iGEM 2021 — Team AFCM-Egypt

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# Team:AFCM-Egypt/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:AFCM-Egypt/Engineering

Software

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* [Home](https://2021.igem.org/Team%3AAFCM-Egypt)
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* Project
  + [Description](https://2021.igem.org/Team%3AAFCM-Egypt/Description)
  + Engineering
  + [Directed Evolution](https://2021.igem.org/Team%3AAFCM-Egypt/Directed-Evolution)
  + [Contribution](https://2021.igem.org/Team%3AAFCM-Egypt/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AAFCM-Egypt/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AAFCM-Egypt/Proof_Of_Concept)
  + [Enterpreneurship](https://2021.igem.org/Team%3AAFCM-Egypt/Entrepreneurship)
  + [Modeling](https://2021.igem.org/Team%3AAFCM-Egypt/Model)
  + [Design](https://2021.igem.org/Team%3AAFCM-Egypt/Design)
* Parts
  + [Basic Parts](https://2021.igem.org/Team%3AAFCM-Egypt/Parts#basic)
  + [Composite Parts](https://2021.igem.org/Team%3AAFCM-Egypt/Parts#composite)
  + [Characterized Parts](https://2021.igem.org/Team%3AAFCM-Egypt/Parts#char)
  + [Improved Parts](https://2021.igem.org/Team%3AAFCM-Egypt/Parts#improved)
  + [New Parts](https://2021.igem.org/Team%3AAFCM-Egypt/Parts#new)
* [Software](https://2021.igem.org/Team%3AAFCM-Egypt/Software)
* Human Practices
  + [Human Practices](https://2021.igem.org/Team%3AAFCM-Egypt/Human_Practices)
  + [Education & science communication](https://2021.igem.org/Team%3AAFCM-Egypt/Communication)
  + [Collaboration](https://2021.igem.org/Team%3AAFCM-Egypt/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AAFCM-Egypt/Partnership)
  + [Integrated Human Practices](https://2021.igem.org/Team%3AAFCM-Egypt/integrated)

Home

# Engineering

List Of Contents

* [Introduction](#title1)
* [DESIGNING OF PARTS](#title2)
* [Designs](#title3)
* [Logical Engineering](#logical)

## Vaccine Engineering

This year, iGEM AFCM-Egypt 2021 team stated 5 main Goals to be achieved via engineering the vaccine through developing the previously designed TNBC DREP-based Multi-epitopes vaccine which are :

* Cell specificity.
* Making logical decisions.
* Effectively deliver the antigenic message and elicit an immune response.
* Controllable and regulatable (system sensitive).
* Ensuring safety.

Our team was able to achieve these goals through the usage of different parts and logic gates.

### DESIGNING OF PARTS

**CMV**: This constitutive promoter has proved its efficacy in Adenovirus-Associated Vectors (AAV) which directs a high level of transient gene expression in many types of cells (1),(2).

Actually, The efficacy of CMV as a constitutive promoter has been proved after quantification of Green Fluorescence Protein (GFP) signals in infected cells which demonstrated that the CMV promoter produced the highest GFP expression in the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus MV, simian virus 40, and herpes simplex virus thymidine kinase) and maintained relatively high GFP expression as shown in fig (1).(3)

![](https://static.igem.org/mediawiki/2021/thumb/2/2b/T--AFCM-EGYPT--eng1.png/384px-T--AFCM-EGYPT--eng1.png)

Fig.1 CMV has highest expression among the six constitutive promoters (3)

**Subgenomic promoters (SGPs)**: A subgenomic promoter is a promoter that is introduced to a virus for the exclusive purpose of producing mRNA for a specific heterologous gene. These subgenomic mRNAs (sgRNA) are produced by many positive-sense RNA, and they often transcribe late viral genes. Actually, they are essential in our vaccine design as we use NSPs in our circuit. That is why we used a set of SGPs (SGP0, SGP1, and SGP2).

![](https://static.igem.org/mediawiki/2021/thumb/a/a2/T--AFCM-EGYPT--eng3.png/120px-T--AFCM-EGYPT--eng3.png)
![](https://static.igem.org/mediawiki/2021/3/31/T--AFCM-EGYPT--engi4.png)

**Non-structural proteins (NSPs)**: We used 4 NSPs from Equine Encephalosis (EEV) and Semliki Forest (SFV) alphaviruses which contribute to the self-amplification ability of RNA replicons. Therefore increasing in the yield of the vaccine via increasing transcription of downstream structures to ensure enhanced neo-antigens uptake by immune cells.

![](https://static.igem.org/mediawiki/2021/7/71/T--AFCM-EGYPT--engi5.png
)

**Riboswitches**: AFCM-Egypt 2021 team designed 2 riboswitches to be tested. A riboswitch is a regulatory region of a messenger RNA molecule that binds to a smaller molecule and causes a variation in the production of the proteins encoded by the mRNA in molecular biology. As a result, an mRNA with a riboswitch is directly involved in regulating its own activity in response to its effector molecule's concentrations.

**o dCas13-L7Ae**: which is a deactivated Cas13 used in cojugation with L7Ae protein (that has an inhibitory effect on the transcription by binding to its **kink-turn**) by Gly Ser linker. The system simply recognizes and binds to mRNA in the cancerous environment which leads to consumption of L7Ae protein. Therefore, not binding to kink-turn to inhibit the inhibitory effect on the transcription which finally leads to stimulation of the transcription in order to increase the yield of the vaccine. That is why, it is a **cell specific** design by binding to mRNA of **PD-L1** which has an immune evasion role in the cancerous environment especially TLCs.

![](https://static.igem.org/mediawiki/2021/2/26/T--AFCM-EGYPT--engi6.png
)

**IntraAb-L7Ae**: This system resembles the previous one with using intracellular antibodies or intrabodies that are produced in the cell, and binds to an antigen within the same cell. So, for example, when the intracellular antibody binds to PD-L1 present in the cancerous environment, it consumes the L7Ae protein, therefore removing its inhibitory effect on transcription which causes the circuit to proceed in the transcription process to produce more copies of the vaccine in order to be recognized by immune cells to combat cancer cells.

![](https://static.igem.org/mediawiki/2021/d/d8/T--AFCM-EGYPT--engi7.png
)

**DD-MS2**: Another designed system includes a different riboswitch which consists of the MS2 protein that binds with small nuclear ribonulceoprotein U2-snRNP to inhibit transcription. In our design MS2 is fused to destabilizing domain (DD) by Gly Ser linker which could be stabilized after administering Trimethoprim (TMP). TMP which acts as a **small molecule inhibitor** will free the MS2 after stabilizing DD. Free MS2 binds to U2-snRNP which inhibit the circuit so as to be able to control the transcription process in extreme conditions as cytokine storm or unpredictable results.

![](https://static.igem.org/mediawiki/2021/7/7c/T--AFCM-EGYPT--engi8.png
)

Toehold switches: To make our system more sensitive, we introduced Toehold switches downstream and upstream to the vaccine.

**ToeholdOnMotif**: It can detect and bind to miRNAs from the cancerous environment whether those indicate tumor suppression or tumor growth which will give the opportunity to make decisions as in case of increased miRNAs that indicate tumor growth. A complex consists of the binding of a ribosome to the ribosomal binding site (RBS) on the Toehold switch which will destabilize the transcripts of L7Ae protein, therefore removing its inhibitory effect which allows continuous transcription at RNA levels to increase the yield of the vaccine.

![](https://static.igem.org/mediawiki/2021/d/df/T--AFCM-EGYPT--engi9.png)

**ToeholdDegMotif**: This is a downstream switch which binds to miRNA that indicates tumor suppression. It is modified by replacing the liker with malate 1 triplex and adding 30X degradation motifs downstream. This enables degradation of the transcripts to decrease the yield of the vaccine after suppressing the tumor.

![](https://static.igem.org/mediawiki/2021/5/5a/T--AFCM-EGYPT--engi10.png)

To test the efficacy of the vaccine our team constructed a set of designs to be tested:

#### Design (1)

![](https://static.igem.org/mediawiki/2021/7/72/T--AFCM-EGYPT--engi11.png)

This design simply illustrates the usage of NSPs to perform the self-amplification function that increases the vaccine yield and results in efficient neo-epitopes uptake in addition to using SGPs and Riboswitches that consists of intracellular antibody fused to L7Ae protein by GLY Ser linker that is previously illustrated.

#### Design (2)

![](https://static.igem.org/mediawiki/2021/thumb/a/a5/T--AFCM-EGYPT--eng14.png/800px-T--AFCM-EGYPT--eng14.png)

The system acquired more safety procedures after introducing the Riboswitch fused with destabilizing domain (DD) that is controllable by TMP administration that acts as small molecule inhibitor to inhibit transcription if the circuit is uncontrollable.

#### Design (3)

![](https://static.igem.org/mediawiki/2021/5/51/T--AFCM-EGYPT--engi13.png
)

This version illustrates a system sensitive circuit via the usage of ToeHold switches upstream and downstream the vaccine (4). The ToeholdOnmotif switch upstream to vaccine binds to miRNA indicating increased tumor growth in order to remove the inhibitory effect of the Riboswitch to make more copies of transcripts to combat cancer cells. Another downstream ToeholdDegmotifs that binds to suppressor miRNA therefore, degrades transcripts to decrease vaccine copies.

| TMP | ToeHoldOn | Antibody | ChimVac |
| --- | --- | --- | --- |
| 1 | 0 | 0 | 0 |
| 1 | 1 | 1 | 0 |
| 1 | 1 | 0 | 0 |
| 1 | 0 | 1 | 0 |
| 0 | 1 | 1 | 1 |
| 0 | 0 | 1 | 1 |
| 0 | 1 | 0 | 1 |
| 0 | 0 | 0 | 0 |

![](https://static.igem.org/mediawiki/2021/4/42/T--AFCM-EGYPT--engi14.png)

The truth tables briefly depicts the logic gates used in our design that are diagramed using the logisim tool software. It represents the decision made in the whole system which consists of 2 (AND) gates and finally, an (OR) gate. The first AND gate has 2 inputs from TMP (through a NOT gate) and Toehold On switch while the second AND gate has inputs from Riboswitch and TMP (through NOT gate) because in case of TMP presence, the circuit must be stopped in all ways.

By passing through OR gate, Transcription will be induced by absence of TMP and removing the inhibitory effect of Riboswitches or binding of miRNA to upstream Toehold switch.

### Design (4)

![](https://static.igem.org/mediawiki/2021/e/ec/T--AFCM-EGYPT--engi15.png)

To guide dcas13 for the specific mRNA from the cancerous environment for PD-L1 as an example, crRNA is introduced to circuit. Therefore, being specific for dCas13 guidance that results in consumption of L7Ae to remove the inhibitory effect of riboswitch in order to continue transcription.

| TMP | ToeHoldOn | mRNA | vacc |
| --- | --- | --- | --- |
| 1 | 0 | 0 | 0 |
| 0 | 0 | 1 | 1 |
| 0 | 1 | 0 | 1 |
| 0 | 0 | 0 | 0 |
| 0 | 1 | 1 | 1 |

This truth table represents the logic decisions used in design (4) in 5 rows. 1st raw represents the possibility of administering TMP which will result in the circuit will ceasing transcription in all conditions. 2nd, 3rd, 4th, and 5th rows represents OR gate that transcription only continued in cases of Toehold on or after removing the inhibitory effect of riboswitch or with both.

### Design (5)

![](https://static.igem.org/mediawiki/2021/b/b7/T--AFCM-EGYPT--engi16.png)

Design 5 has an added feature of oncolytic activity of the vaccine when administered intra-tumorally to combat TNBC cells in parallel with vaccine activity. This is achieved mainly by hBax or FADD to the circuit or both of them to be tested.

**Hbax mutant:** Our TNBC vaccine is supposed to be injected intra-tumorally. That is why an oncolytic activity is added to the circuit to perform an apoptotic function which helps in both combating cancer cells and apoptotic bodies which release neo-epitopes to be recognized by immune cells.

**FADD:** Fas-associated protein with death domain (FADD) is an adaptor protein that bridges members of the tumor necrosis factor receptor superfamily. It is inserted into our circuit linked to the vaccine or to L7Ae protein which has a role as apoptotic activity.

### Design (6)

![](https://static.igem.org/mediawiki/2021/9/9b/T--AFCM-EGYPT--engi17.png)

In this design, a Toggle-Switch and a Feed-Forward Loop (positive autoregulation feedback) between dCas13 and miRNA-On binding toehold switch is inserted. It has a regulatory tool which increases transcripts via inhibition of ToeholdOnmotif in order to remove the inhibitory effect of L7Ae to make more copies.

### Design (7)

![](https://static.igem.org/mediawiki/2021/c/cb/T--AFCM-EGYPT--engi18.png
)

A repressilator is added to this design in order to regulate transcription(5). It is achieved via inhibiting ToeholdOnmotif. In the system, MS2 inhibits u2-sn-RNP, L7Ae inhibits kt1, and miRNA-On inhibits ToeholdOnmotif.

### Design (8)

![](https://static.igem.org/mediawiki/2021/3/37/T--AFCM-EGYPT--engi19.png
)

DREP (HBV-based) launching antigen-coated VLPs. External epitopes can be inserted into the N-terminus of HBcAg, allowing for the insertion of up to 50 amino acids and inducing specialized antibody responses. Another location for insertion is at the C terminus's end, around amino acids 144 and 127. It is feasible to insert epitopes at amino acids 144, 149, 153, 163, and 169 at the C-terminus.

#### References

1. Foecking M, Hofstetter H. Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene. 1986;45.
2. Damdindorj L, Karnan S, Ota A, Takahashi M, Konishi Y. Assessment of the long-term transcriptional activity of a 550-bp-long human beta-actin promoter region. Plasmid. 2012;68.
3. Damdindorj L, Karnan S, Ota A, Hossain E, Konishi Y, Hosokawa Y, et al. A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors. PLoS One [Internet]. 2014 Aug 29 [cited 2021 Sep 29];9(8):e106472. Available from: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0106472
4. Toehold Switches for Synthetic Biology [Internet]. [cited 2021 Oct 2]. Available from: https://wyss.harvard.edu/technology/toehold-switches-for-synthetic-biology/
5. Elowitz MB, Leibler S. A synthetic oscillatory network of transcriptional regulators. Nat 2000 4036767 [Internet]. 2000 Jan 20 [cited 2021 Oct 2];403(6767):335–8. Available from: https://www.nature.com/articles/35002125

## Logical engineering

As a result of a desire to construct a biological circuit with engineering precision, synthetic biology emerged. That's why this year we are trying to display our circuit from multiple views to increase our ability in controlling the final outcome. One of them is logical engineering. In order to integrate logical engineering with cell biological functions we developed a logic-gate scheme for this purpose. It is based on a truth table where the inputs and outputs values varying between 0/1. We converted the truth table into Boolean Formula and applied relevant algorithms to find a digital circuit scheme as simple as possible in terms of electrical engineering where the inputs are determined to be either 0 or 1 based on the availability of oncogenic and tumor-suppressive micro-RNAs in the cellular environment.

In our design we used 5 inputs, each with a specific function, as follows:
If the results were unpredictable, TMP, a small molecule inhibitor, could be used. As a result, the NOT gate is designed to ensure that if the input is 1, the DD-MS2 will not be stabilized, resulting in a 0 output and termination of the circuit.

![](https://static.igem.org/mediawiki/2021/c/cb/T--AFCM-EGYPT--_logic11.png)

![](https://static.igem.org/mediawiki/2021/8/80/T--AFCM-EGYPT--_logic2.png)

Fig.1 shows the effect of TMP on the circuit using a NOT gate.

Additionally, there are intracellular antibody and dcas13. Adding either of them consumes L7AE, thus disinhibiting the circuit. Because of this, we designed a NOR gate, which means that the circuit cannot be terminated except when both inputs are 0.

![](https://static.igem.org/mediawiki/2021/c/c2/T--AFCM-EGYPT--_logic3.png)
![]( https://static.igem.org/mediawiki/2021/d/d7/T--AFCM-EGYPT--_logic4.png)

Fig.2 shows the effect of dcas13 and intracellular Ab. on the circuit using a NOR gate.

On top of that, we have a toehold on and a toehold deg. Motifs. Taking a look at their names, you can see that they appear to serve opposite functions. Therefore, they work in toggle switch fashion in a competitive manner where the one with the highest score dominates. For this reason we chose a Nimply gate where the circuit could be only activated when the Toehold value equals 1 and Toehold degradation motifs equals 0.

![](https://static.igem.org/mediawiki/2021/e/e5/T--AFCM-EGYPT--_logic5.png)
![]( https://static.igem.org/mediawiki/2021/8/81/T--AFCM-EGYPT--_logic6.png)

Fig.3 shows the effect of toehold on and toehold deg. motifs on the circuit using a NOR gate.

Finally, all these input data are gathered through their logic gates, where their outputs are all collected representing inputs for a final And gate controlling the copies of the vaccine construct as shown in fig.4.

![](https://static.igem.org/mediawiki/2021/a/aa/T--AFCM-EGYPT--_logic1.png)

Fig.4 This diagram shows the relation between dCAS13, toehold on, toehold deg.,TMP, and intracellular Ab. represented by Boolean logic gates.

![](https://static.igem.org/mediawiki/2021/d/dc/T--AFCM-EGYPT--_logic8.png)

Fig.5 shows a truth table displays the relationship between dCAS13, Toehold on and Toehold deg., TMP, and intracellular Ab. as the following:

* 0 means no input in the input columns.
* 1 means there is input in the input columns.
* 0 means no output in the output column.
* 1 means there is output in the output column.

![](https://static.igem.org/mediawiki/2021/3/39/T--AFCM-EGYPT--_logic9.png)

Fig.6 This diagram shows the relation between dCAS13, toehold on and toehold deg. represented by an AND gate and Nimply gates.

![](https://static.igem.org/mediawiki/2021/a/a8/T--AFCM-EGYPT--_logic10.png)

Fig.7 shows a truth table displays the relationship between dCAS13, Toehold on and Toehold deg. as the following:

* 0 means no input in the input columns.
* 1 means there is input in the input columns.
* 0 means no output in the output column.
* 1 means there is output in the output column.

![](https://static.igem.org/mediawiki/2021/9/91/T--AFCM-EGYPT--_logic12.png)

Fig.8 This diagram shows the relation between dCAS13 and toehold deg. represented by a Nimply gate.

![](https://static.igem.org/mediawiki/2021/7/7c/T--AFCM-EGYPT--_logic13.png)

Fig.9 shows the relationship between dCAS13 and toehold deg. as the following:

* 0 means no input in the input columns.
* 1 means there is input in the input columns.
* 0 means no output in the output column.
* 1 means there is output in the output column.

![](https://static.igem.org/mediawiki/2021/c/c4/T--AFCM-EGYPT--_logic14.png)

Fig.10 represents the relationship between dCAS13 and Toehold On by AND gate.

![](https://static.igem.org/mediawiki/2021/2/23/T--AFCM-EGYPT--_logic15.png)

Fig.11 displays the relationship between dCAS13 and Toehold on as the following:

* 0 means no input in the input columns.
* 1 means there is input in the input columns.
* 0 means no output in the output column.
* 1 means there is output in the output column.

![](https://static.igem.org/mediawiki/2021/e/e1/T--AFCM-EGYPT--_logic16.png)

Fig.12 This diagram shows the relation between TMP and toehold on represented by an Nimply gate.

![](https://static.igem.org/mediawiki/2021/d/d3/T--AFCM-EGYPT--_logic17.png)

Fig.13 shows the relationship between TMP and toehold on as the following:

* 0 means no input in the input columns.
* 1 means there is input in the input columns.
* 0 means no output in the output column.
* 1 means there is output in the output column.

#### Modularity

Our logical inputs could be used to design different logic gates which could be used to design different circuits or constructing an engineering platform in order to be optimized and tested.

![](https://static.igem.org/mediawiki/2021/6/6e/T--AFCM-EGYPT--_logic19.png)

Fig.14, illustrates our inputs corresponding to their logic gates in various relationships.

![](https://static.igem.org/mediawiki/2021/5/50/T--AFCM-EGYPT--logo.png)

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# Team:AFCM-Egypt/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:AFCM-Egypt/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)