iGEM 2021 — Team UParis_BME

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

# Team:UParis BME/Notebook

* [Home](https://2021.igem.org/Team%3AUParis_BME)
* Project
  + [Description](https://2021.igem.org/Team%3AUParis_BME/Description)
  + [Engineering](https://2021.igem.org/Team%3AUParis_BME/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AUParis_BME/Implementation)
  + [Proof Of Concept](https://2021.igem.org/Team%3AUParis_BME/Proof_Of_Concept)
  + [Contribution](https://2021.igem.org/Team%3AUParis_BME/Contribution)
  + [Collaborations](https://2021.igem.org/Team%3AUParis_BME/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AUParis_BME/Partnership)
* Lab
  + [Lab Journal](https://2021.igem.org/Team%3AUParis_BME/Notebook)
  + [Wet Lab](https://2021.igem.org/Team%3AUParis_BME/Wetlab)
  + [Dry Lab](https://2021.igem.org/Team%3AUParis_BME/Drylab)
  + [Results](https://2021.igem.org/Team%3AUParis_BME/Results)
  + [Parts](https://2021.igem.org/Team%3AUParis_BME/Parts)
  + [Safety](https://2021.igem.org/Team%3AUParis_BME/Safety)
* Human Practices
  + [Integrated Human Practices](https://2021.igem.org/Team%3AUParis_BME/Human_Practices)
  + [Lifestory/iGEMathon](https://2021.igem.org/Team%3AUParis_BME/LifeStory)
* Communications
  + [Communication](https://2021.igem.org/Team%3AUParis_BME/Communication)
  + [Education & Public engagement](https://2021.igem.org/Team%3AUParis_BME/Education)
  + [Social Media](https://2021.igem.org/Team%3AUParis_BME/SocialMedia)
* Team
  + [Team](https://2021.igem.org/Team%3AUParis_BME/Team)
  + [Attributions](https://2021.igem.org/Team%3AUParis_BME/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3AUParis_BME/Sponsors)
* Awards
  + [Prize](https://2021.igem.org/Team%3AUParis_BME/Prize)
  + [Medal Criteria](https://2021.igem.org/Team%3AUParis_BME/Medals)

[ ]

![page illustration](https://static.igem.org/mediawiki/2021/3/3a/T--UParis_BME--fondCelia_essaitaille.png)

# Lab journal

* [Brainstorming sessions!](#paragraphe1)
* [Let’s find teammates and a place to work!](#paragraphe2)
* [Time to meet the Lab!](#paragraphe3)
* [Our lab becomes our new home](#paragraphe4)
* [Scientific mediation and back to school](#paragraphe5)

## **Brainstorming sessions!**

#### **January-February**

Literature review, meetings, discussion… Until we all agreed to work on early cancer diagnosis using de-novo-designed riboregulators, toehold switches!

---

## **Let’s find teammates and a place to work!**

#### **February-March**

We are a brand new team in our Faculty. We presented our project to the research teams from the Basic and Biomedical Sciences Faculty from Université de Paris. We met the researcher Pierre Nioche and his team for the first time, they gladly accepted to host our team to conduct experiments.
We were initially students from the Biomedical Engineering Paris master degree, Célia and Kévin joined the team to help with the dry lab and wiki!

---

## **Time to meet the Lab!**

#### **April/June**

We wrote the first version of our SwitchMi Designer! Let’s build the toehold switch candidates in the lab...
First experiments to build our toehold switches using Splicing for Overlap Extension Polymerase Chain Reaction and first fails that make our strategy evolve!

---

## **Our lab becomes our new home**

#### **July-August**

While we are thinking about molecular cloning protocols to build our toehold switches, we discovered inaccuracies in our SwitchMi Designer. We slept on it to improve the workflow as much as we can!
We succeeded in cloning EGFP, the repressor gene for our toehold switches, in our plasmid and received the other part of our toehold switch candidates previously designed.
We tried to clone the toehold switch part into our plasmid expressing GFP using classical molecular cloning and golden gate cloning strategies, without success.

---

## **Scientific mediation and back to school**

#### **September-October**

Back to school stopped our experiments in the lab. Most of the team members are now enrolled in their last year before starting a PhD.
We focused on giving lectures at highschool and shared our iGEM experience through conferences with Université de Paris and Cité des Sciences et de l’Industrie.

---

## Contact Us !

 : Contact us via email!

## A Special Thanks To

![sponsors](https://static.igem.org/mediawiki/2021/4/49/T--UParis_BME--UP.png)

![sponsors](https://static.igem.org/mediawiki/2021/b/bd/T--UParis_BME--BME_Master.png)

![sponsors](https://static.igem.org/mediawiki/2021/d/d3/T--UParis_BME--iGEM.png)

![sponsors](https://static.igem.org/mediawiki/2021/f/f5/T--UParis_BME--Integrated.png)

![sponsors](https://static.igem.org/mediawiki/2021/e/ea/T--UParis_BME--Promega.png)

![sponsors](https://static.igem.org/mediawiki/2021/1/15/T--UParis_BME--PCR.png)

![sponsors](https://static.igem.org/mediawiki/2021/8/8e/T--UParis_BME--New_England.png)

![sponsors](https://static.igem.org/mediawiki/2021/6/6d/T--UParis_BME--Ligue.png)

![sponsors](https://static.igem.org/mediawiki/2021/6/6c/T--UParis_BME--Frederick.png)

©2021 Copyright: iGEM UParis\_BME, author: Kévin DANG
 Source code released under MIT License
 Made using Bootstrap, jQuery and FontAwesome

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

# Team:UParis BME/Engineering

* [Home](https://2021.igem.org/Team%3AUParis_BME)
* Project
  + [Description](https://2021.igem.org/Team%3AUParis_BME/Description)
  + [Engineering](https://2021.igem.org/Team%3AUParis_BME/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AUParis_BME/Implementation)
  + [Proof Of Concept](https://2021.igem.org/Team%3AUParis_BME/Proof_Of_Concept)
  + [Contribution](https://2021.igem.org/Team%3AUParis_BME/Contribution)
  + [Collaborations](https://2021.igem.org/Team%3AUParis_BME/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AUParis_BME/Partnership)
* Lab
  + [Lab Journal](https://2021.igem.org/Team%3AUParis_BME/Notebook)
  + [Wet Lab](https://2021.igem.org/Team%3AUParis_BME/Wetlab)
  + [Dry Lab](https://2021.igem.org/Team%3AUParis_BME/Drylab)
  + [Results](https://2021.igem.org/Team%3AUParis_BME/Results)
  + [Parts](https://2021.igem.org/Team%3AUParis_BME/Parts)
  + [Safety](https://2021.igem.org/Team%3AUParis_BME/Safety)
* Human Practices
  + [Integrated Human Practices](https://2021.igem.org/Team%3AUParis_BME/Human_Practices)
  + [Lifestory/iGEMathon](https://2021.igem.org/Team%3AUParis_BME/LifeStory)
* Communications
  + [Communication](https://2021.igem.org/Team%3AUParis_BME/Communication)
  + [Education & Public engagement](https://2021.igem.org/Team%3AUParis_BME/Education)
  + [Social Media](https://2021.igem.org/Team%3AUParis_BME/SocialMedia)
* Team
  + [Team](https://2021.igem.org/Team%3AUParis_BME/Team)
  + [Attributions](https://2021.igem.org/Team%3AUParis_BME/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3AUParis_BME/Sponsors)
* Awards
  + [Prize](https://2021.igem.org/Team%3AUParis_BME/Prize)
  + [Medal Criteria](https://2021.igem.org/Team%3AUParis_BME/Medals)

[ ]

![page illustration](https://static.igem.org/mediawiki/2021/9/99/T--UParis_BME--project_background.png)

# Engineering Cycle

* [Engineering as a problem-solving philosophy](#paragraphe1)
* [Dry-Wet-Dry cycle](#paragraphe2)
* [Human Practices cycle](#paragraphe3)
* [Communications cycle](#paragraphe4)

## **Engineering as a problem-solving philosophy**

Since the core of our team is composed of Biomedical Engineering Master’s students, we found it only natural to employ a problem-oriented approach throughout the project. For us, it was almost second nature to adopt the “engineering cycle” philosophy outside of the immediate dry and wet lab context. This ensured that the prototype design was not only guided by the dry and wet lab results, but also informed by the outcomes of the human practice initiatives, insights received from other iGEM teams, feedback from the more global scientific community, etc. Furthermore, the said outcomes of the lab work generated additional questions, which were quickly reflected by our integrated human practices, communications and changes to the overall strategy, as showcased by the [implementation section](https://2021.igem.org/Team%3AUParis_BME/Implementation).

Since almost the entire team was involved across multiple areas of the project, this integration was almost seamless, allowing us to work as a well-tuned mechanism - wittily represented by the rainbow gears (Although we could never agree whether it is pronounced .gif or .gif ...). To fully reflect our philosophy, we decided to split the mechanism into separate cycling gears, showcasing several examples of how the engineering mindset was generating ideas and insights both inside and outside our laboratory work.

![cycle](https://static.igem.org/mediawiki/2021/3/3e/T--UParis_BME--Engineering.gif)

---

## **Dry-Wet-Dry cycle**

![picture](https://static.igem.org/mediawiki/2021/3/37/T--UParis_BME--Model.png)

[iGEM’s introduction to the engineering process](https://2021.igem.org/Engineering/Design) describes the design stage as the entry point of the engineering cycle. Yet, we felt like in the broader context of engineering as a problem-solving philosophy, it is crucial to start with learning the limitations of the existing solutions to the problem. For instance, previous teams have already designed several algorithms that generate toehold switch candidates for a target messenger RNA. However, these algorithms were not working for much shorter microRNA targets.
Still, the access to their source code allowed us to better understand the approaches utilised for toehold design and quicker construct our updated model. Furthermore, depending on the packages and libraries employed, different code will produce different secondary structure and Gibbs free energy estimates. Both of these factors helped inform our first design, as we selected the candidates that performed well with different estimation methods. Finally, we tested our candidate libraries with external algorithms to double-check the result with an external model. This is where we discovered some inaccuracies in our code. For instance, our RNA energy estimates included the T7 transcriptional promoter sequence, which is obviously not right. We also had some trouble accounting for the reporter gene RNA impact on our genetic machine (a little diagram on the right is there to visualise the source of confusion), but eventually we managed to generate perfect candidate libraries for several target miRNAs. Once satisfied with the test, we could proceed to ordering the toehold sequences and preparing the wet lab experiments, which in turn informed the microfluidic design in the downstream dry lab efforts.

![cycle](https://static.igem.org/mediawiki/2021/3/39/T--UParis_BME--dry_wet_dry_cycle_pt1.gif)

Of course, the dry lab cycle wouldn’t be as impactful in isolation. We chose two candidate libraries (toeholds targeting miRNA-141 and miRNA-21) and utilised two different methods to ligate the toehold sequences onto our plasmids. Our system design accounted for antibiotic selection, transfection markers, and selecting the bacteria capable of expressing genes under a T7 promoter. Unfortunately, neither of the ligation techniques worked properly, preventing us from generating sufficient data and achieving the PoC. Alas, as we were limited by the time remaining until the end of the competition, we could not continue the engineering cycle. Yet, we have designed the downstream steps, which involve a different selection of the restriction enzymes, a different bacterial strain, and using the candidate libraries redesigned with the improved dry lab algorithms. In the meantime, our experiments on exosome isolation from cancer cell culture and target miRNA quantification work have been more fruitful - feel free to learn more in the [Results section!](https://2021.igem.org/Team%3AUParis_BME/Results)

![cycle](https://static.igem.org/mediawiki/2021/a/a3/T--UParis_BME--dry_wet_dry_cycle_pt2.gif)

---

## **Human Practices cycle**

The key values of the Human Practices - [Reflection, Responsibility and Responsiveness](https://igem.org/Human_Practices) - line-up almost seamlessly with the iGEM engineering cycle. Reflection, the process of thinking through the team’s motivation and goals, is essential to the first steps of ***learning*** about the problem. Responsibility takes the spotlight whenever we ***design*** a new plasmid, ***build an educational workshop for a school***, or consider the best implementations of our final product, as we aim to improve the lives of cancer patients around the world. We always ensure that the team stays faithful to the key iGEM values - respect, honesty, fairness, and others - as everyone works to minimise risks if having a negative impact. And, finally, we must be responsive to any problem arising from ***testing***, and adapt the strategy based on the insights. For example, we spent a long time editing any communications aimed at the patients to remove potentially insensitive questions or messages. If you want to learn more about the impact of the human practice insights, and about the adaptations we have made throughout the project, please give the relevant sections a read - [Human Practices](https://2021.igem.org/Team%3AUParis_BME/Human_Practices) and [Implementation](https://2021.igem.org/Team%3AUParis_BME/Implementation).

![cycle](https://static.igem.org/mediawiki/2021/6/61/T--UParis_BME--Human_Practices_Cycle.gif)

---

## **Communications cycle**

Social media, team meetups, science communication, educational work, and much more - communication behind an iGEM project is both extensive and diverse. Different key messages and target audiences require a tailored approach, which offers a large opportunity for practicing the engineering cycle approach. From the number of “likes” to the questions asked by potential sponsors, there is no shortage of feedback and insights to guide further design and building of the outreach strategies for different audiences. We learned how to adapt our presentations for different occasions, as well as what our social media followers want to see on our Instagram and Facebook pages. And, of course, the format and the content of our messages kept evolving throughout the iGEM journey. Don’t hesitate to check out our [social media](https://2021.igem.org/Team%3AUParis_BME/SocialMedia), [education](https://2021.igem.org/Team%3AUParis_BME/Education) and [communication](https://2021.igem.org/Team%3AUParis_BME/Communication) sections to see how our presence has evolved both online and during in-person events!

![cycle](https://static.igem.org/mediawiki/2021/d/de/T--UParis_BME--Communications_cycle.gif)

---

## Contact Us !

 : Contact us via email!

## A Special Thanks To

![sponsors](https://static.igem.org/mediawiki/2021/4/49/T--UParis_BME--UP.png)

![sponsors](https://static.igem.org/mediawiki/2021/b/bd/T--UParis_BME--BME_Master.png)

![sponsors](https://static.igem.org/mediawiki/2021/d/d3/T--UParis_BME--iGEM.png)

![sponsors](https://static.igem.org/mediawiki/2021/f/f5/T--UParis_BME--Integrated.png)

![sponsors](https://static.igem.org/mediawiki/2021/e/ea/T--UParis_BME--Promega.png)

![sponsors](https://static.igem.org/mediawiki/2021/1/15/T--UParis_BME--PCR.png)

![sponsors](https://static.igem.org/mediawiki/2021/8/8e/T--UParis_BME--New_England.png)

![sponsors](https://static.igem.org/mediawiki/2021/6/6d/T--UParis_BME--Ligue.png)

![sponsors](https://static.igem.org/mediawiki/2021/6/6c/T--UParis_BME--Frederick.png)

©2021 Copyright: iGEM UParis\_BME, author: Kévin DANG
 Source code released under MIT License
 Made using Bootstrap, jQuery and FontAwesome

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

# Team:UParis BME/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:UParis BME/Results

* [Home](https://2021.igem.org/Team%3AUParis_BME)
* Project
  + [Description](https://2021.igem.org/Team%3AUParis_BME/Description)
  + [Engineering](https://2021.igem.org/Team%3AUParis_BME/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AUParis_BME/Implementation)
  + [Proof Of Concept](https://2021.igem.org/Team%3AUParis_BME/Proof_Of_Concept)
  + [Contribution](https://2021.igem.org/Team%3AUParis_BME/Contribution)
  + [Collaborations](https://2021.igem.org/Team%3AUParis_BME/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AUParis_BME/Partnership)
* Lab
  + [Lab Journal](https://2021.igem.org/Team%3AUParis_BME/Notebook)
  + [Wet Lab](https://2021.igem.org/Team%3AUParis_BME/Wetlab)
  + [Dry Lab](https://2021.igem.org/Team%3AUParis_BME/Drylab)
  + [Results](https://2021.igem.org/Team%3AUParis_BME/Results)
  + [Parts](https://2021.igem.org/Team%3AUParis_BME/Parts)
  + [Safety](https://2021.igem.org/Team%3AUParis_BME/Safety)
* Human Practices
  + [Integrated Human Practices](https://2021.igem.org/Team%3AUParis_BME/Human_Practices)
  + [Lifestory/iGEMathon](https://2021.igem.org/Team%3AUParis_BME/LifeStory)
* Communications
  + [Communication](https://2021.igem.org/Team%3AUParis_BME/Communication)
  + [Education & Public engagement](https://2021.igem.org/Team%3AUParis_BME/Education)
  + [Social Media](https://2021.igem.org/Team%3AUParis_BME/SocialMedia)
* Team
  + [Team](https://2021.igem.org/Team%3AUParis_BME/Team)
  + [Attributions](https://2021.igem.org/Team%3AUParis_BME/Attributions)
  + [Sponsors](https://2021.igem.org/Team%3AUParis_BME/Sponsors)
* Awards
  + [Prize](https://2021.igem.org/Team%3AUParis_BME/Prize)
  + [Medal Criteria](https://2021.igem.org/Team%3AUParis_BME/Medals)

[ ]

![page illustration](https://static.igem.org/mediawiki/2021/3/3a/T--UParis_BME--fondCelia_essaitaille.png)

# Results

* [A plasmid to produce any toehold switch candidates](#paragraphe1)
* [Production of complete toehold switches](#paragraphe2)
* [MCF-7 and HeLa cell lines Exhibit Exosomal miRNAs](#paragraphe3)
* [Perspectives](#paragraphe4)

The aim of ExoSwitch is to create an early cancer diagnosis tool using toehold switches to detect and quantify exosomal miRNAs from blood samples. We modeled a microfluidic device which allows exosome separation from the blood, followed by exosomes lysis and exosomal miRNAs detection using toehold switches in a cell-free system.

Specific miRNAs signatures are correlated with cancer initiation and spreading. The aberrant expression profile of certain miRNAs indicates cancer setup and progression. This means that these expression profiles can hold a diagnostic and prognostic value1.
For our proof-of-concept, we first modeled a bank of toehold switches targeting specific miRNAs using [SwitchMi Designer](https://github.com/igemsoftware2021/UParis_BME_2021) (a software we have designed). We then decided to focus on the detection of two miRNAs, miR-21-5p and miR-141-5p. The miR-141-5p is overexpressed in cancer patients such as in the case of breast or prostate cancer2. The miR-21-5p is commonly found in abnormal quantities in patients suffering from cancer and it is overexpressed in cervix and breast cancer cell lines’ exosomes, such as HeLa and MCF-73-5. We tried to produce the banks of toehold switches targeting these two miRNAs in order to characterize them (phase 1 of our proof-of-concept).
At the same time, we cultured HeLa and MCF-7 cell lines to extract their exosomes and isolate the exosomal miRNAs to fit phase 2 of our proof-of-concept.

## **Phase 1**

## **A plasmid to produce any toehold switch candidates**

Pierre Nioche kindly provided us with a GFP tagged protein and pET24d(+) plasmid. The first step was to build a pET24d(+)\_GFP plasmid by assembling GFP and pET-24d(+) using molecular cloning with restriction enzymes. (**Figure 1**)

![picture](https://static.igem.org/mediawiki/2021/4/4a/T--UParis_BME--wiki-results-scheme.png)
**Figure 1: Assembly of the pET-24d(+)\_GFP plasmid with molecular cloning method using restriction sites.** Created with BioRender.

We first amplified the GFP fragment from the GFP tagged protein via polymerase chain reaction (PCR) to get a higher quantity of DNA for molecular cloning. At each step, samples were analyzed by gel electrophoresis to validate the step. **Figure 2** shows that after PCR we have obtained a unique fragment (one band in the red box) that is slightly below the 800bp band from the NEB 100bp ladder. This fragment corresponds to the expected EGFP fragment with the two restriction sites EcorI and XhoI at its extreme sides, which is 738bp long.

![picture](https://static.igem.org/mediawiki/2021/1/1b/T--UParis_BME--Fig2.png)
**Figure 2: GFP fragment amplified via PCR controlled on an agarose gel.**
Samples and NEB 100bp ladder were run on a 1.2% agarose gel for 30min. The NEB 100bp ladder was loaded in the first well. The DNA fragment encoding for GFP is labeled in the red box, with a size between 800 and 700 bp (expected size= 738bp).

We next aimed to integrate this PCR amplified GFP fragment into the plasmid pET-24d(+). GFP fragments and pET-24d(+) are thus digested using the restriction enzymes EcorI and XhoI overnight. The efficiency of digestion was checked by electrophoresis. **Figure 3** shows that pET-24d(+) was successfully digested: the well loaded with the digest samples exhibit a unique band corresponding to linearized DNA (open and digested plasmid), compared to the well loaded with an undigested plasmid that shows two bands for the two forms of a plasmid.

![picture](https://static.igem.org/mediawiki/2021/c/c0/T--UParis_BME--wiki-resukts-pet24disgested.png)
**Figure 3: Digested pET-24d(+) controlled on a gel.**
Samples were run on a 1.2% agarose gel for 30min. The two first wells are loaded with pET24d(+) plasmid which was previously digested using EcorI and XhoI restriction enzymes and show the band of linearized DNA. The third well has been loaded with undigested pET24d(+) plasmid, showing the two bands of circular and supercoiled forms.

The GFP fragment was then integrated into the linearized pET-24d(+) plasmid using 10 minute ligation protocol with the NEB T4 ligase. To amplify the plasmid, Bl21(DE3) *E. coli* were transformed with the ligated product using heat shock protocol. Since pET-24d(+) plasmid contains the kanamycin resistance gene, bacteria were cultured overnight on kanamycin LB (Luria Broth) to select the *E. coli* who have integrated the plasmid. From this transformation, we selected 8 clones that we're able to grow on our kanamycin media and form colonies. After DNA extraction from these 8 clones, we have made a PCR to amplify the GFP fragment to verify the integration of the GFP fragment into the plasmid. **Figure 4** shows that PCR has amplified a fragment of more than 500pb from DNA of the colonies 2, 4, 5,6 and not from other colonies. This fragment presented the expected size of GFP, suggesting the success of the ligation. To confirm the good construction of our plasmid, we sent it to sequencing (Eurofins Genomics) and clones 2,4,6 were positive with our new construction (**Figure 4**).

![picture](https://static.igem.org/mediawiki/2021/2/25/T--UParis_BME--pET24LigationResults2.png)
**Figure 4: pET-24d(+) and EGFP Ligation results.**
1.2% Agarose Gel with NEB Ladder 100Bp. This figure presents the results of PCR performed on the different clones obtained through the transformation.

![picture](https://static.igem.org/mediawiki/2021/9/94/T--UParis_BME--wiki-results_vectormap.png)
**Figure 5: Vector map of pET-24d(+)\_GFP created by assembly of GFP insert and pET-24d(+) (BBa\_K3878000).** Created with SnapGene with the sequencing results.

We named this newly engineered plasmid pET-24d(+)\_GFP and it contains the part [BBa\_K3878000](http://parts.igem.org/Part%3ABBa_K3878000) (EGFP followed by a T7terminator); its vector map is detailed on **Figure 5**. This plasmid pET-24d(+)\_GFP is the receiving vector to insert the variable part of the toehold switch. The plasmid keeps the T7 promoter and most of the restriction site. One point that can be addressed is the disparition of the EcorI site.

---

## **Production of complete toehold switches**

We tried several methods (detailed in /[wetlab](https://2021.igem.org/Team%3AUParis_BME/Wetlab)) to get the toehold switch directly fully synthesized or the variable part of the toehold switch (**Figure 6**) ready to be assembled into a plasmid that already encodes the repressed gene, GFP.

![picture](https://static.igem.org/mediawiki/2021/c/c1/T--UParis_BME--toehold.png)
**Figure 6: Scheme of a toehold switch.** In our project ExoSwitch, we define the variable part of the toehold switch the part that differs depending on the target RNA. Indeed, the Toehold part is complementary to the target miRNA. Thus, the complementary nucleotide at the root of the hairpin differs with the target miRNA.

#### DNA synthesis by SOE PCR

SOE PCR is the first technique we tried to synthesize one of the full Toehold Switch sequences with. Three sets of backward primers were designed and ordered to enable the step-by-step assembly. The size of the primers depends on the set but is on average from 40 to 70 bps.

SOE with the first set of primers: The PCR1 performed with the first backward primer leads to a successful elongation of the starting sequence (EGFP) with the right expected size (**Figure 7A**). Indeed, electrophoresis gel migration reveals the bands corresponding to the PCR1 product being placed slightly higher than the band corresponding to the GFP migration (**Figure 7A**). The same is true for the PCR2 performed using the product from the PCR1 with the second backward primer together (**Figure 7B**). The PCR2 product still migrates less compared to the PCR1 and is then of higher molecular weight (**Figure 7B**). However, the PCR3 performed with the PCR2 product together with the third backward primer did not complete the Toehold Switch synthesis. The PCR3 product is of very low quantity, and moreover, it seems to be placed lower than the PCR2 product, which means that the sequence has not been elongated… To solve the problem we reproduced the three PCRs several times, but the PCR3 always leads to confusing results. We then decide to redesign the primers to solve the problem.

SOE with the second and third sets of primers: Second and third sets of primers were ordered and delivered. However, only the first two PCRs (PCR1 and PCR2) have been ever successful. The new primers used have not changed the results - after each PCR we were obtaining again something similar to the data represented in **Figure 7C**. We decided to abandon the strategy after several weeks of attempts.

![picture](https://static.igem.org/mediawiki/2021/4/4a/T--UParis_BME--siki-results-SOE.png)
**Figure 7: Electrophoretic analysis of the SOE PCR products obtained with the first set of backward primers.** EGFP is used as the starting sequence for the Toehold Switch synthesis. NEB Ladder 1KBp is used as a reference for all three gels (A,B and C). Three consecutive SOE PCRs were performed: PCR1 (taking GFP as the starting sequence, Xho1 as a forward primer, and the first backward primer), PCR2 (taking PCR1 product as the starting sequence, Xho1 as a forward primer, and the second backward primer), PCR3 (taking PCR2 product as the starting sequence, Xho1 as a forward primer, and the third backward primer). **A: Post-PCR1 gel electrophoresis.** The starting sequence (GFP) is successfully elongated. **B: Post-PCR2 gel electrophoresis.** The PCR1 product is successfully elongated. **C: Post-PCR3 gel electrophoresis.** The PCR2 seems to be of higher molecular weight compared to the PCR3 product, no elongation of the PCR2 product was observed.

Hypotheses on what went wrong: We found out that the first set of primers is indeed inaccurate. We hypothesize that the last (third) primer is not able to fix correctly to the amplified DNA due to the specificity of its secondary structure. But at the same time, second and third sets of primers are designed correctly and do not lead to the right PCR3 product formation either. That’s why we still suppose that the secondary structure somehow makes the SOE PCR synthesis challenging.

Moreover, we noticed that a long particular region of the Toehold sequence is rich in GC bases. This makes the pairing of the strands very strong. Thus, we suppose that despite the opening of the DNA during a PCR cycle, the third primer does not have enough time to fixate to the corresponding place. This is what we have decided to call a competition between primers and the toehold sequence, and what can be one of the reasons for challenges we met on this step.

#### Molecular cloning using restriction enzymes

The synthesized fragments, the variable part of the toehold switch targeting miR-141-5p, are received into plasmids from IDT and amplified with 10-BETA *E. coli* bacteria transformation. Plasmids with the variable part of the toehold switch are then successfully digested overnight using BamHI and BglII restriction enzymes. **Figure 8** shows a unique band slightly smaller than 300pb, corresponding to the purified and digested fragment (between 260 to 290bp depending on the candidate).

![picture](https://static.igem.org/mediawiki/2021/4/40/T--UParis_BME--Fig8.png)
**Figure 8: Purification of the digested variable part of toehold switch targeting miR-141-5p confirmed on a gel.**  1.2% agarose gel loaded with the NEB 100bp ladder on the first well and the 6 candidates.

The assembly of the variable part of the toehold switch into the plasmid pET-24d(+)\_GFP was performed as previously but did not work as shown after sequencing of the plasmids with Eurofins Genomics (**Figure 9**).
Hypotheses on what went wrong: We obtained a very low amount (~40 ng/μL) of plasmid and insert after the purification step, too low a concentration of both components significantly decreased our chance to get the good assembly product. In addition, BglII and BamHI generate the same sticky ends, meaning that the plasmid can close itself easily. We should have used a higher concentration of plasmids and inserts and better optimized our strategy to use other restriction enzymes that do not generate the same overhangs.

![picture](https://static.igem.org/mediawiki/2021/c/cd/T--UParis_BME--wiki-results-GGtoehold.png)
**Figure 9: Sequencing of the pET-24d(+) plasmid after the ligation step does not show the correct insertion of the variable part of the toehold switch candidates targeting miRNA\_141\_5p.** Created with Snapgene.

#### Molecular cloning using golden gate assembly

In parallel with the restriction enzyme method, we worked on another method using the NEB® Golden Gate Assembly Kit (BsaI-HF®v2). With the help of Manish Kushwaha, we designed the fragments coding for the variable part of the toehold switches targeting miR-21-5p preceded by T7 promoter. iGEM Evry kindly gave us the BBa\_K3453101 as a plasmid for the golden gate assembly. The variable parts of the toehold switch are synthesized by IDT and received into a plasmid. We successfully amplified the fragment via PCR to get a high quantity of the fragments (between 100 and 200 ng/μl).
The amplified fragment was inserted into the BBa\_K3453101 plasmid using NEB® Golden Gate Assembly Kit (BsaI-HF®v2). *E.coli* was transformed with the golden gate product. Since receiving plasmid contains the Tetracycline resistance gene, bacteria were cultured overnight on kanamycin LB (Luria Broth) to select the *E.coli* who have integrated the plasmid. We obtained 200 colonies per candidate. To confirm the success of the ligation, we performed a PCR amplification on the variable part of the toehold switch fragments. **Figure 10** shows a similar band for each candidate, even for the negative control. We concluded that our construction is not a success and that none of the plasmids contains the variable part of the toehold switch. In addition, we sequenced some plasmids to the sequencing, and the results confirmed the non-assembly of the construct. None of them had the fragment cloned into the plasmid, as indicated in the gel run with PCR amplified fragment front.

![picture](https://static.igem.org/mediawiki/2021/1/1d/T--UParis_BME--Fig10.png)
**Figure 10: The ligation of the variable part of the toehold switch targeting miR-21-5p into the receiving plasmid did not worked.** 1.2% Agarose Gel run on the PCR product performed on clones that grow after the transformation with Golden Gate Protocol. Well 1 and 16 were loaded with the negative control (no fragment) and wells 2 to 15 were run with the samples.

Hypotheses on what went wrong: We tried the golden assembly only once without optimizing the protocol. We hypothesize that we may not have well designed our fragments or that the ratio fragment/plasmid must be improved.

---

## **Phase2**

## **MCF-7 and HeLa cell lines Exhibit Exosomal miRNAs**

In this part of the project, we aimed to verify that MCF-7 and HeLa cell lines are expressing abnormal quantities of exosomal miRNAs. These cell lines have been selected because they are well-characterized cancer cells, for human breast cancer cells and human cervical cancer, respectively (see our [wetlab](https://2021.igem.org/Team%3AUParis_BME/Wetlab) section for more details)5.
MCF-7 and HeLa cells were grown separately at a 70% of confluence as we can see in **Figure 11**.

![picture](https://static.igem.org/mediawiki/2021/7/7e/T--UParis_BME--wiki-results-confluency.png)
**Figure 11: HeLa cells at 70% confluency.**

#### Exosome extraction from HeLa cells and MCF-7 cells:

Extraction of small vesicles was performed on the two cell lines using a protocol based on differential centrifugation in order to obtain pure exosomes free from all types of residues and contamination.
Due to the transparency of the vesicles, a BiCinchoninic acid Assay (BCA) test has been performed to evaluate the amount of total proteins in our samples. Vesicle membrane contains proteins, thus we can correlate the concentration of proteins with the concentration of small vesicles.

**Table 1: protein concentrations in the samples of MCF-7, HeLa cells culture respectively after exosome extraction.**

| n° | Sample | Protein concentration (µg/mL) |
| 1 | MCF-7 Extract A | 555,90 |
| 2 | MCF-7 Extract B | 660,07 |
| 3 | Exosomes HeLa | 350 |

BCA results show protein concentration between 350 and 660 µg/mL, as described in Table 1. These concentrations are decent, leading to the assumption that we had vesicles.

A western blot has been conducted to confirm the presence of exosomes in the vesicle extract, using CD9+ and CD63+ as surface markers and Calnexin as a negative control (non-exosomal protein). As shown on **Figure 12**, we observed a band for the CD63+ markers for the extracts from HeLa cells but not from MCF-7 cells. The negative control was The negative control Calnexin was expressed. Calnexin is an abundant 90kDA chaperone protein that resides in the membrane of the endoplasmic reticulum, and is not supposed to be expressed in exosomes. Calnexin was detected in samples from MCF-7 cells (A and B), meaning that it may not be pure exosomes. None of the vesicle extracts exhibit CD9+. This result is not that surprising because CD9 expression on exosomes vary with the cell type and the physiological conditions6-7. CD63+ was detected but not Calnexin for the sample from HeLa cells, we concluded that the vesicle extract from HeLa cells contained exosomes.

![picture](https://static.igem.org/mediawiki/2021/f/f7/T--UParis_BME--calnexin.png)
**Figure 12: Vesicles extracted from HeLa and MCF-7 cell media.** Western blot analysis of CD9, CD63 and calnexin protein respectively in the MCF-7 and HeLa exosomes

Thus, we performed miRNAs extraction and quantification from HeLa exosomes. We first isolate miRNAs from HeLa exosome sample.

To quantify the targeted miRNA, we performed reverse transcription on the RNA sample we have and obtained the complementary DNA (cDNA). PolyA-tag was added to the miRNAs and we performed a qRT-PCR on these polyA-tagged miRNAs to quantify the miR-141-5p and miR-21-5p.

Comparison of the variation of Cq using U6 primers, specific miRNA primers (targeting miR-34a, miR-141-5p and miR-21-5p), and between water and cDNA sample was done. H2O was added in place of cDNA sample as control value.
We analyzed the difference between H2O and sample Cq, which is the number of cycles necessary to extract fluorescence from the noise of the qRT-PCR. Low Cq means high RNA quantity. U6 was the positive control of the well functionning of the qRT-PCR reaction. H2O Cq was higher than our cDNA Cq meaning that no errors have been done following the protocol. The Cq measured for miR-34a for the cDNA sample is equal to the Cq for the water. This indicates that miRNA-34a was not present in the exosomes extracted from HeLa cells, which was expected as this miRNA selected as a negative control for assessing miR-21-5p and miR-141-5p presence in cancer derived exosomes.
The Cq measured for the miR-141-5p and miR-21-5p are lower in the cDNA sample than in the water (**table 2**), meaning that these miRNAs were contained within exosomes from the HeLa cells.

**Table 2: Final results of qRT-PCR with mean Cq from each miRNA.**
We calculate the mean Cq of the triplicate of each miRNA with their primers in order to compare them with their respective blank.

| Sample | Cq(miR-141-5p) | Cq(miR21-5p) | Cq(miR34a) | Cq(miU6) |
| H2O | 29,37 | 17,65 | 25,18 | 37,17 |
| cDNA sample from Hela cells' Exosomes | 19,85 | 14,85 | 24,71 | 21,98 |

We were able to demonstrate that exosomal extracts from HeLa cell lines contain miRNAs such as miR-141-5p and miR-21-5p. However, we were not able to quantify these miRNAs. In order to find the precise concentration of our initial sample, we will need to compare our Cq value with a range of dilution of synthetic miRNA.

---

## **Perspectives**

Toehold switches exhibit a very specific secondary structure that makes their synthesis particularly difficult. Nevertheless, we are convinced that the construction of the plasmid encoding for the complete toehold switch can be successfully done with more time and protocol optimisation.
Once the plasmid encoding for the complete toehold switch is produced we will double transform BL21(DE3) *E. coli* with a miRNA (either the trigger one or a negative control with another miRNA) to demonstrate the specificity and sensibility of the toehold switch candidates.
After this step, we hope that at least one candidate per toehold switch bank targeting a specific miRNA will give satisfactory results.
We will characterize the toehold switch with a cell-free system. We will measure the fluorescence of :

* the toehold switch without trigger miRNA to confirm the toehold switch does not produce any fluorescence,
* the toehold switch with another miRNA that is not its target to confirm its specificity,
* the miRNA alone to confirm that signal was not from autofluorescent proteins produced from the miRNA
* the toehold switch with a predefined quantity of trigger miRNAs to confirm if the quantity of fluorescence is correlated with the quantity of miRNAs and how.

We hope these results will tell us if our [SwitchMi Designer](https://github.com/igemsoftware2021/UParis_BME_2021) is well able to model toehold switches to detect and quantify specific miRNAs and that we may be able to improve it.
The toehold switch that gave the best results on the characterization experiment will be tested on a cell-free system with the miRNAs extracted from our MCF-7 and HeLa cell lines previously isolated. We will compare the results given by the toehold switch with the one we obtained using the classical qRT-PCR method.

---

1. ***Yerukala Sathipati S, Ho S-Y. Identifying a miRNA signature for predicting the stage of breast cancer. Sci Rep. 2018;8(1):16138. doi:10.1038/s41598-018-34604-3***
2. ***Wang W-T, Zhao Y-N, Yan J-X, et al. Differentially expressed microRNAs in the serum of cervical squamous cell carcinoma patients before and after surgery. J Hematol Oncol. 2014;7(1):6. doi:10.1186/1756-8722-7-6***
3. ***Sabit H, Cevik E, Tombuloglu H, et al. miRNA Profiling in MCF-7 Breast Cancer Cells: Seeking a New Biomarker. Journal of Biomedical Sciences. 2019;8(2). Accessed October 20, 2021. https://www.jbiomeds.com/abstract/mirna-profiling-in-mcf7-breast-cancer-cells-seeking-a-new-biomarker-24417.html***
4. ***Li J, Zhang Z, Du M, et al. microRNA-141-3p fosters the growth, invasion, and tumorigenesis of cervical cancer cells by targeting FOXA2. Archives of Biochemistry and Biophysics. 2018;657:23-30. doi:10.1016/j.abb.2018.09.008***
5. ***Han Y, Xu G-X, Lu H, et al. Dysregulation of miRNA-21 and their potential as biomarkers for the diagnosis of cervical cancer. Int J Clin Exp Pathol. 2015;8(6):7131-7139.***
6. ***Mimori K, Mori M, Shiraishi T, et al. Expression of ornithine decarboxylase mRNA and c-myc mRNA in breast tumours. Int J Oncol. Published online March 1, 1998. doi:10.3892/ijo.12.3.597***
7. ***Reduced motility related protein-1 (MRP-1/CD9) gene expression as a factor of poor prognosis in non-small cell lung cancer. Lung Cancer. 1996;14(2-3):387. doi:10.1016/0169-5002(96)87780-4***

## Contact Us !

 : Contact us via email!

## A Special Thanks To

![sponsors](https://static.igem.org/mediawiki/2021/4/49/T--UParis_BME--UP.png)

![sponsors](https://static.igem.org/mediawiki/2021/b/bd/T--UParis_BME--BME_Master.png)

![sponsors](https://static.igem.org/mediawiki/2021/d/d3/T--UParis_BME--iGEM.png)

![sponsors](https://static.igem.org/mediawiki/2021/f/f5/T--UParis_BME--Integrated.png)

![sponsors](https://static.igem.org/mediawiki/2021/e/ea/T--UParis_BME--Promega.png)

![sponsors](https://static.igem.org/mediawiki/2021/1/15/T--UParis_BME--PCR.png)

![sponsors](https://static.igem.org/mediawiki/2021/8/8e/T--UParis_BME--New_England.png)

![sponsors](https://static.igem.org/mediawiki/2021/6/6d/T--UParis_BME--Ligue.png)

![sponsors](https://static.igem.org/mediawiki/2021/6/6c/T--UParis_BME--Frederick.png)

©2021 Copyright: iGEM UParis\_BME, author: Kévin DANG
 Source code released under MIT License
 Made using Bootstrap, jQuery and FontAwesome