iGEM 2020 — Team AUC-EGYPT

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# Team:AUC-EGYPT/Notebook

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# 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)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:AUC-EGYPT/Engineering

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* [Home](/Team%3AAUC-EGYPT)
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  + [Implementation](/Team%3AAUC-EGYPT/Implementation)
  + [Model](/Team%3AAUC-EGYPT/Model)
  + [Software](/Team%3AAUC-EGYPT/Software)
* Parts ▾
  + [Basic Parts](/Team%3AAUC-EGYPT/Parts)
  + [Composite Parts](/Team%3AAUC-EGYPT/Parts)
* Human Practices ▾
  + [Human Practices](/Team%3AAUC-EGYPT/Human_Practices)
  + [Integrated Human Practices](/Team%3AAUC-EGYPT/Human_Practices)
  + [Entrepreneurship](/Team%3AAUC-EGYPT/Entrepreneurship)
  + [Education](/Team%3AAUC-EGYPT/Education)
  + [Collaborations](/Team%3AAUC-EGYPT/Collaborations)
  + [Partnership](/Team%3AAUC-EGYPT/Partnership)

![](https://static.igem.org/mediawiki/2020/9/94/T--AUC-EGYPT--eng.png)

[Delivery System](#DS)

[Toehold switches](#ts)

[Mammalian vs Prokaryotic toehold switches](#mp)

[Our Engineered Toehold Switches](#oe)

[Our Toeholds](#ot)

[Experimental design](#ed)

[Toehold Switch—GAL4BD-VP16—UAS (TGU) device characterization](#tsg)

[Experimental design for siRNA](#eds)

[References](#ref)

In the current COVID-19 pandemic, people are trying to adapt by changing their usual daily routines, practices, used tools, etc. As we know, the research practices were affected as well. However, our team decided to take the challenge and work on a therapeutic treatment for COVID-19 without having access to the lab. An important question to be asked is how we were able to evaluate our proposed therapy. The secret lies in our modified Engineering Design Process (EDP) that allowed us to achieve engineering success, integrate our human practices, and create a modular system with thoroughly analyzed parts. In figure (1), our 12 phases EDP is shown where certain stages can be repeated more than twice! The main modifications we included are applying the modular approach and using qualitative analysis. The modular approach allows us to evaluate and analyze each single unit/device in the system separately. Accordingly, modifications in the system can be easily handled, problems can be easily identified, testing can be more specific, and feedbacks can be easily integrated. For the qualitative analysis, it allowed us to double check the modules of our system. In other words, applying a SWOT analysis depending on the literature, comparison to the design criteria and the integrated human practices is our first level of testing before using modeling or wet lab. In this manner, our team was able to enhance each unit in the system several times without wasting materials or efforts. The application of this engineering process and the corresponding time frame will be further discussed for both our delivery system and Toehold-siRNAs construct.

![](https://static.igem.org/mediawiki/2020/a/a4/T--AUC-EGYPT--engr1.jpg)

Diagram (1): Our accustomed Engineering Design process.

## Delivery System:

After deciding on the components of our designed therapeutic therapy, the question that was asked is “How will our construct be delivered into the patient cells?”. Exploring the strengths and drawbacks of the prior solutions in the literature, we found that the main constrains of delivery system are entry into the cell, endosomal escape, lysosomal degradation, nuclear uptake, unsafe replication, integration with the cell’s genome, production limitations, loading capacity and lacking specific targeting [1]. Accordingly, we were able to identify the design requirements for our delivery system to have the following criteria: specific targeting, non-replicating, safe, high production, suitable loading capacity, high transduction rates, easy testing for cell entry, and affordable.
Based on these criteria, we started our research to select a suitable delivery system. At the beginning, we chose the Virus-like particles (VLPs) system. The VLPs are nanostructures that acted as safe and helpful tools for biotechnology starting from the 1980s in order to mimic the conformation and behavior of the native viruses with the special characteristic of lacking the viral genome [2]. Accordingly, they cannot replicate, and the possibility of harmful rearrangements is eradicated. The production of hundreds of VLPs from different origins as microbes, plants, mammals and insects and over 35 virus families took place to help not only in the production of therapeutic vaccines, but also in gene therapy and drug delivery [2,3,4,5]. The reason behind the success of the VLPs is that they are formed from functional viral proteins, so their cell entry propensity is an important characteristic for a cell-specific targeting delivery system [5]. The VLPs are also capable of delivering peptides, drugs and nucleic acid [2,6,7]. In the literature, the VLPs were able to encapsulate Plasmid DNA with sizes starting from 4 kb up to 17.7 [8,9]. Accordingly, as synthesis of VLPs for viruses from the Coronaviridae family as TGEV, SARS, MHV and IBV was successfully implemented [3,10], we decided to develop SARS-CoV-2 particles that targets the ACE2 receptor expressed only on the cells that are most likely to be infected by the virus.
After selecting the virus-like particles as our delivery system, we conducted a SWOT analysis. It was found that the VLPs have the following strengths: specific targeting, non-replicating, lack the viral genome, mimic the virus cell entry and escape lysosomal degradation. However, it has one main weakness that it was mainly used as a vaccine not a vehicle for gene therapy or drug delivery. Accordingly, the ability to load out DNA circuit into the SARS-CoV-2 like particles were not tested before in the literature. This weakness takes us to the threat of failing to have successful assembly of the VLPs if the plasmid carrying our construct was co-infected with the plasmids coding for the membrane, envelope and spike of our virus-like particle. On the other hand, we still have an opportunity which is utilizing only the spike of the decoy SARS-CoV-2 like particles in order to maintain the specific targeting characteristic.
As a result, we repeated the research and brainstorming phase to improve our delivery system. We decided to pseudotype lentivirus vectors with the spike of SARS-CoV-2 [11]. Three variations of the spike protein were developed. First, a normal codon-optimized spike for mammalian cells. Second, a variant that has two amino acid mutations to basic residues in Spike’s cytoplasmic tail (K1269A and H1271A) as these mutations were found to improve the plasma membrane expression of Spike through eliminating the endoplasmic reticulum retention signal. Third, a variant with a cytoplasmic tail from influenza hemagglutinin (HA) as it was previously reported for SARS-CoV-1 that replacing or deleting the Spike’s cytoplasmic tail improves the pseudotyping efficiency [11]. However, we didn’t use the SARS-CoV-1 spike to have higher levels of successful cell entry for our particles as it was assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity [11]. Furthermore, we decided to use the third-generation lentivirus vectors as they are self-inactivating and requires the minimal virus genes for assembly which are gag, pol and rev only [12]. Following this approach, we will be able to have safe pseudo viruses with biosafety level 2 instead of the biosafety level of SARS-CoV-2 which increases the accessibility of testing our delivery system. These pseudo viruses can be used for other application than the focus of our project as the assays used to measure the effect of antibodies on the spike-mediated viral infections or any other immunity-related tests involving SARS-CoV-2.
Hence, we moved to the analyze phase again. Doing the SWOT analysis, we found that we maintained the strengths of the VLPs system, but we added an advantage of a highly efficient loading system thanks to the lentivirus vectors. These vectors showed promising results in gene therapy and drug delivery as reported in the literature [13]. The biosafety threats were eliminated as well through two practices, First, using the 3’ and 5’ LTRs in the third-generation lentivirus vectors [12,13]. Second, removing the genes coding for the tat genes and the envelope glycoprotein (replaced by SARS-CoV-2 spike) as they won’t be needed in the assembly of the vectors. However, one weakness is that we have limited production in the mammalian cell cultures. One opportunity that we spotted was the capacity to have recombinant plasmids where we load our lentivirus vectors to the donor plasmids of an expression system with higher production.
As a result, we targeted a baculovirus-mediated lentivirus vectors’ production [12]. The baculovirus expression system was used to produce a vaccine for influenza in 1.5 months which would have been 7-9 months with other platforms [2]. Accordingly, as mentioned in our project description, the baculovirus technology will help us in obtaining a scalable, fast, easy and safe vectors production during the implementation phase of our project [12]. We will use the baculovirus expression system in mammalian cultures to maintain the needed post translational modifications of our pseudo-viruses while maintaining a high production rate [12]. The efficiency of the transduction of the selected genes through the lentivirus vectors with recombinant baculovirus reaches more than 90% in the mammalian cell cultures [12].
Afterwards, we have integrated all of these modifications together to create our delivery system. The lentiviruses vectors with recombinant baculoviruses can act as a modular platform for gene therapy or drug delivery where the envelope and spike proteins can be changed to target different receptors. To reach the complete image of our system, we have communicated our project and integrated lots of feedbacks as we got the chance to present our graphic abstract in MIT Mammalian meetup back on the 27th of August, have an interview with Molecular Cloud and GenScript on the 9th of October, and the final feedback was received through presenting our work in the first African Meetup held on the 16th of October. The final stage in our engineering design process is creating a future plan for the testing and improvement of our project till we conduct a successful proof of concept and be able to meet our design criteria.
For the test plan of our delivery system, first, to test the transduction efficacy of our baculovirus mediated-lentivirus vectors, four different baculoviruses into which the lentivirus elements will be cloned should be produced in insect cells. The baculovirus production rates can be tested through an immunoblot analysis that involves an antibody that detects the major envelope protein of the baculovirus [12]. Then, end-point titer determination should be used to control the multiplicity of infection (MOI) in lentivirus production. The lentivirus vectors will be produced through transduction to 293T mammalian cells. Different MOI and numbers of baculovirus will be used in the transduction to determine the optimal settings of the experiment [12]. Second, to confirm the efficiency of the assembly into functional vectors and that the used genes are the minimal required for lentiviruses production, the lentiviruses production will be tested omitting on of the baculoviruses containing either the gag-pol genes, rev genes or the VSV-G genes. The VSV-G gene is the normal envelope glycoprotein of the lentivirus vectors which will be used in this stage to confirm the functionality of our modular high production and loading capacity modular vector before being replaced with the spike protein of SARS-COV-2 in the upcoming experiments.
Third, the loading efficiency of our vectors will be tested through constructing 4 recombinant plasmids where three of them are essential for the production of functional self-inactivating lentivirus vectors and one of them will be a transfer plasmid where the gene of insert to be loaded should be inserted. In this test, we will use the GFP as our gene of interest, so that we can detect it to determine the transduction efficacy [12]. Fourth, the transduction efficacy and production tests will be repeated after replacing the plasmid coding for the lentiviruses’ envelope with that coding for the SARS-CoV-2 spike [11]. Fifth, the specific-targeting capacity of our pseudo-viruses will be tested through confocal microscopy. Two sets of cells will be prepared, one will be cells expressing the ACE2 receptor and the other set will be the same type of cells after going through ACE2 depletion by using PMA [3]. The expected results of this test are the transduction of our pseudo viruses only to the cells expressing the ACE2 receptor.
Finally, the loading test will be repeated through inserting the genes coding for our toehold-switch and siRNA construct into our SARS-CoV-2 like particles. If we got high transduction rates at this experiment, we will be ready to conduct our proof of concept experiment where the functionality of our designed construct will be tested after being successfully loaded into the target cells. In the following sections, the experimental plan for the Toehold-switches and the siRNAs will be further explained.

## What are Toehold switches?

Toehold switches are a class of riboregulators that can be rationally designed to sense complementary trigger RNAs, regulating the expression of a downstream gene. The regulated gene is repressed in cis by the switch. Specifically, the hairpin structure of the toehold switch sequesters the ribosomal binding site (RBS) and the start codon of the downstream gene, blocking access to the translation machinery of the cells. Acting as a trans-activator, the trigger binds the free, unpaired toehold domain, initiating strand displacement and opening the hairpin structure. The binding between the toehold switch and the trigger exposes the RBS and the start codon and, hence, activates the translation of the downstream gene (Fig 1) [14].

![](https://static.igem.org/mediawiki/2020/f/fc/T--AUC-EGYPT--engr2.jpg)

Figure 1 Toehold Switch Activation
Retrieved from Green et al., (2014)

## Mammalian vs Prokaryotic toehold switches

Toehold switches were originally designed to function in prokaryotic and cell free expression systems [14], [15]. The loop of the hairpin featured a prokaryotic RBS and the bulge in the middle of the stem domain featured the start codon. In Eukaryotes, the RBS, typically the Kozak consensus sequence, is not separate from the start codon. Wang et al., (2019) redesigned toehold switches by replacing the original RBS with a Kozak sequence [16]. They designed the switches to detect miRNAs in HeLa and Human embryonic kidney (HEK) cells. Results were a proof of concept that toehold switches can be functional in mammalian contexts. The dynamic range of these toehold switches was not satisfying as they could not get beyond a level of two. We hypothesize we can enhance the dynamic range of these toehold switches by following the series B design scheme implemented by Pardee et al., (2016) which proved to be more efficient than design of the first generation toehold switches by Green et al.,(2014) (Fig 2).

![](https://static.igem.org/mediawiki/2020/b/bd/T--AUC-EGYPT--engr3.jpg)

Figure 2 First Generation Vs Series B toeholds
Mainly the loop size of series B is larger, and the b domain which unfolds on the trigger constitutes the whole stem region in First generation toeholds whereas it constitutes only the bottom of the bulge in Series B Toeholds.

## Our Engineered Toehold Switches

![](https://static.igem.org/mediawiki/2020/9/9b/T--AUC-EGYPT--engr4.jpg)

Figure 3 First Generation-like and Series B-like
Series B-like differs in that the b domain does not constitute the full stem. Rather, there’s a conserved sequence on the top of the stem. Additionally, the loop size is larger. This is mimicking series B.

Using our enhanced tool [(see Software)](https://2020.igem.org/Team%3AAUC-EGYPT/Software), we generated two sets of mammalian toehold switches: series B-like and first generation-like (Fig 3) [(See Parts)](https://2020.igem.org/Team%3AAUC-EGYPT/Parts). One important aspect in series B design is the conserved 4 weak base pairs (A-T). They were not translated in the original toeholds as they preceded the start codon [15]. Now that the start codon is the last three basses of the loop (being part of the Kozak sequence), these bases are translated. We changed the sequence of the first three nucleotides as they translated a stop codon, yet we preserved the logic of having them as weak base pairs.

## Our Toeholds

We designed toehold switches against triggers in the S2 subunit of the spike mRNA. From a design perspective, the S2 domain is conserved among Sars-Cov-2 strains and, hence, is a good candidate for therapeutic development[17]. Scanning this 1.8 kb region using our developed version of Toeholder [(see Software)](https://2020.igem.org/Team%3AAUC-EGYPT/Software), we generated 143 series B-like candidate toehold switches. Based on our free energy criteria of what defines a good switch [(see Modeling)](https://2020.igem.org/Team%3AAUC-EGYPT/Model), we identified potential 15 toehold switches to be tested in our Phase II project. From the Thermodynamic model we concluded that there is no correlation between the number of accessible nucleotides and the free energy of binding between the toehold switch and the trigger. Accordingly, we generated a new set of toeholds, resetting the minimum number of unpaired bases to zero since there is no longer a correlation. Using a simple python script, we changed the loop sequence (the main difference between both classes of toeholds) of the series B-like toeholds to the corresponding first generation-like toeholds.

## Experimental design

The two classes of Toehold switches will be constitutively expressed using a CMV promotor [18]. U2OS cells will be transfected with a CMV—ToeholdSwitch—GFP construct and a CMV—trigger construct (Fig 4). It is expected that the toehold switch will bind to the trigger, expressing the GFP reporter. The degree of fluorescence will be recorded, and this is considered the ON signal level. Serving as a control, a cell line will be transfected with only a CMV—ToeholdSwitch—GFP construct. In this setup, the toehold switch is expected to remain in the off state. The degree of fluorescence will be recorded, and this is considered the OFF state. The degree of fluorescence will be measured with a plate reader. Toehold switches in each class will be ranked based on their dynamic range—the ON/OFF ratio. The top toehold switches that display the highest dynamic range will be chosen as candidates for the next experimental setup. We expect the series B-like toehold switches to have a higher dynamic range.

![](https://static.igem.org/mediawiki/2020/5/5b/T--AUC-EGYPT--engr5.jpg)

Figure 4 Toehold Characterization Constructs

## Toehold Switch—GAL4BD-VP16—UAS (TGU) device characterization

Our system is supposed to control the ON/OFF state of GAL4BD-VP16 transactivator. Toehold switches that displayed the highest dynamic range will be ligated to GAL4BD-VP16. All in one vector, the CMV—TGU—GFP construct will characterize of the dynamic range of the TGU devices. U2OS cells will be transfected with the CMV—TGU—GFP construct and a CMV-Trigger construct (Fig 5). In this setup, the toehold is supposed to activate the translation of GAL4BD-VP16 which will bind to its downstream UAS promoter, thereby inducing the expression of GFP. The degree of fluorescence in recorded as the ON state. In a control setup, cells are transfected with only the CMV—TGU—GFP construct. In this setup, the toehold is supposed to remain in the OFF state, blocking the translation of GAL4BD-VP16. Hence, GFP is not expected to be expressed. The degree of fluorescence will be measured and recorded as the OFF state. TGU devices that display the highest dynamic range will be selected as candidates for therapeutic development.

![](https://static.igem.org/mediawiki/2020/c/cd/T--AUC-EGYPT--engr6.jpg)

Figure 5 TGU Device Characterization Constructs

## Experimental design for siRNA:

To test the efficacy of our siRNA, 3 Types of vectors will be constructed: one as a negative control and the other two will be our tested sequences for the target gene (one for each sequence). The control vector will be designed to have no known target in the cells being used for distinguishing sequence-specific silencing from non–specific effects and reflect a baseline cellular response that can be compared to the levels in cells treated with target-specific siRNA. The vector will contain ON-TARGETplus siRNA (X-siRNA) along with a GFP sequence to report the sufficient entry of the vector into the cell and it’s expression. On the other hand, for testing, the vector will be designed twice for each siRNA-R, separately. The plasmid will have the siRNA sequence, targeting our gene of interest, attached to the downstream GFP. The targeted miRNA amounts will be detected after applying the 3 types of vectors, and the efficiency of knockdown by our candidate siRNA sequences will be verified through a comparison between them and the control. The test will be repeated 3 times at least to then calculate the average and the standard deviation of the resulted viral mRNA transcripts using a qPCR. (Construct containing the target gene)
After verifying the efficiency of our candidate sequences, we will integrate the two siRNA sequences into one plasmid as multiplexed siRNAs with the GFP using endogenous intronic primary microRNAs (pri-miRNAs) as a scaffold located in the green fluorescent protein (GFP) as a marker protein for higher efficiency and low cost. This multiplexed intronic siRNA – GFP platform is mainly designed to co-express multiple small RNAs within the polycistronic cluster from a Pol II promoter at more moderate levels to reduce potential vector toxicity. The intronic siRNAs are co-transcribed with a precursor GFP mRNA as a single transcript and presumably cleaved out of the precursor-(pre) mRNA by the RNA splicing machinery, spliceosome. The spliced intron with siRNA will be further processed into mature siRNAs capable of triggering their silencing effect, while the ligated exons become a mature messenger RNA for the translation of the functional GFP protein to report their entry into the cell [19]. By this, we anticipate our siRNA to silence SARS-CoV-2 replicase mRNA and thus decrease the number of those transcripts by the virus, inhibiting virus replication.
If the amount of the replicase mRNA transcripts didn't decrease compared to the control, we will try to use another siRNA candidate, designed by both siDirect and Oligowalk software tools, and select the matches. Then we will repeat the previous experiment. If they didn't work, we will understand that the siRNA sequences are not problematic, instead, we could apply different concentrations of siRNAs to allow more interaction with the SARS-CoV-2 mRNA.

## References

[1]K. Jain, “An Overview of Drug Delivery Systems,” Drug Delivery Systems Methods in Molecular Biology, pp. 1-54, 2019. doi:10.1007/978-1-4939-9798-5\_1
[2] A. Roldão, A. C. Silva, M. C. M. Mellado, P. M. Alves, and M. J. T. Carrondo, “Viruses and Virus-Like Particles in Biotechnology: Fundamentals and Applications,” Compr. Biotechnol., p. 633, 2017, doi: 10.1016/B978-0-12-809633-8.09046-4.
[3] A. Naskalska et al., “Novel coronavirus-like particles targeting cells lining the respiratory tract,” PLoS One, vol. 13, no. 9, p. e0203489, Sep. 2018, doi: 10.1371/journal.pone.0203489.
[4] W. A. Rodríguez-Limas, K. Sekar, and K. E. J. Tyo, “Virus-like particles: The future of microbial factories and cell-free systems as platforms for vaccine development,” Current Opinion in Biotechnology, vol. 24, no. 6. Elsevier Current Trends, pp. 1089–1093, Dec. 01, 2013, doi: 10.1016/j.copbio.2013.02.008.
[5] M. Zdanowicz and J. Chroboczek, “Virus-like particles as drug delivery vectors,” Acta Biochim. Pol., vol. 63, no. 3, pp. 469–473, 2016, doi: 10.18388/abp.2016\_1275.
[6] N. G. Cortes-Perez et al., “Rotavirus-Like Particles: A Novel Nanocarrier for the Gut,” J. Biomed. Biotechnol., vol. 2010, p. 317545, 2010, doi: 10.1155/2010/317545.
[7] Y. Fu and J. Li, “A novel delivery platform based on Bacteriophage MS2 virus-like particles,” Virus Research, vol. 211. Elsevier B.V., pp. 9–16, Jan. 04, 2016, doi: 10.1016/j.virusres.2015.08.022.
[8] R. L. Garcea and L. Gissmann, “Virus-like particles as vaccines and vessels for the delivery of small molecules.,” Curr. Opin. Biotechnol., vol. 15, no. 6, pp. 513–517, Dec. 2004, doi: 10.1016/j.copbio.2004.10.002.
[9] C. Kimchi-Sarfaty and M. M. Gottesman, “SV40 pseudovirions as highly efficient vectors for gene transfer and their potential application in cancer therapy.,” Curr. Pharm. Biotechnol., vol. 5, no. 5, pp. 451–458, Oct. 2004, doi: 10.2174/1389201043376670.
[10] E. Mortola and P. Roy, “Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system,” FEBS Lett., vol. 576, no. 1–2, pp. 174–178, Oct. 2004, doi: 10.1016/j.febslet.2004.09.009.
[11] K.H.D. Crawford, R. Eguia, A.S. Dingens, A.N. Loes, K.D. Malone, C.R. Wolf, H.Y. Chu, M.A. Tortorici, D. Veesler, M. Murphy, D. Pettie, N.P. King, A.B. Balazs, & J.D. Bloom, “ Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays,” Viruses, vol. 12, pp. 513. 2020. doi: https://doi.org/10.3390/v12050513
[12] H. Lesch, S. Turpeinen, E. Niskanen et al. “Generation of lentivirus vectors using recombinant baculoviruses,” Gene Therapy, Vol. 15, pp.1280–1286. 2008 https://doi.org/10.1038/gt.2008.76
[13] M.C. Milone, & U. O’Doherty, “Clinical use of lentiviral vectors,” Leukemia, Vol. 32, pp. 1529–1541. 2018. doi: https://doi.org/10.1038/s41375-018-0106-0
[14] A. A. Green, P. A. Silver, J. J. Collins, and P. Yin, “Toehold Switches: De-Novo-Designed Regulators of Gene Expression,” Cell, vol. 159, no. 4, pp. 925–939, Nov. 2014, doi: 10.1016/j.cell.2014.10.002.
[15] K. Pardee et al., “Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components,” Cell, vol. 165, no. 5, pp. 1255–1266, May 2016, doi: 10.1016/j.cell.2016.04.059.
[16] S. Wang, N. J. Emery, and A. P. Liu, “A Novel Synthetic Toehold Switch for MicroRNA Detection in Mammalian Cells,” ACS Synth. Biol., vol. 8, no. 5, pp. 1079–1088, May 2019, doi: 10.1021/acssynbio.8b00530.
[17] N. Kaushal, Y. Gupta, M. Goyal, S. F. Khaiboullina, M. Baranwal, and S. C. Verma, “Mutational Frequencies of SARS-CoV-2 Genome during the Beginning Months of the Outbreak in USA,” Pathogens, vol. 9, no. 7, p. 565, Jul. 2020, doi: 10.3390/pathogens9070565.
[18] D. R. Thomsen, R. M. Stenberg, W. F. Goins, and M. F. Stinski, “Promoter-regulatory region of the major immediate early gene of human cytomegalovirus,” Proc. Natl. Acad. Sci. U. S. A., vol. 81, no. 3, pp. 659–663, Feb. 1984, doi: 10.1073/pnas.81.3.659.
[19] A. Seyhan, "A multiplexed miRNA and transgene expression platform for simultaneous repression and expression of protein-coding sequences," Molecular BioSystems, vol. 12, no.1, pp. 295-312. 2016.

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# Team:AUC-EGYPT/Experiments

![](https://static.igem.org/mediawiki/2020/a/a2/T--AUC-EGYPT--GIF_1.gif)

# 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

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:AUC-EGYPT/Results

![](https://static.igem.org/mediawiki/2020/a/a2/T--AUC-EGYPT--GIF_1.gif)

# 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.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)