer and autoimmune disorders. Their compact size and stability also make them promising candidates for targeted drug delivery and other biomedical applications. | BBa_K5036015 | AFCM Egypt 2024 |
| MMP-9 Nano body2 | Nanobodies are a unique type of antibody derived from camelids. These single-domain antibodies are significantly smaller and more stable, making them ideal for applications in diagnostics and therapeutics. Nanobodies can be used to develop highly sensitive and specific tests for a variety of diseases, including cancer and autoimmune disorders. Their compact size and stability also make them promising candidates for targeted drug delivery and other biomedical applications. | BBa_K5036016 | AFCM Egypt 2024 |
| MMP-9 Nano body3 | Nanobodies are a unique type of antibody derived from camelids. These single-domain antibodies are significantly smaller and more stable, making them ideal for applications in diagnostics and therapeutics. Nanobodies can be used to develop highly sensitive and specific tests for a variety of diseases, including cancer and autoimmune disorders. Their compact size and stability also make them promising candidates for targeted drug delivery and other biomedical applications. | BBa_K5036017 | AFCM Egypt 2024 |
| MCP | This MS2 coat protein(MCP) exhibits a strong affinity for a specific stem-loop structure (MS2). This binding ability make 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. | BBa_K5036013 | AFCM Egypt 2024 |
| NSP3A | It is a non structural large and multifunctional protein which has high affinity to the cap binding proteins such as eIF4GI. | BBa_K5036018 | AFCM Egypt 2024 |
| MS2-(x24)HHR | MS2 is a small viral protein which forms the outer shell of the MS2 bacteriophage. Its ability to bind to specific RNA sequences has made it a valuable tool for studying RNA biology and gene expression and it is frequently used in combination with the MS2 system to purify and analyze RNA-protein complexes. this part contains 24 repeats of MS2. while HHR is a type of self-catalytic RNA molecule that has been engineered to cleave specific RNA targets which is essential for various biological processes, such as gene regulation and viral replication. | BBa_K5036019 | AFCM Egypt 2024 |
| MS2-(x16)HHR | MS2 is a small viral protein which forms the outer shell of the MS2 bacteriophage. Its ability to bind to specific RNA sequences has made it a valuable tool for studying RNA biology and gene expression and it is frequently used in combination with the MS2 system to purify and analyze RNA-protein complexes. this part contains 16 repeats of MS2. while HHR is a type of self-catalytic RNA molecule that has been engineered to cleave specific RNA targets which is essential for various biological processes, such as gene regulation and viral replication. | BBa_K5036023 | AFCM Egypt 2024 |
| MS2-(x12)HHR | MS2 is a small viral protein which forms the outer shell of the MS2 bacteriophage. Its ability to bind to specific RNA sequences has made it a valuable tool for studying RNA biology and gene expression and it is frequently used in combination with the MS2 system to purify and analyze RNA-protein complexes. this part contains 12 repeats of MS2. while HHR is a type of self-catalytic RNA molecule that has been engineered to cleave specific RNA targets which is essential for various biological processes, such as gene regulation and viral replication. | BBa_K5036024 | AFCM Egypt 2024 |
| MS2-(x8)HHR | MS2 is a small viral protein which forms the outer shell of the MS2 bacteriophage. Its ability to bind to specific RNA sequences has made it a valuable tool for studying RNA biology and gene expression and it is frequently used in combination with the MS2 system to purify and analyze RNA-protein complexes. this part contains 8 repeats of MS2. while HHR is a type of self-catalytic RNA molecule that has been engineered to cleave specific RNA targets which is essential for various biological processes, such as gene regulation and viral replication. | BBa_K5036025 | AFCM Egypt 2024 |
| YAP-1 | It is a transcriptional coactivator that plays a crucial role in various biological processes, including organ development, tissue regeneration, and cancer progression. It is a key component of the Hippo pathway which is a signaling pathway that regulates cell growth, proliferation, and differentiation. Furthermore, YAP-1 has a function in controlling stem cell activity. Ensuring that there is a generation of new cells and their specialization to maintain a steady cell supply. | BBa_K5036020 | AFCM Egypt 2024 |
| Three repeated gRNA operators | This gRNA binds to dCas9-VPR and targets a paired gRNA operator reporter to activate a downstream gene of interest so to increase expression of the gRNA operator reporters is to have repeated guide operator sequences. | BBa_K1875009 | BostonU 2016 |
| miniCMV promoter | The minimal CMV (miniCMV) promoter is a low expression synthetic promoter in mammalian cells. Recognition sequences for regulatory proteins can be placed upstream of the miniCMV promoter to generate high expression of downstream genes. | BBa_K1875000 | BostonU 2016 |
| PolyA tail | This is simply a 100-base polyA tail. The length of the PolyA tail does affect expression, though the data on the relative efficiencies may be said to be inconclusive at the very least. The reason this tail is so essential is that the cell is not performing the concomitant tailing after transcription, and hence this must be done by us, either post-transcriptionally, or it must be incorporated in the plasmid in use. | BBa_K1875006 | BostonU 2016 |
Conclusively , our design prevents the spontaneous translation of YAP-1. The activation of this switch is conditioned upon elevated levels of MMP9, a protein often associated with tissue injury, as it binds to the MMP9 Nanobodies triggering the circulation of the mRNA transcript and ,ultimately, leading to the translation of YAP-1. To prevent spontaneous binding between poly A tail and the mRNA cap, HHR performs self-cleavage at the poly A tail end, controlling the switch’s basal activity. As the wound healing process progresses and MMP-9 declines, our engineered switch is deactivated ensuring that YAP-1 levels remain optimally controlled.
| Part Name | Description | ID | Designer |
|---|---|---|---|
| MCP-MMP9 Nanobody1 | Our engineered switch contains a nanobody that recognizes MMP9, an enzyme elevated in injured cells. This nanobody is linked to MCP protein, which is attached to MS2 from the other end because of its high affinity for MS2. | BBa_K5036045 | AFCM Egypt 2024 |
| MCP-MMP9 Nanobody2 | Our engineered switch contains a nanobody that recognizes MMP9, an enzyme elevated in injured cells. This nanobody is linked to MCP protein, which is attached to MS2 from the other end because of its high affinity for MS2. | BBa_K5036046 | AFCM Egypt 2024 |
| MCP-MMP9 Nanobody3 | Our engineered switch contains a nanobody that recognizes MMP9, an enzyme elevated in injured cells. This nanobody is linked to MCP protein, which is attached to MS2 from the other end because of its high affinity for MS2. | BBa_K5036047 | AFCM Egypt 2024 |
| MMP9 Nanobody1,NSP3A | Our engineered switch contains a nanobody that recognizes MMP9, an enzyme elevated in injured cells. This nanobody is linked to NSP3A protein, which is attached to the cap from the other end. | BBa_K5036048 | AFCM Egypt 2024 |
| MMP9 Nanobody 2,NSP3A | Our engineered switch contains a nanobody that recognizes MMP9, an enzyme elevated in injured cells. This nanobody is linked to NSP3A protein, which is attached to the cap from the other end. | BBa_K5036049 | AFCM Egypt 2024 |
| MMP9 Nanobody3, NSP3A | Our engineered switch contains a nanobody that recognizes MMP9, an enzyme elevated in injured cells. This nanobody is linked to NSP3A protein, which is attached to the cap from the other end. | BBa_K5036050 | AFCM Egypt 2024 |
| YAP-1-MS2(x24)-HHR-poly A tail | This composite part includes the YAP-1 coding region, followed by 24 copies of MS2 which in turn is followed by the HHR enzyme, and then a poly A tail. This structure ensures that the switch is activated only in response to tissue damage, as indicated by elevated MMP9 levels, resulting in the production of YAP-1 protein. | BBa_K5036041 | AFCM Egypt 2024 |
| YAP-1-MS2(x16)-HHR-poly A tail | This composite part includes the YAP-1 coding region, followed by 16 copies of MS2 which in turn is followed by the HHR enzyme, and then a poly A tail. This structure ensures that the switch is activated only in response to tissue damage, as indicated by elevated MMP9 levels, resulting in the production of YAP-1 protein. | BBa_K5036042 | AFCM Egypt 2024 |
| YAP-1-MS2(x12)-HHR-poly A tail | This composite part includes the YAP-1 coding region, followed by 12 copies of MS2 which in turn is followed by the HHR enzyme, and then a poly A tail. This structure ensures that the switch is activated only in response to tissue damage, as indicated by elevated MMP9 levels, resulting in the production of YAP-1 protein. | BBa_K5036043 | AFCM Egypt 2024 |
| YAP-1-MS2(x8)-HHR-poly A tail | This composite part includes the YAP-1 coding region, followed by 8 copies of MS2 which in turn is followed by the HHR enzyme, and then a poly A tail. This structure ensures that the switch is activated only in response to tissue damage, as indicated by elevated MMP9 levels, resulting in the production of YAP-1 protein. | BBa_K5036044 | AFCM Egypt 2024 |
MSCs have the ability to communicate with surrounding cells through exosomes. For that, we have made use of this feature by delivering our mRNA switch which carries YAP-1 sequence to the viable cells within the wound.
We have implemented a novel loading system which consists of:
| Part Name | Description | ID | Designer |
|---|---|---|---|
| MCP | A MS2 Coat Protein binds to MS2 aptamer forming an MS2-MCP-gRNA complex that is directed to the editing site. Additionally, it is a specific tagging system for MS2 aptamer that helps researchers understand how RNA behaves in living cells. | BBa_K5036013 | AFCM Egypt 2024 |
| CD63 | It is a protein which is found on the cell surface and it is a member of the tetraspanin family.that can aid in cell signaling and also regulate movement of vesicles within cells. In addition, it can help in our immune response to various infections. | BBa_K5036014 | AFCM Egypt 2024 |
Our exosomes are modified with CD63 and MCP as our loading system. In response to injury, MSCs release these exosomes containing our YAP-1-engineered switch. MS2, with its high affinity for MCP, binds to it and facilitates the loading of YAP-1 into the exosomes.
| Part Name | Description | ID | Designer |
|---|---|---|---|
| Loading system(CD63,MCP) | We've engineered exosomes to carry a specific delivery system which is composed of CD63 and MCP. CD63 is membrane-specific for exosomes while MCP MS2 Coat Protein and it is a specific tagging system for MS2 Protein that helps researchers understand how RNA behaves in living cells and other types of cells. | BBa_K5036038 | AFCM Egypt 2024 |
Our team is eager to contribute significantly to the IGEM this year by improving 3 parts. By refining these parts, we aim to increase our control over them and introduce a new feature that could be valuable to other IGEM teams., potentially expanding the reach and impact of our project.
| improved(old) part ID | Our Part ID | Part Name | improvement |
|---|---|---|---|
| BBa_K4818026 | d-CAS9(C) BBa_K5036004 dCas9(N) Ba_K5036009 |
dCas9 | These two parts are improvement of dCas9(BBa_K4818026) which was designed by (iGEM23_INSAENSLyon1). We have divided dCas9 into two non functional domain and we have integrated it into our novel synthetic receptor(dCas9(C/N)-TF Syn-VEGFR-1\2) which gives us control over the release of dCas9 preventing gene off targeting effects.our receptor is conditioned by VEGF concentration within the microenvironment of our engineered cells so upon binding of VEGF to our receptor, it cause dimerization of the two receptor chains mediating release of the two non-functional domains into the cytoplasm and spontaneous assembly of the two fragments into an effector complex which then will be guided by NLS and our gRNA to its target gene. |
| BBa_K3139012 | C-TEV BBa_K5036001 N-TEV BBa_K5036007 |
TEV | These two parts are improvement of dCas9(BBa_K3139012)which was designed by (iGEM19_NAU-CHINA.)We partitioned TEV into two nonfunctional domains and incorporated them into our innovative synthetic receptor, dCas9(C/N)-TF Syn-VEGFR-1/2 that enables us to regulate the release of TEV, thereby mitigating unintended gene silencing effects.This conditional activation mechanism significantly enhances the safety and precision of TEV protease-mediated protein release.VEGF dependant dimerization mediate assembly of the two domains into a catalytically active enzyme which will act by cleavage of the two receptor chains mediating release of dCas9. |
| BBa_K2923011 | BBa_K5036013 | MCP | This part is improvement of (BBa_K2923011) which was designed by (iGEM19_Strasbourg). We could enhance MCP by incorporating a novel feature that would benefit other IGEM teams. While MCP's high affinity for MS2 is advantageous for various functions, we have integrated it into our Translation Initiation Device (TID) switch by attaching a specific sensor (nanobody) that recognizes MMP9 so that our switch could turn from off to on state only in response to tissue damage.This modification transforms MCP into an intracellular protein sensor, enabling it to detect the protein of interest through its interaction with the corresponding nanobody. |
By examining the parts of our project, we find that they can be adapted to other applications, as we use biosensor with gRNA to target gene and we use Switch for the specific gene, and by making some minor changes to our parts or use one part only, we can use them in useful ways in different applications.
ARMED FORCES COLLEGE OF MEDECINE
AFCM EGYPT iGEM 2024
