Parts
This year, our team added collections of new basic, composite, and improved parts to the iGEM registry to be used by other teams in the future in their biological circuits.
Twelve basic parts:
| Part ID | Name |
|---|---|
| BBa_K4586000 | CCP1 |
| BBa_K4586003 | ZF21.16 minCMV promoter |
| BBa_K4586004 | Cas12k |
| BBa_K4586005 | MCP-ADAR2 |
| BBa_K4586006 | Guide RNA |
| BBa_K4586007 | Sensor |
| BBa_K4586008 | MS2 |
| BBa_K4586009 | LAMP2b |
| BBa_K4586014 | CX43 |
| BBa_K4586017 | U6-promoter |
| BBa_K4586030 | Citrullinated vimentin |
| BBa_K4586032 | Anti CD19 |
-Ten composite parts:
| Part ID | Name |
|---|---|
| BBa_K4586022 | ZF21.16-VP64 |
| BBa_K4586024 | Synthetic notch(CD8 alpha-his tag-vimentin-mouse notchcore-ZF21.16\VP64) |
| BBa_K4586025 | Cargo (guide RNA - switch- Cas12k- MCP/ADAR) |
| BBa_K4586027 | Booster genes (SDC4-STEAP3-NadB) |
| BBa_K4586026 | Loading system(CD63-L7Ae) |
| BBa_K4586028 | Exosomal receptor (vimentine, Lamp2b) |
| BBa_K4586029 | DART-V-ADAR (tissue specific switch) |
| BBa_K4586031 | ZF21.16 minCMV promoter-Cre recombinase |
| BBa_K4586033 | Anti CD19-Exosomes receptor |
| BBa_K4586034 | Anti CD19 Syn-notch receptor |
Improvement
1-DART-V-ADAR (tissue specific switch)
we optimized that system design by improving a mutant form of ADAR enzyme designed by iGEM18_NTU-Singapore by conjugating its catalytic portion to MS2 coat protein (MCP), which has a high binding affinity to MS2 RNA hairpin structure and replaces the unidentifiable dsRNA- interaction domains of the natural ADAR enzyme. Therefore, the flanking of our sensor by two MS2-RNA hairpins enhances the recruitment of the improved form of ADAR2 (MCP-ADAR) toward the sensor, increasing the dynamic range and the frequency of sensor editing and amplifying the signal of natural ADAR.
2-Booster genes (SDC4, STEAP3, NadB)
We enhanced the efficiency and safety of this translational (BBa_K2796028) unit designed by iGEM18_LZU-CHINA to increase the default level of exosomes secretion. Taking advantage of the recombination ability of the Cre loxP system to express our booster genes conditionally under the control of Cre recombinase enzyme activity that deletes stop sequence preventing the translation of booster genes.
That modification is done firstly by adding loxP-(STOP)-loxP upstream to the coding sequence of our booster genes and secondly by regulating the Cre recombinase enzyme expression through our conditional promoter ZF21.16 minCMV promoter.
Exosomes production modeling:
1-Unconditioned expression of booster genes:
2-Conditioned expression of booster genes:
Safety
In 2023, AFCM-Egypt not only provided a single theoretical innovation for safety but also provided a persistent perspective safety platform through multiple layers to ensure safety. And for iGEM teams who will work on cell-based therapies or want to implement a safety approach to their design in the future, they can use these safety approaches. This year, we implemented multiple layers of safety to provide a safe and complete cure for rheumatoid arthritis by targeting the auto-reactive B-cells. We are more specific in targeting auto-reactive B-cells. This is achieved through a multi-layer safety system consisting of three additions and a suicidal gene :
DART-V-ADAR (tissue-specific switch)
This year, our team added this new composite part (BBa_K4586029) to the registry which is considered the most effective approach in our safety measures that will be used to develop new smart therapeutic approaches based on RNA delivery and reduce off-target effects to minimize possible risk factors.
(Detection and Amplification of RNA Triggers via Adenosine Deaminase Acting on double-stranded RNA) - (DART-V-ADAR) system, which is a programmable modular single-stranded RNA sensor that is designed to be complementary to specific mRNA within the target cell.
This sensor contains a stop codon (UAG) with a mismatched adenosine group that prevents the translation of our therapeutic agent.This mismatched adenosine group is converted to an inosine group(UIG) via ADAR enzyme hybridization or deamination activity after the binding between the sensor and its complementary mRNA within the target cell. As a result of the base editing, the stop codon becomes a sense codon that doesn't block translation. Therefore, the expression of our therapeutic cargo (Cas12k) will be done effectively. This system can be easily reproduced by other iGEM teams by following the instructions within our step-by-step tutorial for designing DART-V-ADAR sensors with MS2-RNA hairpins.
>>>for more details visit safety page by clicking Here!
SYN-notch receptor
This receptor consists of three domains: extra-cellular, trans-membrane, and intra-cellular domains. The external domain is the most important part, as it is responsible for the specificity of the receptor. Thus, we designed it to be CCP-1 which has a high affinity for the B-cell receptor (BCR), which is specific for each B-cell colony. This will make the activation of the system conditioned by the presence of auto-reactive B-cells.
Exosomal surface receptor
Similarly, we designed the exosomal surface receptor to be CCP-1 which acts as a second safety gate after the activation of the SYN-notch receptor and the release of exosomes in the media.
Suicidal gene
Last but not least, we added a suicidal gene (IC9 system) that is considered the last line of safety in our project. It is activated upon administration of a chemical inducer of dimerization that induces apoptosis of the transferred MSCs by the IC9 system.
for more information, visit the Design page by clicking Here!
AFCM Safety Guide
Welcome to our Safety guidebook on laboratory safety in the field of SynBio. Developed
specifically for iGEM teams, this resource aims to provide information and guidelines to ensure
safety and minimize risks when working in laboratories.
Reason behind its creation
Synthetic biology has the potential to change the way we design and engineer systems, offering
solutions to some of the pressing global challenges. As iGEM enthusiasts, we understand the
impact this field can have across industries like healthcare and agriculture. However, with great
advancements come great responsibilities, and safety must always remain a top priority.
We have created this guidebook to address the need for safety measures in biological
laboratories. While iGEM teams are known for their creativity and innovative spirit, often pushing
boundaries and exploring cutting edge concepts, it is vital that these endeavors are carried out
responsibly and safely. Our goal is to equip iGEM teams with the knowledge and guidelines to
mitigate risks while conducting their experiments with utmost caution and care.
Why is it important?
Working in laboratories involves handling living organisms, chemicals and dangerous
substances. If not handled with care, these environments can pose risks to both individuals and
the surrounding community. Neglecting safety protocols can lead to injuries or even
contamination of the environment.
This guidebook offers iGEMers a resource to structure their protocols and procedures based on
established practices that have been developed through years of laboratory research. It
provides an overview of hazards and risks along with effective strategies to prevent accidents,
minimize exposure, and respond promptly during emergencies.
PDF for General safety principles
Troubleshooting
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Firstly, our project idea was dependent on mesenchymal stem cells due to their direct and indirect immune modulatory functions, self-replication, high differentiation, neuro-protection,...etc.However, we faced difficulties finding MSCs despite their multiple sources. Moreover, it needs specialized laboratories with expensive requirements. As a result, we shifted to another applicable cell line called the HEK-293T cell line. HEK-293T was available, but it does not survive in hot weather, and this was somehow raising anxiety as Egypt is hot and is facing a recurrent power outage. Finally, we decided to use the wi-38 cell-line due to its suitable needs and convenient circumstances.
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At the beginning, we were aiming to use CRISPR-Cas9 to target the BAFF-R gene in the auto-reactive B-cell. We found that CRISPR-Cas9 has more than 3000 base pairs that are incompatible with IDT ordering conditions. Thus, we replaced it with Cas12k, which is shorter in sequence and similar in function.
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Despite MSCs' multiple usages, natural killer cells were inspiring and were able to perform the same task needed by the MSCs. Thus, we started to add the cytolytic enzymes (perforins and granzymes) inside the exosomes to target the auto-reactive B-cells. When referring back to our instructors, they informed us that these cytolytic enzymes have very low specificity, which will lead to off-targeting and harming many other innocent cells.
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Initially, our thinking was limited to the use of MSCs only. We were trying to gain the maximum benefit out of them, so we targeted the JAK-STAT 3 pathway by increasing the expression of P13K to trigger the immunomodulatory functions of MSCs. However, this approach has limited specificity, short-term hindering effect on the B-cell, and only potentiates immune-modulatory functions with no explicit sign of B-cell death. By contacting many experts, we found that we can add exosomes that will have a longer impact on target B-cells. Exosomes will not only improve the duration, but they will also insert apoptotic signals that will ensure cell death. Moreover, we can increase the specificity by adding exosomal surface specific receptors.
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We were designing the external domain of the mesenchymal stem cells and the exosomes to be BCR (B-cell receptor).Unfortunately, we found difficulty to isolate auto-reactive B-cells from patients.Thus, we changed the BCR to be CD19,which is found on the surface of B-cells and specifically, overexpressed on the auto-reactive B-cells' surface.
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We have changed the sensor target of the switch to be BAFF RNA (responsible for B-cells survival) rather than ACPA RNA(more specific for auto-reactive B-cells colony). This was due to the unavailability of auto-reactive B-cells from patients.
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We found low concentration of the ordered parts after gel electrophoresis.Thus, we decided to amplify them through PCR.Similarly, the PCR yielded low concentration.So, we searched for help from Dr.Dalia Mostafa. She informed us with PCR alternatives as TA cloning to increase our parts concentration.
Software
This year, we're providing a conjugation between clinical and bioinformatics software tools.
Firstly, we’ve developed a bioinformatics tool that can predict the top ranking Rheumatoid Arthritis epitope in an individual, which can vary from one person to another according to each individual’s unique genetic background and collection of etiological exposures. The user can input a CDR3-β sequence, and the tool can predict whether the input sequence is associated with RA or with another condition. Moreover, the tool can predict the binding between the CDR3-β sequence and a list of RA epitopes in order to choose the top ranking complex. This is followed by isolating the top ranking epitope to be integrated in the structure of our Syn-Notch receptor, which later can be visualized using AlphaFold2.
Furthermore, we’ve developed a clinical X-ray classification model that can predict whether the user’s input x-ray is normal or shows any rheumatoid change. This can be used as a follow-up method for assessing the effectiveness of treatment options, including our own approach. It can enable physician to detect early rheumatoid changes to take the necessary measures to stop the condition’s progress
Modeling
We have constructed two novel user-friendly interfaces for our models that could help other iGEM teams to gain the optimum outcome of their design . The first model offers sets of differential equations that represent the interaction and binding at the cellular and molecular level. This model’s ODEs are useful for other teams working on designing artificial receptors as this model allows them to compare the binding stability and affinity between the target molecule or cell and the ligand binding domain as well as providing them with the parameter values that could be manipulated to fit into their design. The second model provides all needed equations to simulate the effect of any transcriptional module present in the internal domain of any artificial receptor, thus any other teams can manipulate this model to fit into their design and do their iteration to gain the maximum pre-lab results. Therefore, we made these two models accessible to anyone through designing an online user-friendly tool or interface that allows the user to change the parameter values according to the measurement of their parts and devices to be suitable for their receptor design.
This tool is considered as a pre-wet lab tool that can guide other teams to compare different choices available to engineer any type of artificial receptor by providing the kinetics of the internal circuit and the binding between the external domain and the target molecule.
You can visit these tools by clicking Here!.
Human practices
The human practices part was a very important part of the contributions, as we made several efforts throughout the year to serve as a guide for the rest of the teams in the future. Our project was about rheumatoid arthritis, so preparing a guide to the disease that contains basic information about the disease and illustrations in a simple and easy style and language to present it to people was very important.
The educational part was very important. Since the beginning of the year, we have held periodic meetings via the Internet for individuals of all ages and fields from all over the Republic and provided them with guidance on synthetic biology. We were also always available to provide assistance to any team wishing to join the competition and provide them with sufficient information, whether inside or outside Egypt. We also organize workshops and lectures suitable for all ages, levels, and fields throughout the year. Synbio sprouts for secondary school students was one of the events we hosted where we teached them the basics of synthetic biology so that they have a foundation on which to rely in the future at the university level. Our efforts did not stop at the purely educational part, as we taught them basic skills such as digital marketing and marketing analysis.
References
- Silman AJ, MacGregor AJ, Thomson W, Holligan S, Carthy D, Farhan A, Ollier WE. Twin concordance rates for rheumatoid arthritis: results from a nationwide study. Br J Rheumatol. 1993 Oct;32(10):903-7.
- Qiu M, Zhou XM, Liu L. Improved Strategies for CRISPR-Cas12-based Nucleic Acids Detection. J Anal Test. 2022;6(1):44-52. doi: 10.1007/s41664-022-00212-4. Epub 2022 Feb 28. PMID: 35251748; PMCID: PMC8883004.
- Gayet, R.V., Ilia, K., Razavi, S. et al. Autocatalytic base editing for RNA-responsive translational control. Nat Commun 14, 1339 (2023). https://doi.org/10.1038/s41467-023-36851-z
- N Momin, E., Vela, G., A Zaidi, H. and Quiñones-Hinojosa, A., 2010. The oncogenic potential of mesenchymal stem cells in the treatment of cancer: directions for future research. Current immunology reviews, 6(2), pp.137-148.
- Zhang, Y., Liu, Y., Liu, H. and Tang, W.H., 2019. Exosomes: biogenesis, biologic function and clinical potential. Cell & bioscience, 9(1), pp.1-18. https://cellandbioscience.biomedcentral.com/articles/10.1186/s13578-019-0282-2