iGEM 2023 — Team afcm-egypt

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# Notebook

Overview

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# Engineering Success

Overview

- [Introduction](#Introduction)

- [Iteration 1](#Iteration1)

- [Iteration 2](#Iteration2)

- [Iteration 3](#Iteration3)

- [Iteration 4](#Iteration4)

- [Iteration 5](#Iteration5)

- [Iteration 6](#Iteration6)

- [Iteration 7](#Iteration7)

- [Iteration 8](#Iteration8)

- [Iteration 9](#Iteration9)

- [Iteration 10](#Iteration10)

- [Iteration 11](#Iteration11)

- [Iteration 12](#Iteration12)

- [Iteration 13](#Iteration13)

- [Iteration 14](#Iteration14)

- [Iteration 15](#Iteration15)

- [References](#references)

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## Introduction

To tackle one of the most widely spread autoimmune diseases in our region, our journey has gone through various phases of Design and Research-Build-Test-Learn. We started with a general study of the different autoimmune diseases prevalent in our society. After a lot of hard work and long hours of deep research, we came up with a long list of a variety of serious and widely spread diseases. On top of that list came Rheumatoid Arthritis (RA).

RA is one of the oldest yet the most common diseases in our society. Its unknown etiology and lack of terminal specific cure made it our undisputed target in 2023.

Therefore, we started working with one goal in mind, and that is to create an effective treatment for Rheumatoid Arthritis . On our adventure against this disease, we came across a lot of challenges that have only inspired us to keep going.

Here, we will take you on a quick tour through the different stages of our project.

![](https://static.igem.wiki/teams/4586/wiki/transformed-banner/afcm-egypt-1080-x-2000-px-1080-x-1080-px-2-new.png)

## Iteration 1:

![](https://static.igem.wiki/teams/4586/wiki/transformed-banner/1new.png)

### Design and research:

Our literature search demonstrated that macrophages play an important role in the pathophysiology of Rheumatoid arthritis by inducing inflammation and increasing disease activity, which can also be one of the factors that can cause joint destruction.

They can also act as antigen-presenting cells, which can stimulate the activity of other inflammatory pathways, resulting in further tissue destruction.

### Build

Our first iteration was to genetically engineer the macrophages to make them able to suppress the immune reaction. This was achieved by switching them from the M1 Inflammatory phenotype to the immunoregulatory phenotype M2, which regulates and suppresses other immune cells’ activity while promoting tissue regeneration, thus limiting the progression of the disease.

![](https://static.igem.wiki/teams/4586/wiki/11.png)

### Test

Through a deeper search, we found that macrophages are not the only cells responsible for the damage caused by Rheumatoid Arthritis.

So the effect of the phenotypic switch won't be enough to significantly control the disease activity.

### Learn

We wanted to have a broader look for other factors damaging the joints and corrupting the immune response, such as Fibroblasts-like synoviocytes (FLS), autoreactive B-cell antibodies, and T cytotoxic cells. So we decided to work on another approach targeting one of these main factors.

## Iteration 2:

![](https://static.igem.wiki/teams/4586/wiki/pastel-product-life-cycle-chart-with-abstract-shape-instagram-post-2.png)

### Design and research:

Our research proved that the pathophysiology of Rheumatoid arthritis is multifactorial, not just affected by one or two pathways; there are multiple factors affecting the disease pathophysiology, such as T-cells, B-cells, macrophages, and fibroblasts like synoviocytes (FLS), etc.

![](https://static.igem.wiki/teams/4586/wiki/10.png)

### Build

Our second iteration was to modify one of the most commonly engineered cell-based therapies, which is T-cells. These modifications will take place through genetic engineering to make T-cells able to detect and suppress the immune reaction. This was achieved by targeting and destroying the auto-reactive B-cells that produce ACPA antibodies that induce inflammation and destruction, both local and systemic, thus limiting the progression of the disease.

![](https://static.igem.wiki/teams/4586/wiki/transformed-banner/1.png)

This Figure illustrates the design of CAR T-cell targeting the autoreactive B-cell.

### Test

Through considerable exploration, we found that the auto-reactive B-cells are not the only cells responsible for the damage caused by Rheumatoid Arthritis.

On the other hand, using T-cell-based therapy results in ramping up the immune response. This means that T-cells could increase the inflammatory response and also suppress normal healthy cells, not only the auto-reactive B cells.

Therefore, this design of the therapeutic approach wouldn't reach the intended optimum results in terms of achieving best specificity and controlling disease activity, as well as having many undesirbale side effects.

![](https://static.igem.wiki/teams/4586/wiki/transformed-banner/8.png)

### Learn

We decided to look for other cells that can be used for cell-based therapy. But this time, we will look for cells that can regulate more than one pathway and -at the same time- have a few adverse effects on immunity. We made patient’s safety and drug’s specificity our top priority. Hence, we started looking for another approach that could control disease activity without significant unwanted effects on other normal healthy systems.

## Iteration 3:

![](https://static.igem.wiki/teams/4586/wiki/3.png)

### Design and research:

Our search was conducted to find a vector or a cell that has wide control over most of the factors responsible for the pathophysiology of rheumatoid arthritis. After going through many papers and consulting many experts, we finally decided to take advantage of Stem cells as an innovative therapeutic approach, specifically Mesenchymal Stem Cells (MSCs).

![](https://static.igem.wiki/teams/4586/wiki/10mscsnew.png)

This figure illustrates the role of MSCs in regulating the immune cells in RA

### Build

Our third iteration was to modify MSCs; to make them directed specifically towards the site of interest. Therefore, we had to add an extracellular receptor, making MSCs able to detect the fibroblast-like synoviocyte (FLS). As a result, activated MSCs act by regulating autoimmune cells' function and suppressing their pathogenic effect on joints by secreting their built-in exosomes that regulate the FLS to decrease disease activity. Besides that, MSCs also have an amazing feature; they naturally contribute to the regeneration of damaged tissues in the joints.

![](https://static.igem.wiki/teams/4586/wiki/transformed-banner/9.png)

This figure illustrates the role of FLS in disturbing the immune cells in RA

### Test

Despite all the great aspects of MSCs and their contribution to control the disease, we found that their activity remains non-specific and uncontrollable. In addition, there is no significant effect on the disease activity, as the FLS is not the only cell contributing to the pathophysiology of the disease. Our design also lacks specific targeting of the FLS, which reduces its potential as a great treatment option for RA.

### Learn

We concluded that we need to increase the effectiveness and the specificity of our approach. Furthermore, we wanted to target one of the main cells responsible for the pathophysiology of the disease , rather than just depending on the great built-in functions of the MSCs.

## Iteration 4:

![](https://static.igem.wiki/teams/4586/wiki/4.png)

### Design and research:

Through a deeper search and long brainstorming sessions, the idea of using exosomes as a vector synthesized by MSCs came to light. Exosomes are a class of cell-derived extracellular vesicles of endosomal origin; they also play an important role in intercellular communication.

### Build

In the fourth iteration, we wanted to isolate the exosomes first from MSCs and modify these exosomes to carry the apoptotic signal called BID to initiate the apoptotic pathways in the T-cells and B-cells. We also added a targeting receptor on the exosomes to ensure that the cargo reaches and suppresses the auto-immune cells.

![Ihela](https://static.igem.wiki/teams/4586/wiki/transformed-banner/cd19.png)

This Figure illustrates 1-isolation method of exosomes, 2-the exososme carrying the BID siganl.

### Test

The application of this design proved that the BID signal carried by Exosomes is still not specific enough to the targeted auto-immune cells. In fact, there are more than 200 normal peptides targeting various cells, making them victims of apoptosis due to off-targeting of this signal. Also, exosomes have quite a short half life, they require multiple doses to achieve their purpose.

### Learn

We found that our last design needs more modification in order to prevent the off-targeting effect on other normal immune cells caused by the multiple peptides on the surface of the exosomes. We also needed to focus on developing the BID apoptotic signal, making it more selective for the targeted B cells. This could be done by either substituting this signal with another one or genetically modifying this signal with safety switches.

## Iteration 5:

![](https://static.igem.wiki/teams/4586/wiki/eng/success-5.png)

### Design and research:

After contacting Immunology & Rheumatology experts and meeting with Stem cell specialists, we learned that the auto-reactive B-cell is considered one of the main cells causing the pathogenesis of RA. Since the concentrations of B-cell produced ACPA titre correlate with the severity of the condition. We also found out that the inflammatory effects of ACPA are not just limited to joints but also reach out for other tissues resulting in severe damage.

### Build

In our fifth iteration, we went back to working on MSCs. Hence, we started to modify the extracellular domain of the MSCs’ receptor to directly recognize auto-reactive B-cells. This was achieved through using one of the citrullinated antigens found in the literature and testing their binding affinity to our engineered receptor by simulating it through software tools.

![](https://static.igem.wiki/teams/4586/wiki/14.png)

### Test

We found that the binding affinity of MSCs with autoreactive B-cells was not high enough, which causes the lack of specificity of this design. We also faced the same problem in the previous iteration where we depended mainly on the natural effects of MSCs in enhancing the immunity and regulating the inflammatory response.

### Learn

We discovered that we need to make more modifications to our last design to increase the specificity by finding another ligand. We also need to integrate another system into our design making it more specific to the auto-reactive B-cell and regulating its activity. On another hand, we felt that we could benefit much more from the various potentials of MSCs by modifying them with genetic engineering.

## Iteration 6:

![](https://static.igem.wiki/teams/4586/wiki/eng/success-6.png)

### Design and research:

During our search, we tried to simulate the binding between our receptor and its target to test the stability inside the MSCs. Therefore, the literature taught us that there are different types of synthetic receptors that can be used on MSCs with a modifiable extracellular domain. The type of receptors depend on the nature of the target which may be a cell or a molecule.

### Build

Our sixth iteration was to modulate and choose a type of receptor suitable for our design, so we went through six types of receptors from GPCRs, CAR, CAAR, MESA, GEMs, and others. After prolonged research and various tests, we finally settled on the SYN-Notch receptor.

[Click Here for more details about the binding affinity](https://2023.igem.wiki/afcm-egypt/model#Model1)

### Test

We found that the SYN-Notch receptor was the most suitable for our design. This receptor is characterized by its ability to sense cells in the extracellular environment. Adding to that, its modular ability to regulate the extra and intra cellular domains. It is also considered a stable receptor thanks to its Mouse Notch Intermembrane domain.

![](https://static.igem.wiki/teams/4586/wiki/design/design/desgin-6.jpg)

### Learn

After we settled on the type of receptor, we wanted to improve our approach by increasing the binding ability of the receptor and also modifying our genetic parts, both by using directed evolution. All along, we were eager to reach the best form of our design either by applying more modifications or merging different approaches or new designs together.

## Iteration 7:

![](https://static.igem.wiki/teams/4586/wiki/eng/success-7.png)

### Design and research:

After our successful attempts to modify the receptor and genetic parts, we needed to settle on a better approach that could guide us to an optimum design. Therefore, we had the idea of merging our prior approaches together (MSCs and Exosomes), which led to the third integrated design in our project.

### Build

Our seventh iteration was the integration of the prior approaches, MSCs and Exosome-based therapy. We developed MSCs capable of secreting exosomes containing a BID signal that initiates apoptosis within the autoreactive B-cell. In addition, we implemented booster genes in our circuit in order to increase the expression of exosomes from the MSCs.

### Test

Our integrated approach has greatly improved our platform. As a result, our design can widely control and treat RA by regulating the multiple factors responsible for the pathogenesis.

![Ihela](https://static.igem.wiki/teams/4586/wiki/transformed-banner/2.png)

This Figure illustrates the integrated approach using both MSCs and Exosomes

Moreover, we modeled the kinetics of using booster genes to amplify the exosome production as shown in the following graph.

![Ihela](https://static.igem.wiki/teams/4586/wiki/design-3.png)

This Figure illustrates the booster genes used for increase the production of the exosomes

However, we still needed to make some modifications to our platform to assure the applicability of our therapeutic approach as an effective drug for RA.

### Learn

We decided to modify the BID signal inside our exosomes in order to become more targeted, specific and stable. We were also keen on enhancing the signal’s ability to inhibit the auto-reactive B-cells.

## Iteration 8:

![](https://static.igem.wiki/teams/4586/wiki/eng/success-8.png)

### Design and research:

After many unfortunate attempts to modify the BID signal of the exosomes, we came up with another idea of using another system instead of BID. This new system should be more specific and effective in inhibiting the auto-reactive B-cells.

### Build

In our eighth iteration, we replaced the BID apoptotic signal as we developed exosomes with a CRISPR-Cas system that knocks down the B cell-activating-factor-receptor (BAFF-R) gene, which is an essential factor for B-cell survival.

[Click Here for more details about the final design](https://2023.igem.wiki/afcm-egypt/design#Cargo)

### Test

This design was considered the peak of our therapeutic journey as it has become much more effective and selective than its priors. However, we still need to assure the safety and feasibility of our treatment by sparing normal B cells from the silencing effect of the CRISPR-Cas system, while at the same time, suppressing the inflammatory cells implemented in the pathogenesis of RA.

### Learn

We thought about adding a new modification into our circuit in order to reach a safer version of our integrated design. This modification would aim to regulate the disease activity and its progression while preventing the silencing effect of the CRISPR-Cas system on the normal B-cells.

## Iteration 9:

![](https://static.igem.wiki/teams/4586/wiki/experiments/pastel-product-life-cycle-chart-with-abstract-shape-instagram-post-6.png)

### Design and research:

To increase the safety of our approach, we decided to choose a new family of the CRISPR system and we also wanted to regulate the expression of our engineered exosomes. Hence, we decided to look for a method that can control the translation of the Cas9\g\mRNA ACPA system. As a result, we could regulate the binding of the Syn-Notch receptor with its targeted autoreactive B-cell.

### Build

In our ninth iteration, we decided to choose the CRISPR-Cas12k system as our new CRISPR family and adding a new layers of safety to our approach. Therefore, we implemented the cre-recombinase system to the booster genes to control their expression. Secondly, we inserted a new tissue specific switch in our circuit called DART-V-ADAR (Detection and Amplification of RNA Triggers via ADAR). This switch will guarantee a conditioned translation for our system, based on the availability of ACPA’s mRNA in the targeted cells.

[Click Here for more details about the final design](https://2023.igem.wiki/afcm-egypt/design#Cargo)

### Test

Our design has reached a high level of specificity and selectivity with the Cas12k\g\mRNA ACPA system controlled by the tissue-specific switch. As a result, our platform can deplete the autoreactive B cells safely with a minimal off-targeting effect.

![Ihela](https://static.igem.wiki/teams/4586/wiki/asset-2cas12k.png)

A high level of specificity and selectivity with the Cas9\g\mRNA ACPA system controlled by the tissue-specific switch

Thanks to the modeling, we found that booster genes added to our circuit would increase exosomes production 1.2 times the normal release in absence of booster genes.

### Learn

We decided to adapt this last integrated approach and start our Wet Lab work as soon as possible.

## Iteration 10:

![](https://static.igem.wiki/teams/4586/wiki/10neww.png)

### Design and research:

Fortunately, the people responsible in the airport agreed to help us by speeding up the process of the delivery so we could receive our order in the shortest time possible .

### Build

After more than one month from our order, we finally received our parts and materials. Hence, without any further delay, we directly arranged all the lab requirements and launched our wet lab phase. We started working on resuspension, amplification and digestion protocols and -at the same time- bacterial transformation for our vector.

### Test

We tried to resuspend all the g-Blocks, amplify using PCR, and start the digestion protocol. We also began running the parts on gel to see if the digestion process would go well.

### Learn

Unfortunately, we found that the concentration of all the DNA parts is too low to go through the ligation process. troubleshooting may be due to:

* Provided parts( inserts) were not ordered in cloning vectors that are known for easier restriction and ligation, while linear fragments have too many issues.
* Parts were provided in low concentration.
* Primers were not designed as recommended.
* It is better to do the assembly using Gibson Assembly , Overlap PCR or 3A assembly.
* The provided restriction enzymes are of low efficiency.

## Iteration 11:

![](https://static.igem.wiki/teams/4586/wiki/11neww.png)

### Design and research:

We reached out to some doctors and experts to help us improve and fix this issue. Therefore, they advised us to adopt another methods such as TA cloning method or increasing the PCR reaction.

### Build

In this iteration, we ordered the new essential kits for this suggested approach in order to start implementing them in our design. However, we were running out of time as we had only a couple of weeks for the wiki freeze, so we decided to increas the PCR reaction untill recieving the kits .

### Test

In order to use TA cloning, we had to make our order as fast as possible from the available resources and running PCR amplification with higher PCR reaction that result in increase the purified concentrations of DNA parts.

### Learn

We found that we will face the same problem concerning the delay of the delivery process of the order from the airport. We also had to find alternatives to all the cell lines that were not available in our country, and beginning the transformation step.

## Iteration 12:

![](https://static.igem.wiki/teams/4586/wiki/12neww.png)

### Design and research:

After receiving some of the parts and materials needed, we faced a new problem which is the difficulty to extract and isolate mesenchymal stem cells (MSCs). This is due to the lack of antibodies and growth media essential for its characterization.

### Build

We found another cell line that could be used instead of MSCs which is called Human Embryonic Kidney 293 cells(HEK 293). This cell line is considered an effective way to use viruses to evolve target genes and transfer them into the cells.

### Test

Unfortunately, the HEK 293 cells could not survive in our hot temperatures even in the lab incubators. So we had to find another alternative to resume our lab work. After a quick but deep research, we finally replaced the HEK 293 with WI-38 which is a diploid human cell line composed of fibroblasts derived from lung tissue.

### Learn

We finally settled on the WI-38 cell line as a good replacement for MSCs. However, we still needed to isolate the autoreactive B-cells in order to apply our design on them.

## Iteration 13:

![](https://static.igem.wiki/teams/4586/wiki/13neww.png)

### Design and research:

We found that it is impossible to isolate the autoreactive B-cells due to many reasons as:

* Ethical consideration of taking direct samples from patients.
* Lack of lab protocols needed for the isolation of only autoreactive B-cells while sparing normal B-cells.

### Build

In our fourteenth iteration, we decided to make a proof of concept on anti-CD19 instead of CCP1 which is the self-antigen attacked by ACPA of the autoreactive B-cells.

### Test

The anti-CD19 binded to the CD19 receptor that is overexpressed on the surface of the autoreactive B-cells. This activated the internal domain of the Syn-Notch receptor to produce the engineered exosomes. These exosomes contain the CRISPR system responsible for suppressing B-cells.

### Learn

We can finally prove our concept on the CD19 receptor, as it is available and easier to implement in the lab. We now have to assure the effectiveness of DART-V-ADAR safety switch.

## Iteration 14:

![](https://static.igem.wiki/teams/4586/wiki/pastel-product-life-cycle-chart-with-abstract-shape-instagram-post-5.png)

### Design and research:

We made the DART-V-ADAR switch sensitive for ACPA mRNA, which makes it specific for autoreactive B-cells only and also incorporates the cre-recombinase system to the exosomes circuit.

### Build

Due to the difficulty of isolating the autoreactive B-cells mentioned in the previous iteration, we had to make some modifications to our safety switch. Hence, instead of targeting ACPA mRNA, the switch was directed towards BAFF-R mRNA, which is found in all the B-cells, and also integrates the cre-recombinase system to control the expression of exosomes.

### Test

On testing the new switch activity, we found that it is sensitive for all B-cells. This means that its specificity was decreased by our last modification. However, this was intended to happen in order to compare the sensitivity of the safety switch with other cells other than B-cells, and also we controlled the expression of exosomes in relation with binding of MSCs with the auto-reactive B-cell.

### Learn

We tested the DART-V-ADAR switch sensitivity by comparing between its function on B-cells and on other types of cells, and also our new approach that control the exosome expression.

## References

* Kojima, R., Bojar, D., Rizzi, G. et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat Commun 9, 1305 (2018). https://doi.org/10.1038/s41467-018-03733-8.
* Sgodda M, Alfken S, Schambach A, Eggenschwiler R, Fidzinski P, Hummel M, Cantz T. Synthetic Notch-Receptor-Mediated Transmission of a Transient Signal into Permanent Information via CRISPR/Cas9-Based Genome Editing. Cells. 2020 Aug 20;9(9):1929. doi: 10.3390/cells9091929. PMID: 32825374; PMCID: PMC7563181.
* Li, M., Fan, YN., Chen, ZY. et al. Optimized nanoparticle-mediated delivery of CRISPR-Cas9 system for B cell intervention. Nano Res. 11, 6270–6282 (2018). https://doi.org/10.1007/s12274-018-2150-5
* 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

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# Experiments

Overview

- [Plan](#Plan)

- [Phase 1](#phase1)

- [Day 1](#day1)

- [Day 2](#day2)

- [Day 3](#day3)

- [Phase 2: Molecular lab](#mol)

- [Preservation of pcDNA3.1](#pre)

- [DH5α competent cells preparation-chemical methods](#dh5)

- [pcDNA transformation](#pc)

- [Alkaline lysis miniprep](#alk)

- [Resuspension](#res)

- [PCR Amplification](#pcr)

- [Digestion](#dig)

- [Ligation](#lig)

- [Colony PCR](#col)

- [Gel electrophoresis](#gel)

- [Phase 3: Cell lab:](#phase3)

- [Subculture](#sub)

- [Lipofectamine transfection](#lipo)

- [References](#references)

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## Plan

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/1.jpeg)

We have a lot of work to do on our project regarding the wet lab. This work needs to be regulated and well planned, so we divided it into 3 main phases. Each phase will contain a group of experiments that relate to each other to get the results that will be used in the next step:

* **Phase 1 : Safety Practice**
  Before starting our wet lab phase, the wet lab team must refresh their information about lab safety and technique to keep them safe during this phase.

**Phase 2 : Cell Lab**
This step will focus on cell culture and growth to produce the mature cells that will be ready to be transfected.

* **Phase 4 : Validation**
  It concerns the part of DNA from resuspension , digestion, ligation, and gel electrophoresis.

**Phase 3 : Molecular lab**
This phase concerns validation of the expression of the transfected part with specific antibodies and tagging it with specific tagging for each antibody.

## Phase 1: Safety practice

During this phase, the wet lab team went to the lab to revise the general safety rule to prevent any disaster from happening during the wet lab work. Also, they were trained in the basic lab techniques to improve the efficacy of the work, prevent material and time waste, and reduce mistakes resulting from a lack of experience.

### Day 1: Identification of safety signs and lab equipments

Recorder: Omar Fouad
Attendance: All team members

Firstly, we have learned that missing a single procedure, such as labelling the products, can ruin months and years of work. Either in clinical or biological labs, labels are crucial and can be variable according to your needs. Furthermore, they can be used for:

* Organization: you can organize your products, materials,or equipment by names, colors,date of expiration,or even barcodes. Printed IDs or barcodes help you know where the item is, what steps it has undergone,and what steps are left.
* Accuracy: The easier you can collect your data and work, the more accurate your results are.
* Safety: Any mistake in labels can lead to a catastrophe. For example, mixing incompatible agents can result in unpredictable reactions or, clinically, replace patient results.

Similarly, the safety signs have the same importance. We have learned some signs related to our laboratory work, such as no food or drinks permitted, personal protective equipment related to our biosafety level, biohazard signs, and carcinogenic signs.

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/2.png)

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/3.png)

Moreover, we have learned how to use some of the laboratory devices , such as PCR, centrifuges, etc…

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/4.png)

### Day2: Precautions when handling hazardous materials

Recorder: Youssef amir
Attendance: All team members
We have learned that hazardous chemicals are substances or mixtures that have the potential to cause adverse side effects, damage the surroundings,or cause injury. They can be presented in gas, liquid, or solid forms and cause explosions, corrosion, toxicity, etc There are many worldwide organizations that people follow to know about hazardous chemicals, such as OSHA's Hazard Communication Standard (HCS), the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals in Australia, mostly work health and safety (WHS) duties, etc. Moreover, there are material safety data sheets associated with the products to state the risks associated with them and the safety measures required when dealing with them specifically.
Some precautions are taken into consideration when dealing with hazardous substances.

* The chemicals are inside a sealed, labelled safe container
* Some materials are harmless on their own. However, when mixed with another chemical,they release toxic fumes
* Some materials are harmless on their own. However, when mixed with another chemical,they release toxic fumes
* If there is a potential for splashing, the user has to wear eyeglasses
* The user has to be in a well-ventilated place if using corrosive or flammable chemicals

### Day 3: Safe by practice

Recorder: Ahmed Wael
Attendance: All team members
Finally, we started to learn how to use some equipment properly under the supervision of experts. In addition, how to irrigate the eyes or any body part if exposed to corrosive substances. As medical students, we also know how to use the first aid box and how to prevent further complications.

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/5.jpeg)

## Phase 2: Molecular lab

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/6.png)

During the molecular lab phase, we aim to prepare the plasmids carrying our genetic circuits for transfection in the cell lab phase. We started our molecular phase by resuspension of the dried genetic parts ordered from IDT. Then it was digested by the restriction enzymes mentioned below. After that, we transform the plasmid vector pcDNA 3.1- into bacteria for amplification to prepare the proper amount of the plasmid vector to carry our circuits. Then the genetic circuits were ligated with the plasmid vector to form our plasmids. In addition to that, we have run gel electrophoresis after digestion and ligation to be sure about the length of the plasmid vector and the genetic parts and to ensure that they were added to the right place.

### Preservation of pcDNA3.1(-)

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/7.png)

### DH5α competent cells preparation-chemical methods

Recorder: Omar Fouad
Attendance: Hossam algamal , Ahmed wael and Mohamed Aboelghar
Documentation of the day:
Location: AFCM Lab

* Today's experiment in the lab focused on preparation of the DH5α competent cells to be ready for transformation.
* Our goal today is to prepare DH5α competent cells to transform them with pcDNA 3.1-.
* We started our experiments by adding the overnight cultured media to 500 ml of SOC medium in a 2 L flask.
* Then we let the cell grow to an OD600 between 0.4 or 0.6.
* Then we cooled the cells for 10 min.
* Then we transfer the culture to a sterile 500 mL centrifuge tube then we centrifuge them at a sterile 500 mL.
* Then we resuspend the cells into 150 mL of cold CMG buffer.
* Then we incubated them on ice for 15 min.
* Then We centrifuged the precipitated cells again at 4000 g for 5 min at 4 °C.
* After that we resuspended the cells in 36 mL of cold CMB buffer and transferred them to a 50 mL disposable centrifuge tube.
* We added 1.26 mL of high quality DMSO again, and mixed well, then incubated them on ice for 5 min.
* Then we incubated the cells’ suspension on ice for 5 min.
* We added 1.26 mL of high quality DMSO again, and mixed well, then incubated them on ice for 5 min.
* Then we dispensed the suspension into sterile microcentrifuge tubes and we froze them in a liquid nitrogen.
* Finally we stored the cells at -80.

### pcDNA transformation into DH5α and culture on ampicillin plates

Recorder: Ahmed Wael
Attendance: Hossam algamal , Ahmed wael , Mohamed Aboelghar
Documentation of the day:
Location: AFCM Lab

* We removed the cells from the -80 C freezer and thawed in your hands.
* We added 1-5 µl containing 1 pg-100 ng of pcDNA 3.1 to the cells and carefully flicked 4-5 times.
* We placed the mixture on ice for 2 min.
* We heated it at 42 C for 30 sec.
* We placed it on ice for another 2 min.
* We added 950 µl of room temperature SOC to the mixture, then immediately spreaded 50-100 µl onto a selection plate and incubated overnight at 37-42°C.

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/8.png)

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/9.png)

### Alkaline lysis miniprep

Recorder: Mohamed Aboelghar
Attendance: Hossam algamal , Ahmed wael , Mohamed Aboelghar
Documentation of the day:
Location: AFCM Lab

* We prepared the following solution:
  + Solution-1:
    - 25 mM Tris-HCl (pH 8)
    - 50 mM glucose
    - 10 mM EDTA

    \*Stored at 4 C
  + Solution-2: Denaturing buffer
    - 0.2 N NaOH
    - 1.0% SDS

    \*Stored at room temperature
  + Solution-3: Renaturing buffer
    - 120 mL 5M Potassium acetate
    - 23 mL glacial acetic acid
    - 57 mL of dH2O

    \*Stored at 4 C

  * We cultured 1.5 ml of the overnight bacteria Then we added to the 1.75 ml centrifuge tube.
  * We centrifuged the tube at 10,000 g for 30 ml.
  * We removed the supernatant and resuspended the bacteria in 100 μL of the resuspension buffer.
  * We vortexed the solution for 2 min until the bacteria were fully resuspended
  * We added 200 μL of the denaturing solution to the resuspended bacteria, and we inverted 5 times to mix the content.
  * We incubated the solution on ice for 5 min.
  * We added 150 μL of the renaturing solution to the denatured bacteria, then we mixed it by inverting the tube several times.
  * We incubated the solution on ice for 5 min.
  * We centrifuged the solution at 12,000 g for 5 min.
  * We pipetted the supernatant into a new tube.
  * We added 700 μL of cold 100% ethanol to the solution.
  * We poured out the supernatant.
  * We washed the solution with 70% ethanol to remove excess salt.
  * We dried the pellet on air for 20-30 min
  * We resuspend the pellet with 25-50 μL of TE.

### Resuspension

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/10.png)

Recorder: Khalid Hela
Attendance: Hossam algamal , Omar Fouad , Emad Hamdy
Location: AFCM Lab
Documentation of the Day:

* Today we aim to work on our gBlocks Gene Fragments Under the guidance of Dr. Ahmed, the expert in molecular biology,as we aim to resuspend the anticipated gBlocks Gene Fragments from IDT.
* The gBlocks contain genetic material waiting for transformation into a liquid form to be suitable for laboratory experiments. We knew that we must carefully follow each step of the resuspension process to ensure the best efficiency of the samples.
* With our gloves on and our minds focused, we prepared the equipment.as we used a trusty centrifuge, a reliable heat block, and a powerful vortexer, Additionally, we gathered pipettes, specifically a P20 and a P200, to handle the delicate genetic material.
* We repeat quantification of each sample twice using a Nanodrop instrument because this ensures that there is nothing in the water or buffer that absorbs at A260 and would artificially inflate our sample readings

**Methods:**

* Before opening the tube, We spun it down in a microcentrifuge for 3–5 seconds to ensure the DNA is in the bottom of the tube.

  \*Note: The pellet can become statically charged and, without this step, can either fly out of the tube or remain in the cap, resulting in loss of yield.
* We added molecular grade water, to reach a final concentration of 10 ng/µL.

  \*Note: Our experiments have shown that storage concentrations <1 ng/µL result in loss of material due to adherence to the plastic tube in the absence of a carrier such as tRNA.
* We vortexed it briefly.
* We incubated the tube at approximately 50°C for 15–20 min. Then we heated the tube to ensure the solvent comes in contact with the tiny pellet, even if it is stuck to the side of the tube. Thus, this step will increase the likelihood that the entire pellet will be resuspended.
* We briefly vortexed the tube and centrifuged it.
* We verified the final concentration immediately by nanodrop. We used an elution buffer to blank.

  \*Notes: Repeat quantification of each sample twice. When using a Nanodrop instrument, test the sample resuspension solution alone between each sample measured. This ensures that there is nothing in the water or buffer that absorbs at A260 and would artificially inflate your sample readings

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/11.png)

### PCR Amplification

* We placed a thin wall PCR tube that was placed on ice.
* The following reaction was settled:
  + 2 μL Template DNA (10 ng-500 ng)
  + 5 μl 10X Taq buffer with MgCl2
  + 1 μl dNTP mix (10 mM each nt)
  + 2.5 μL Forward Primer (10 μM stock)
  + 2.5 μL Reverse Primer (10 μM stock)
  + 0.2 μL Taq DNA Polymerase (5 units/μL)
  + 36.8 μL Sterile dH2O (variable)
* The reaction tubes were placed in a PCR machine.
* The annealing temperature was set 5°C below the primer melting temperature(TM).
* The extension step was set at 1-2 min per kilobase of product.
* The initial denaturation for 2 min at 94°C.
* Then denatured for 30 sec at 94°C.
* The primer was annealed for 30 sec at 5 Anneal primers for 30 seconds at 5°C below TM.
* The DNA was extended for 2 min at 72°C.
* The steps from 8-10 were repeated for 25-30 cycles.
* The final extension for 5 min at 72°C.
* 2 μL were tested on gel.

### Digestion

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/12.png)

Recorder: Hossam Bannis
Attendance: Youssef Amir ,Emad Hamdy , Mohamed Aboelghar
Documentation of the Day:

* Our mission Today is to prepare our plasmids for digestion using our specific restriction enzymes. Our engineered plasmids are designed to transform normal Mesenchymal Stem Cells (MSC) into our SUPER-MSCs, with their super powers.
* We are using the Digestion Protocol of NEB and to confirm correct positioning of our genetic parts, we digested each part with specific restriction enzymes. We are using six restriction enzymes due to our biobrick prefix and biobrick suffix.

We designed our genetic parts to be digested using specific restriction enzymes for each part to make sure that it will be placed in its right position in the plasmid vector. We have 3 plasmids that need to be transfected. Each one will be responsible for a specific function we provide to Mesenchymal Stem Cell (MSC) to be a SUPER-MSC. The target orientation we need to reach will be like the following tables:

Plasmid-1

| Part | Length | Restriction enzymes |
| --- | --- | --- |
| Syn-Notch | 2740 bp | * EcoRI * Hind-3 |
| Booster gene-1 | 2490 bp | * Hind-3 * Pst-1 |
| Booster gene-2 | 2725 bp | * Pst-1 * BamHI |
| pcDNA 3.1- (Backbone) | 5427 bp | * EcoRI * BamHI |
| Total Length | 13162 bp |

Table-1: Plasmid-1 consists of a pcDNA 3.1- plasmid vector ligated with 3 genetic parts : Syn-notch , Booster gene-1, and Booster gene-2. This plasmid is responsible for the expression of the Syn-Notch receptor on the surface of the transfected cell. The total length of the plasmid after ligation is 13162 bp which will be tested after ligation by gel electrophoresis.

Plasmid-2

| Part | Length | Restriction enzymes |
| --- | --- | --- |
| Loading System | 1483 bp | * EcoRI * Hind-3 |
| Exosomal Receptor | 1158 bp | * Hind-3 * Pst-1 |
| CX-43 | 2250 bp | * Pst-1 * BamHI |
| pcDNA 3.1- (Backbone) | 5427 bp | * EcoRI * BamHI |
| Total Length | 10079 bp |

Table-2: Plasmid-2 consists of a pcDNA 3.1- plasmid vector ligated with 3 genetic parts : Loading system, Exosomal receptor, Cx43. This plasmid will be responsible for producing engineered exosomes in the transfected cell. The total length of the plasmid after ligation is 10079 bp which will be tested after ligation by gel electrophoresis.

Plasmid-3

| Part | Length | Restriction enzymes |
| --- | --- | --- |
| Guide RNA-BFP-switch | 2725 bp | * EcoRI * Hind-3 |
| New MS2 | 496 bp | * Hind-3 * Xbal |
| Rest of MS2 and Cas12k | 2484 bp | * Xbal * Kpn-1 |
| MCP-ADAR-RFP | 2065 bp | * Kpn-1 * BamHI |
| pcDNA 3.1 (Backbone) | 5427 bp | * Eco-RI * BamHI |
| Total Length | 12845 bp |

Table-3: Plasmid-3 consists of a pcDNA 3.1- plasmid vector ligated with 4 genetic parts : Guide RNA-BFP-switch, New ms2, Cas12k, and MCP-ADAR-RFP. This plasmid is responsible for producing the killing signal for the auto-reactive B-cell. The total length of the plasmid after ligation is 12845 bp which will be tested after ligation by gel electrophoresis.

**Procedure:**

Preparation:

* We set up the following reaction ( enzymes should be added last) :

| Reagents | Volume |
| --- | --- |
| Nuclease free water | 16u |
| Buffer 2:1 (10X) | 2.5ul |
| DNA | 100-500 ng |
| BSA | 0.5ul |
| Restriction enzyme | 0.5ul |
| Total | 20ul |

* There should be a total volume of 20ul. We mixed well and spun it down briefly.
* We incubated the restriction digest at 37 C for 30 min, and then 80 C for 20 min to heat kill the enzymes. We incubated in a thermal cycler with a heated lid
* We run a portion of the digest on a gel (8ul, 100 ng), to check that both plasmid backbone and part length are accurate.

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/13.jpeg)

### Ligation

Recorder: Hossam Algamal
Attendance: Hossam Bannis, Ahmed wael , Khalid Hela

**Documentation of the Day:**

* Today our goal is to ligate our digested parts into our 3 plasmid vectors in the correct order to be ready for transfection.
* We did a ligation reaction to assemble our three plasmids that were ready for transfection into our Wi38 cells. We followed the ligation protocol from iGEM registry protocols and the reactions went smoothly. We are excited to see the results of the transfection and to continue the work on Wi38 cells.
* We used that restriction enzymes to digest the 3 plasmids backbone and our parts:(EcoRI - SpeI)
* We used a molar ratio of 1:3 of plasmid backbone to parts in the ligation

Ligation is the process of rearranging the digested parts in its right order in the plasmid to be ready for transfection. The genetic parts were ligated using the following protocol in order to produce 3 plasmids ready for transfection to our engineered cell.

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/14.png)

**Methods:**

* We added 2ul of the digested plasmid backbone (25 ng).
* We added an equimolar amount of digested fragment (< 3 ul).
* We added 1 ul of T4 ligase buffer. Note: Do not use quick ligase.
* We added 0.5 ul of T4 ligase.
* We added 10 ul of water.
* We ligated it at 16C for 30 min. Then we heated it at 80 C for 20 min.

### Colony PCR

Recorder: Mohamed Aboelghar
Attendance: Ahmed Attia, Ahmed Wael , Ahmed Shalan
**Documentation of the Day:**

* We prepared the following in sterile 1.5 mL microcentrifuge tube:

| **Component** | **Volume for 10 µL reaction** | **Master Mix volume** |
| --- | --- | --- |
| MilliQ water | 4.6 µL | 12.5 µL |
| OneTaq 2x Master Mix with Standard Buffer | 5 µL | 25 µL |
| Forward primer (10 µM) | 0.2 µL | 62.5 µL |
| Reverse primer (10 µM) | 0.2 µL | 6.25 µL |

* We dispensed the 10 µL of master mix in 0.2 mL PCR tube.
* We touched a single bacterial plate with the tip of the pipette then mixed it into one of the PCR samples.
* We amplified the nucleic acid in a thermocycler according to the annealing temperature and the length of the desired amplicon.
* We visualised the results on agarose gel.

### Gel electrophoresis:

![Ihela](https://static.igem.wiki/teams/4586/wiki/experiments/15.jpeg)

Recorder: Mohamed Aboelghar
Attendance: Ahmed Attia, Ahmed Wael , Ahmed Shalan
 **Documentation of the Day:**

* We arrived in the lab and set up the electrophoresis chamber.
* We prepared the agarose gel, EDTA buffer and EDTA.
* We poured the agarose gel into the electrophoresis chamber.
* We prepared the samples.
* We placed the samples into the wells of the agarose gel.
* After that we filled the electrophoresis chamber with a 1x TBE solution.
* We connected the electrophoresis chamber to the power supply and set the voltage to 120 volts.
* We run the gel
* We took out the gel from the electrophoresis chamber and Applied ethidium bromide to stain it.
* We visualised the fragments on the gel by a gel imager.
* After Analysing the results, we documented it.

## Phase 3: Cell lab:

### Wi-38 culture:

Recorder: Ahmed Attia
Attendance: Hossam Algamal ,Omar Fouad, Youssef Amir
**Documentation of the Day:**

Receipt of Frozen Cells and Starting Cell Cultures:

* We placed the frozen cells immediately in liquid nitrogen storage until it was ready for culture.
* When the cells were ready to culture we placed them in 37 C water until the ice crystal disappeared.
* We swapped the outside surface of the ampule with 70% ethanol, then we dispensed its content into a T75 tissue culture flask with 20mL of warm growth medium.
* We recovered the cells overnight in a 37°C, 5% CO2 humidified incubator.
* The next day, we diluted the DMSO-containing shipping/cryopreservation medium that was aspirated from the cell layer and replaced with fresh medium.

### Subculture

ecorder: Ahmed Attia
Attendance: Hossam Algamal ,Omar Fouad, Youssef Amir
**Documentation of the Day:**

* We propagated the cells until density reached 70-80% confluence.
* Then we aspirate the medium.
* We washed the cell layer with warm 1x PBS.
* We added 15mL of Accutase then we returned the flask to the incubator for 10 -15 minutes.
* We removed the detached cells immediately to a centrifuge tube, and we rinsed the flask with warm 1X PBS to collect residual cells, and centrifuged them at 500 x g for 5 minutes (4°C).
* We resuspended the well pellet gently in a warm growth medium.

### Lipofectamine transfection:

Recorder: Ahmed Gehad
Attendance:Youssef amir, Hossam Bannis , Mohamed Aboelghar
**Documentation of the Day:**

After arriving at the lab we proceeded to prepare the Opti MEM medium.

* After arriving at the lab we proceeded to prepare the Opti MEM medium.
* We diluted the Lipofectamine 3000 reagent in Opti-MEM medium.
* We diluted the plasmids in the Opti-MEM medium.
* We Mixed the diluted plasmids with the diluted Lipofectamine 3000 reagent.
* We Incubated the DNA-lipid complex.
* We Added the DNA-lipid complex to the cells.
* We Incubated the cells at 37°C .

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# Results

Overview

- [Introduction](#Intro)

- [In Silico Validation](#InSilico)

- [1) Molecular Docking](#Docking)

- [2) Molecular Dynamics](#Dynamics)

- [3) Directed Evolution](#DEvolution)

- [4) Mathematical modeling](#MathMod)

- [Lab Validation](#LabValidation)

- [1) Bacterial Transformation](#BacterialTransf)

- [2) PCR Amplification](#PCRAmpl)

- [3) Digestion](#Digestion)

- [4) Ligation](#Ligation)

- [5) Culture & Colony PCR](#CultureColony)

- [6) Lipofectamine 3000 Transfection](#LipoTrans)

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![](https://static.igem.wiki/teams/4586/wiki/results/timeline-result-1.png)

## Introduction

SUPER was designed to be a universal platform for the eradication of autoimmune diseases, taking rheumatoid arthritis as a prototype for our platform, and also to be immunity-friendly by sparing the common side effects of the current treatment, which is immune suppression, so we decided to develop multilayer platform based on Mesenchymal Stem Cells secreting exosomes carrying a CRISPR-Cas system.

On this page, we will discuss the latest results we have reached until this point to validate our approach in the following order:

**1- In-Silico Simulations**

**2- Wet-lab Validation**

## In-Silico validation:

To simulate the different stages of our approach, we had to conduct several in-silico simulations to validate the efficiency of our approach before proceeding to the wet lab phase. These simulations include:

* Conducting docking simulations to determine the best peptide to be included in the design of our Syn-Notch receptor.

* Performing Molecular Dynamics (MD) simulations on our chosen complexes to determine their stability within a physiological environment.

* Using directed evolution approaches to improve the function of our parts.

* Modeling the action of the Syn-Notch using differential equations.

### 1- Molecular Docking

These simulations were conducted between the Anti-Citrullinated Peptide Antibody (ACPA) receptor of B-cells as the main target in association with three different proteins that are known to be targeted by the autoantibody. These proteins are:

* Citrullinated Vimentin (CV).

* The Cartilage Intermediate Layer Protein (CILP).

* The Cyclic Citrullinated Peptide (CCP1).

![](https://static.igem.wiki/teams/4586/wiki/results/gif1.gif)

Figures 1,2,3. Demonstrate the 3D structure of the proteins after completing the docking simulations. These structures were visualized using the PyMOL Molecular Visualization System. (A) ACPA-CV, (B) ACPA-CILP, (C) ACPA-CCP1

![](https://static.igem.wiki/teams/4586/wiki/results/docking-scores.png)

Figure 4. We found that the highest docking score was the ACPA and CCP1, with a score of (-298.80).

These docking simulations were followed by subjecting our proteins to molecular dynamics simulations to analyze their interactions at the atomic and molecular levels, as well as visualize the energy changes of the binding process.

### Molecular Dynamics:

Using the AMBER Molecular Dynamics Package on Google Colab, we’ve performed molecular dynamics simulations on all of the aforementioned complexes to test their stability in a perturbed environment, similar to what they would encounter within the cellular settings.

#### ACPA-CCP1:

The ACPA-CCP1 simulations showed the best results in terms of structural stability and flexibility. After 4 nanoseconds of simulation, the Root Mean Square Deviation (RMSD) was between 1.5-2.5 Å, which means that the deviation between the atoms of both proteins was minimal, indicating stability of the binding between the 2 proteins. The value of the Root Mean Square Fluctuation (RMSF) also ranged between 2-3 Å, which also indicates minimal fluctuation in the position of each of the residues forming the structure of each protein.

![](https://static.igem.wiki/teams/4586/wiki/results/acpa-ccp1-md.gif)

Figure 5. A display of the MD results of the ACPA-CCP1 Complex

#### ACPA-CV:

As for the ACPA-CV complex, the MD results weren’t as good as with the ACPA-CCP1 Complex. It showed an RMSD value that fluctuated between 2 up to 4 Å, showing more deviation between the position of atoms in the 2 proteins and indicating less stable binding than the ACPA-CCP1 complex. The RMSF calculations were also slightly higher than the ACPA-CCP1 complex, fluctuating between 1-4 Å.

![](https://static.igem.wiki/teams/4586/wiki/results/acpa-cv.gif)

Figure 6. A display of the MD results of the ACPA-CV Complex

#### ACPA-CILP:

Finally, the results of the ACPA-CILP showed constant increase in the RMSD value, reaching up to 7 Å. This indicates more deviation in the positions of the molecules that construct both proteins, resulting in a less stable binding process compared to the other two complexes.

![](https://static.igem.wiki/teams/4586/wiki/results/acpa-cilp-md.gif)

Figure 7. A display of the MD results of the ACPA-CILP Complex

All of these simulations have directed our modeling process towards using the CCP1 protein, as it showed the highest docking score, in addition to being the most stable structure when bound with the ACPA, compared to the other proteins.

The simulation of the behavior of the 3 aforementioned complexes was important to determine the best protein that can effectively identify and bind with the ACPA receptor of Auto-reactive B-cells. The selected protein can then be integrated into the design of our Syn-Notch receptor, which can significantly improve the specificity of our approach, while maintaining the stability and flexibility of the binding between the 2 structures. This ensures a high level of specificity of our receptor, which is very important as this represents one of the safety measures of our system that allows for precise and specific targeting of the ACPA-presenting B-cells while avoiding normal B-cells.

#### CD19-CD19 Ligand:

As a proof-of-concept of our approach, our wet lab work was conducted through the binding between the CD19 receptor of B-cells and the CD19-ligand that is integrated in the structure of the Syn-Notch receptor. This is due to the fact that to work with ACPA-presenting cells, we would have to extract them from samples of RA patients, so we decided to test the validity of our approach before proceeding to the next phase.

We’ve also performed docking and MD simulations for the CD19-CD19 Ligand to analyze the binding between them, as well as ensuring the stability of their structures within a simulation of a physiological system. The docking score between the 2 structures was -311.10. However, when the structures were submitted for MD simulations, their RMSD reached a value of 5 Å, as well as the values of their RMSF.

![](https://static.igem.wiki/teams/4586/wiki/results/cd19-cd19-ligand.gif)

Figure 8. The 3D structure of the docking between the CD19 and the CD19 Ligand, visualized using PyMOL

![](https://static.igem.wiki/teams/4586/wiki/results/cd19-cd19-ligand-md.gif)

Figure 9. A display of the MD results of the CD19-CD19 Ligand Complex.

### Directed Evolution:

To improve the features of the proteins used in our project, we turned to the concept of directed evolution, where random mutations can be applied to protein residues in order to direct the protein into expressing better features. The impact of these mutations is then measured in relation to their surrounding residues, and on the protein structure as a whole.

Using an online tool called EVcouplings, we subjected our proteins to generate mutant variants at different positions of the protein. The effect of these mutations is then measured independently, as well as on the global protein structure, which is called epistatic fitness. Both these parameters correspond to the overall evolutionary fitness of the formed mutant variants, which contributes to improving their functions.

Herein, we’re presenting the mutational landscape that was generated by EVcouplings for the proteins that are most important in the performance and safety of our therapeutic approach. We’ve chosen to apply these mutations to 4 proteins that are implemented in different aspects of the therapeutic pathway. These include:

* CD19 Ligand: as it is involved in the primary step of recognizing the auto-reactive B-cells.
![](https://static.igem.wiki/teams/4586/wiki/results/cd19-ligand.png)

Figure 10. Depicts the mutational landscape of the CD19 Ligand, generated by the EVcouplings software. It shows that the mutant variant associated with the highest epistatic fitness was (P152S), while the highest independent score was related to the (P162G).

* The (CD63-L7Ae) loading system: which is designed for loading our therapeutic cargo in the exosomal delivery system once the recognition process is achieved. Afterwards, these exosomes start delivering our cargo to the auto-reactive cells to terminate their activity.
![](https://static.igem.wiki/teams/4586/wiki/results/cd63-l7ae.png)

Figure 11. Depicts the mutational landscape of the CD63-L7Ae, generated by the EVcouplings software. It shows that the mutant variant associated with the highest epistatic fitness was (N117K), while the highest independent score was related to the (L18N).

* The therapeutic cargo itself: represented in the CRISPR/Cas12k system: whose role is to target the B-cell Activating Factor Receptor (BAFF-R) gene which is essential for the survival and differentiation of B-cells. This results in initiating an apoptotic response that destroys the auto-reactive cells.
![](https://static.igem.wiki/teams/4586/wiki/results/cas12k.png)

Figure 12. Depicts the mutational landscape of the Cas12k, generated by the EVcouplings software. It shows that the mutant variant associated with the highest epistatic fitness was (A8C), while the highest independent score was also related to the (A8C).

* Our (DART-VADAR) safety switch: this safety system is designed so that our CRISPR/Cas12k cargo can only perform its action within auto-reactive B-cells, further improving the specificity and safety of our approach.
![](https://static.igem.wiki/teams/4586/wiki/results/adar.png)

Figure 13. Depicts the mutational landscape of the ADAR enzyme, generated by the EVcouplings software. It shows that the mutant variant associated with the highest epistatic fitness was (Q173E), while the highest independent score was also related to the (A182G).

### Mathematical Modelling:

**Binding Model**

The model confirms the use of CCP1( cyclic citrullinated peptide 1) as an external domain for our Syn-Notch receptor as it has the highest docking score for binding to BCR (B-cell receptor) from VIM (vimentin) and CV (citrullinated vimentin) external domains.So, this implies that CCP1 is more stable and has low dissociation rate after binding to BCR; however, VIM and CV show moderate to high dissociation rate after binding to BCR as show in the graphs.

+ CCP1 binding model to BCR
![](https://static.igem.wiki/teams/4586/wiki/results/modeli3.png)

Figure 14.The graph describes the binding between the CCP1 to BCR to form a stable binding state with minimal dissociation.

**Designing an engineered exosomes**

After binding the Syn-Notch receptor to its target, its internal domain will start a cascade of signals to produce our CRISPR system coated with exosomes. The exosomes have a receptor to guide them toward the autoreactive B-cells. This receptor has the same composition of the external domain of the Syn-Notch receptor. But to make sure that we use the suitable initiation signal for exosome secretion we simulate it using:

#### Mathematical modelling:

The model confirms the use of the ZF21.16 VP64 signal, from the internal domain of Syn-Notch receptors, to be effective enough to produce engineered exosomes.
![](https://static.igem.wiki/teams/4586/wiki/results/modeling-40.png)

Figure 15.The graph describes the relation between activation of the internal domain of Syn-Notch receptors and its effect on the level of our engineered exosomes.

By comparing internal domains of other receptors like Chimeric antigen receptor (CAR) ,Antibody scaffold receptor, receptors Activated Solely by Synthetic Ligands (RASSLs), and the exosomes produced after their activation upon binding to their targets, the result was not satisfactory as the signals were not specific to activate our circuit to produce the sufficient amount of our engineered exosomes.

+ Activation model of CAR internal domain and its effect on the level of exosomes.
![](https://static.igem.wiki/teams/4586/wiki/results/modeling11.png)

Figure 16.The graph describes the relation between activation of the internal domain of CAR and its effect on the level of our engineered exosomes.

+ Activation model of Antibody scaffold receptor internal domain and its effect on the level of exosomes
![](https://static.igem.wiki/teams/4586/wiki/results/modeling13.png)

Figure 17.The graph describes the relation between activation of the internal domain of Antibody scaffold receptors and its effect on the level of our engineered exosomes.

+ Activation model of RASSLs internal domain and its effect on the level of exosomes.
![](https://static.igem.wiki/teams/4586/wiki/results/modeling12.png)

Figure 18.The graph describes the relation between activation of the internal domain of RASSLs and its effect on the level of our engineered exosomes.

## Lab Validation

### Step (1) Bacterial Transformation

We chose the pcDNA 3.1- vector to deliver our genetic circuits to the cells. It was ordered from Thermo Fisher with a 20µg size, which was not enough to deliver all the circuits. So, we used bacterial transformation to amplify the vector.
![](https://static.igem.wiki/teams/4586/wiki/results/bacterial-transformation.png)

Figure 19.This figures illustrates the process of bacterial transformation and selected colonies as
Plating of different constructed pCDNA on ampicillin agar plates All plates were incubated at 37 °C for 16 h. The equivalent of 50 µl of bacterial cells after transformation was plated on each plate.

### Step (2) PCR Amplification

In order to amplify all the DNA parts, we used PCR amplification to reach the desired concentration by using specific forward and reverse primers, running the parts on gel electrophoresis, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figures.
![](https://static.igem.wiki/teams/4586/wiki/results/pcr-ampli.png)

Figure 20

0.8 % agarose gel electrophoresis of PCR; each well contain one part in the following order, as shown in the following table:

| ID | Part Name | ID | Part Name |
| --- | --- | --- | --- |
| P1 | Syn-Notch | P6 | CX-43 |
| P2 | Booster-1 | P7 | Guide RNA-BFP-switch |
| P3 | Booster-2 | P8 | First MS2 |
| P4 | Loading System | P9 | Sensor + second MS2 and Cas12k |
| P5 | Exosomes receptor | P10 | MCP-ADAR-RFP |

### Step (3) Digestion

#### pcDNA restriction digestion by Ecor1 and BamH1 :

We used Ecor1 and BamH1 restriction enzymes as our sites in the plasmid for the ligation within the parts, followed by the running of gel as shown in the following figure:
![](https://static.igem.wiki/teams/4586/wiki/results/vector-cut.png)

Figure 21. 0.8% agarose gel electrophoresis of digested pcDNA3.1(-) ; well P1 represents the uncut vector, respectively; well P2 represents the cut vector, respectively; and M represents 1Kb DNA ladder.

#### The amplified genetic parts were labeled with IDs and separated into 3 plasmids:

Plasmid-1

| Part | Length | Restriction enzymes |
| --- | --- | --- |
| Syn-Notch | 2740 bp | + EcoRI + Hind-3 |
| Booster gene-1 | 2490 bp | + Hind-3 + Pst-1 |
| Booster gene-2 | 2725 bp | + Pst-1 + BamHI |
| pcDNA 3.1- (Backbone) | 5427 bp | + EcoRI + BamHI |
| Total Length | 13162 bp |

Table-1: Plasmid-1 consists of a pcDNA 3.1- plasmid vector ligated with 3 genetic parts : Syn-notch , Booster gene-1, and Booster gene-2. This plasmid is responsible for the expression of the Syn-Notch receptor on the surface of the transfected cell. The total length of the plasmid after ligation is 13162 bp which will be tested after ligation by gel electrophoresis.

Plasmid-2

| Part | Length | Restriction enzymes |
| --- | --- | --- |
| Loading System | 1483 bp | + EcoRI + Hind-3 |
| Exosomal Receptor | 1158 bp | + Hind-3 + Pst-1 |
| CX-43 | 2250 bp | + Pst-1 + BamHI |
| pcDNA 3.1- (Backbone) | 5427 bp | + EcoRI + BamHI |
| Total Length | 10079 bp |

Table-2: Plasmid-2 consists of a pcDNA 3.1- plasmid vector ligated with 3 genetic parts : Loading system, Exosomal receptor, Cx43. This plasmid will be responsible for producing engineered exosomes in the transfected cell. The total length of the plasmid after ligation is 10079 bp which will be tested after ligation by gel electrophoresis.

Plasmid-3

| Part | Length | Restriction enzymes |
| --- | --- | --- |
| Guide RNA-BFP-switch | 2725 bp | + EcoRI + Hind-3 |
| New MS2 | 496 bp | + Hind-3 + Xbal |
| Rest of MS2 and Cas12k | 2484 bp | + Xbal + Kpn-1 |
| MCP-ADAR-RFP | 2065 bp | + Kpn-1 + BamHI |
| pcDNA 3.1 (Backbone) | 5427 bp | + Eco-RI + BamHI |
| Total Length | 12845 bp |

Table-3: Plasmid-3 consists of a pcDNA 3.1- plasmid vector ligated with 4 genetic parts : Guide RNA-BFP-switch, New ms2, Cas12k, and MCP-ADAR-RFP. This plasmid is responsible for producing the killing signal for the auto-reactive B-cell. The total length of the plasmid after ligation is 12845 bp which will be tested after ligation by gel electrophoresis.

We performed the double digestion method for the nine parts in the prefix and suffix of each part with its specific restriction enzyme and applied these parts to gel electrophoresis as shown in the following figure.
![](https://static.igem.wiki/teams/4586/wiki/results/digestion-2.png)

Figure 22. 0.8% agarose gel electrophoresis of digested gBlocks; wells P1-P2-P3 represent our first three parts, which are the contents of the first plasmid, respectively; wells P4-P5-P6 represent three parts, which are the contents of the second plasmid, respectively; wells P7-P8-P9-P10 represent four parts of the third plasmid, respectively; and M represents 1Kb DNA ladder.

### Step (4) Ligation

The genetic parts were ligated according to their function into three plasmid vectors. The first plasmid is carrying the Syn-Notch receptor and Booster genes; the second plasmid is carrying the loading system, exosomal receptor, and CX-43, finally, the third plasmid is carrying the CRISPR-Cas system and its safety switch.

Each ligation process was made through sequential ligation.
![](https://static.igem.wiki/teams/4586/wiki/results/plasmid-1-sequence-1-pdf-1.png)

Figure 23

### Step (5) Culture & Colony PCR

After the ligation step, we did culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid.

Plasmid-1

![](https://static.igem.wiki/teams/4586/wiki/results/1.png)

Figure 24.Cell culture plate of transformed pCDNA vector containing insert parts.
This plasmid contains

+ (Syn-notch)
+ (Booster gene-1)
+ (Booster gene-2)

Plasmid-2

![](https://static.igem.wiki/teams/4586/wiki/results/2.png)

Figure 25.Cell culture plate of transformed pCDNA vector 2 containing insert parts.
This plasmid contains

+ (Loading system)
+ (Exosomal receptor)
+ (CX43)

Plasmid-3

![](https://static.igem.wiki/teams/4586/wiki/results/3.png)

Figure 26.Cell culture plate of transformed pCDNA vector containing insert parts.
This plasmid contains

+ (Guide RNA-BFP-switch)
+ (New ms2)
+ (Rest of ms2 and cas12k)
+ (MCP-ADAR-RFP)

Then we selected the best colonies for the colony selection process and screened them with colony PCR to make sure that our parts were correctly ligated with the vector.
![](https://static.igem.wiki/teams/4586/wiki/results/untitled-design-9.png)

Figure 27. This figure illustrates the selection of colonies for our three plasmids. All plates were incubated at 37 °C for 16 h. The equivalent of 50 µl of bacterial cells.

And this image illustrate our plates collection
![](https://static.igem.wiki/teams/4586/wiki/results/555.png)

Figure 28

Then we did Colony PCR to make sure that our plasmid were correctly ligated with its parts, as shown in the following figure:

![](https://static.igem.wiki/teams/4586/wiki/results/p1.png)

Figure 29. 0.8% agarose gel electrophoresis of digested gBlocks; wells P1-P2-P3 represent our first three parts, which are the contents of the first plasmid, respectively; wells P4-P5-P6 represent three parts, which are the contents of the second plasmid; respectively; wells P7-P8-P9-P10 represent four parts of the third plasmid; and respectively M represents 1Kb DNA ladder.

### Step (6) Lipofectamine 3000 Transfection

And we transfected our plasmids into the Wi-38 intended to validate our approach through the following main steps : structural validation to reflect the successful expression of our biological parts structurally to be presented at the intended site, in the desired amount, and at the right time. and functional validation to ensure their ability to perform what was intended to be done and characterize their action and performance.

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