iGEM 2023 — Team toulouse-insa-ups

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## Notebook

This page retraces our iGem adventures from team formation to the wiki freeze!

January
February
March
April
May
June
July
August
September
October

Week 50
Week 51
Week 52

Week 1
Week 2
Week 3
Week 4

Week 5
Week 6
Week 7
Week 8

Week 9
Week 10
Week 11
Week 12
Week 13

Week 14
Week 15
Week 16
Week 17

Week 18
Week 19
Week 20
Week 21
Week 22

Week 23
Week 24
Week 25
Week 26

Week 27
Week 28
Week 29
Week 30

Week 31
Week 32
Week 33
Week 34
Week 35

Week 36
Week 37
Week 38
Week 39

Week 40
Week 41

### Week of January 2nd to 8th

On January the 5th, we had our first meeting with the advisors and the students of the former team.
We were introduced to the competition, and last year’s team explained to us the main roles. It was decided that we would have a regular meeting every Thursday, lasting about one hour and a half during lunchtime.
Throughout the week, we discussed and decided on the ideal roles for each member of the team within the organization and reviewed the projects we worked on for our applications.

![](https://static.igem.wiki/teams/4768/wiki/notebook/january/kick-off-meeting.png)

### Week of January 9th to 15th

Roles have been assigned as follows: Organization (Cléa), Communication (Bayane), Experimentation (Pakindame), Wiki (Koloina), Human Practice (Héloïse), Finances (Mathieu), Modeling (Jeanne) and Entrepreneurship (Marie).

We also discussed three topics we found interesting to develop:

* - biological glue
* - biological light
* - oil spill treatment

From these discussions, some new ideas emerged, such as using biological light to illuminate tissue during an operation to assist the surgeon.

![Weekly zoom meeting with our advisors.](https://static.igem.wiki/teams/4768/wiki/notebook/unnamed.png "Weekly zoom meeting with our advisors")***Weekly zoom meeting with our advisors.***

### Week of January 16th to 22th

This week, we continued brainstorming on the three topics we discussed the week before. Due to exams, it was challenging to find time to explore new subjects. We started creating slideshows to present our ideas to our supervisors.

![Slideshows to present our ideas each week](https://static.igem.wiki/teams/4768/wiki/notebook/diapo.png "Slideshows to present our ideas each week")***Slideshows to present our ideas each week***

### Week of January 23th to 29th

We started looking for new ideas. We abandoned the idea about a biological glue because it had already been done.

### January 30th to February 5th

We started meeting more often every week, including weekends. We created a poster for the open day of INSA.

![](https://static.igem.wiki/teams/4768/wiki/notebook/380406883-254827790312952-7428414981021798788-n.png)

### Week of February 6th to 12th

During the regular meeting, new ideas were discussed. We also continued our discussion about using liposomes to fight cancer cells.

The first team building event was organized, where we enjoyed tartiflette, a French cooking specialty, and played board games together.

![Tartiflette savoring](https://static.igem.wiki/teams/4768/wiki/notebook/repas.png "Tartiflette savoring")***Tartiflette savoring***

On February 11th, we participated in the school's opening day to present the competition and the projects of previous teams to high school students.

![INSA's Opening day](https://static.igem.wiki/teams/4768/wiki/notebook/img-20230211-092611.jpg "INSA's Opening day")***INSA's Opening day***

We also met with Thomas Esquerré, an anesthetist at the University Hospital of Toulouse, to discuss the interest of our idea, which is using liposomes to fight cancer cells. He explained to us the methods currently used during intraoperative procedures.
You can find more information about this interview on our  [IHP page.](https://2023.igem.wiki/toulouse-insa-ups/human-practices)

![Meeting with Thomas Esquerré](https://static.igem.wiki/teams/4768/wiki/notebook/thomas.png "Meeting with Thomas Esquerré")***Meeting with Thomas Esquerré***

### Week of February 13th to 19th

We came close to choose the liposome topic but decided to keep the possibility of finding new ideas for one last week. We discussed the molecular aspect of the project and divided it into three parts to facilitate the research:

* - the binding to cancer cells
* - the signal transduction
* - the production of anticancer drugs

The first meeting to begin organizing a French meet-up in Toulouse was held. The BioEco graduate school is partnering in this project. We got in touch with iGEM Nantes to explore the possibility of collaborating to organize this event.

### Week of February 20th to 26th

This week marks the  **final selection** of our topic on the use of liposomes to kill cancer cells! We continued our research on the molecular aspect of our project, as well as on current treatments for cancer and their limitations.

![](https://static.igem.wiki/teams/4768/wiki/notebook/ezgif-com-optimize.gif)

The first meeting with iGEM Nantes took place, and we decided to collaborate between our teams to organize this mini-jamboree in Toulouse.

![Meeting with iGEM Nantes](https://static.igem.wiki/teams/4768/wiki/notebook/reu-coorga-meet-up-igem-nantes-24-02-23.png "Meeting with iGEM Nantes")***Meeting with iGEM Nantes***

### Week of February 27th to March 5th

We met with Jean-Pascal Capp, a teacher-researcher at Toulouse Biotechnology Institute who used to work in the field of cancer. He confirmed some of our choices regarding our system and provided us with new leads for anticancer drugs.

![Meeting with Jean-Pascal Capp](https://static.igem.wiki/teams/4768/wiki/notebook/img-3799.jpeg "Meeting with Jean-Pascal Capp")***Meeting with Jean-Pascal Capp***

We listed all the different molecular possibilities for each part of our project and created diagrams to illustrate our ideas.

![](https://static.igem.wiki/teams/4768/wiki/notebook/sch-ma.png)

### Week of March 6th to 12th

We met with Dr Ilfad Blazevic, an oncologist in IUCT-Oncopole at Toulouse to have his expertise on our project. During the meeting, we also gained more insights into the various types of treatments for cancer and their limitations.

Please, consult our  [IHP page](https://2023.igem.wiki/toulouse-insa-ups/human-practices)  to get more information regarding this interview.

![Meeting with Ilfad Blazevic](https://static.igem.wiki/teams/4768/wiki/notebook/img-3775.jpeg "Meeting with Ilfad Blazevic")***Meeting with Ilfad Blazevic***

We also started reflecting on a project name.

### Week of March 13th to 19th

A second team building event was organized with our advisors, and everyone showcased their cooking skills!

![](https://static.igem.wiki/teams/4768/wiki/notebook/repas-encadrants-13-03.jpg)

### Week of March 20th to 26th

This week, we had a meeting with iGEM Nantes and Eur BioEco to finalize the date of the meetup! It has been scheduled for the 22nd and 23rd of June.

![Meeting with iGEM Nantes and Eur BioEco](https://static.igem.wiki/teams/4768/wiki/notebook/r-union-meet-up-21-03-2023.png "Meeting with iGEM Nantes and Eur BioEco")***Meeting with iGEM Nantes and Eur BioEco***

We also found the name for our project: **CALIPSO** which stands for Cell-targeting Advanced LIPosomes for Selective Oncotherapy.

![](https://static.igem.wiki/teams/4768/wiki/notebook/titre.png)

### Week of March 27th to April 2nd

This week marked the start of our regular meetings with the iGEM Nantes team to organize the meetup. We discussed the event's outline, including the welcoming speech, conference, and mini-jamboree.

![Meeting with iGEM Nantes](https://static.igem.wiki/teams/4768/wiki/notebook/meeting-nantes.png "Meeting with iGEM Nantes")***Meeting with iGEM Nantes***

We also decided to focus our human practice approach on **patients** to better understand their living conditions.

Additionally, we began discussing the genetic constructions we would need for our experiments.

### Week of April 3rd to 9th

We had a meeting with Elvire Prochilo, an entrepreneurship and management consultant, who provided valuable guidance on conducting our entrepreneurship project. She gave us advice on the business plan and other aspects.

In addition, we created a draft for our logo.

![](https://static.igem.wiki/teams/4768/wiki/notebook/2-2.png)
![](https://static.igem.wiki/teams/4768/wiki/notebook/4.png)
![](https://static.igem.wiki/teams/4768/wiki/notebook/6.png)

### Week of April 10th to 16th

We started familiarizing ourselves with Benchling and searched for the protocols.

Additionally, we designed an Instagram post for the meetup and reached out to the French teams to inform them about the event.

![](https://static.igem.wiki/teams/4768/wiki/collaborations/post-pr-sentation-meet-up-1.png)

### Week of April 17th to 23th

This week, we finalized our modeling strategy, aiming to focus on both the structural and functional aspects of the project.

Visit our  [modeling overview page](https://2023.igem.wiki/toulouse-insa-ups/model)  to find out more!

We also had a team building meal together at a restaurant!

![Burger savouring](https://static.igem.wiki/teams/4768/wiki/notebook/l-atelier-du-burger.jpg "Burger savouring")***Burger savouring***

### Week of April 24th to 30th

This week marks the official beginning of the iGEM adventure with the team’s registration for the competition!

It has been an intense week of work as we started to design our biobricks on Benchling to ensure we can order them on time.
We also prepared a list of everything we would need for the experimental part of our project and secured sponsorship from GeneFrontier to provide us with the PURE system solution.

During this month, we secured sponsorship from the Toulouse Biotechnology Institute (TBI) and Toulouse White Biotechnology (TWB)! We also initiated contact with various companies in France, although we received several negative responses.

![](https://static.igem.wiki/teams/4768/wiki/notebook/gfc-logo-220x220px.png)
![](https://static.igem.wiki/teams/4768/wiki/notebook/tbi-logo.png)
![](https://static.igem.wiki/teams/4768/wiki/notebook/twb-logo-verti-cmjn.png)

***Logos of our new sponsors***

### Week of May 1st to 7th

We started placing orders for certain products, such as lipids, and continued the process of designing our biobricks.
Additionally, we created diagrams to visualize all the steps required to demonstrate the functionality of our system.

Perseo Pharma announced its sponsorship for our project!

![](https://static.igem.wiki/teams/4768/wiki/notebook/perseo-pharma-logo.png)

### Week of May 8th to 14th

We met Philippe Urban, an expert in protein biochemistry, who assisted us in using AlphaFold to determine the structure of the T7 split.

We began to prepare for Exposciences, a scientific event aimed at popularizing science to young people aged 5 to 25 years old. For our participation, we created two posters: one explaining synthetic biology and another detailing different types of treatments. We also prepared activities to explain the fundamentals of synthetic biology, such as transcription and translation. See explanations of the game in our  [Education and Communication page](https://2023.igem.wiki/toulouse-insa-ups/education)

![](https://static.igem.wiki/teams/4768/wiki/notebook/jeu-exposcience-draft-v2-pptx.jpg)

### Week of May 15th to 21th

We had a meeting with a teacher-researcher from our school, Jean-Pascal Capp, to gather more information about cell culture and general safety protocols. We discussed which cell lines would be suitable for our experiments and how to handle them safely.

### Week of May 22th to 28th

Our collaboration with the Catalyseur of Toulouse, a business incubator, has started! We had a meeting with Colette Schenker, who is in charge of the support program for entrepreneurs and start-ups. She provided us with valuable advice to improve our presentation skills.

We also ordered our gBlocks for our parts from IDT!

![](https://static.igem.wiki/teams/4768/wiki/notebook/idt-logo.png)

### Week of May 29th to June 4th

This was the final week of our classes.

On Saturday, we took part in Exposciences. It was a great opportunity to learn how to present our project to the general public. We also recorded an episode of the podcast "Pose ta science" with Radio Francas. You can find more information about this event in our  [Education and Communication page](https://2023.igem.wiki/toulouse-insa-ups/education)

![](https://static.igem.wiki/teams/4768/wiki/notebook/img-20230603-wa0004.jpg)
![](https://static.igem.wiki/teams/4768/wiki/notebook/img-20230603-wa0005.jpg)
![](https://static.igem.wiki/teams/4768/wiki/notebook/img-20230603-wa0020.jpg)

***Photos taken during Exposciences***

### Week of June 5th to 11th

This week marked the beginning of our internship! It was a busy week as we received materials for our experiments, organized our lab and underwent security training for the use of autoclaves.

![First day of lab](https://static.igem.wiki/teams/4768/wiki/notebook/img-8531.jpg "First day of lab")***First day of lab***

On Tuesday, we pitched our project during the annual Comité of the Catalyseur to other entrepreneurs.

![](https://static.igem.wiki/teams/4768/wiki/notebook/img-20230606-wa0011.jpg)
![](https://static.igem.wiki/teams/4768/wiki/notebook/img-20230606-wa0023.jpg)

***Oral presentation at the Catalyseur***

We also spent the week calling companies and public organizations to raise funds. Unfortunately, many of our emails went unanswered.

### Week of June 12th to 18th

To be more efficient in lab, we divided the different responsibilities as follows:

* - Cloning: Pakindame
* - Pure system: Mathieu
* - Liposome preparation: Bayane
* - Cell culture: Jeanne
* - Expression and purification of DhdR: Cléa
* - Expression and purification of anti-Her2 nanobodies: Héloïse
* - orders: Koloina/Marie

We started the cloning process for one construction.

***Progression of our constructions***

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | **DhdR** | **sfGFP** | **TYPH** | **Trastuzumab** | **Pertuzumab** |
| **PCR** |  | X |  |  |  |  |
| **Gel migration** |  | X |  |  |  |  |
| **DpnI treatment** |  | X |  |  |  |  |
| **PCR clean-up** |  | X |  |  |  |  |
| **In-Fusion** |  | X |  |  |  |  |
| **Transformation** |  | X |  |  |  |  |
| **PCR screening** |  | X |  |  |  |  |
| **Inoculation** |  |  |  |  |  |  |
| **Miniprep** |  |  |  |  |  |  |
| **Enzymatic digestion** |  |  |  |  |  |  |
| **Sequencing** |  |  |  |  |  |  |

The Oncopole celebrated its 100th anniversary during which we discussed our project with professionals and made contacts for our IHP.

We finally selected our project’s logo and graphical charter!

![](https://static.igem.wiki/teams/4768/wiki/notebook/logo-1.png)
![](https://static.igem.wiki/teams/4768/wiki/notebook/logo-2.png)

### Week of June 19th to 25th

The cloning process continued.

***Progression of our constructions***

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | **DhdR** | **sfGFP** | **TYPH** | **Trastuzumab** | **Pertuzumab** |
| **PCR** | X | X | X |  |  |  |
| **Gel migration** | X | X | X |  |  |  |
| **DpnI treatment** | X | X | X |  |  |  |
| **PCR clean-up** | X | X | X |  |  |  |
| **In-Fusion** | X | X | X |  |  |  |
| **Transformation** | X | X | X |  |  |  |
| **PCR screening** | X | X |  |  |  |  |
| **Inoculation** |  | X |  |  |  |  |
| **Miniprep** |  | X |  |  |  |  |
| **Enzymatic digestion** |  | X |  |  |  |  |
| **Sequencing** |  |  |  |  |  |  |

This was a really intense week as we had to handle the last-minute details of the meetup.
The meetup took place from Thursday to Friday and everything went well. It was a great opportunity to exchange with other iGEM teams and learn more about their projects.
Visit ourcollaborations page to learn about the course of this event!

![](https://static.igem.wiki/teams/4768/wiki/collaborations/img-4732.jpg)

### Week of June 26th to July 2nd

As researching sponsors proved to be very challenging, we launched our crowdfunding campaign to raise additional funds ([see our Acknowledgment page](https://2023.igem.wiki/toulouse-insa-ups/acknowledgement)).

![](https://static.igem.wiki/teams/4768/wiki/notebook/cagnotte.png)

### Week of July 3rd to 9th

Experimentally, we initiated the production and purification process for DhdR. Additionally, we discussed and tested various strategies for cloning the T7 constructs.

We pitched our project at the Summer Tech day, an event that brings together entrepreneurs and investors.

![Pitch at Summer Tech day](https://static.igem.wiki/teams/4768/wiki/notebook/french-tech-2.jpg "Pitch at Summer Tech day")***Pitch at Summer Tech day***

Finally, we organized several meetings with healthcare personnel as part of our human practice approach. For instance, we met with two advanced practice nurses, Kristell Thomas and Alexandra Lambert, as well as Anne Gauci, a healthcare manager. Please consult our [IHP page](https://2023.igem.wiki/toulouse-insa-ups/integrated-human-practices) to learn more about these interviews.

### Week of July 10th to 16th

We wrote the storyboard of our promotion video and produced drawings for the animation.

![](https://static.igem.wiki/teams/4768/wiki/notebook/video.png)

### Week of July 17th to 23th

We began the production and purification process for anti-Her2 nanobodies while the cloning for the T7 constructs remained ongoing. We restarted the production and purification of DhdR, and it was successful! See our results [here](https://2023.igem.wiki/toulouse-insa-ups/module2).

We also presented our project at the BioSynSys Symposium held on the campus of INSA Toulouse. This provided us with an opportunity to receive feedback from the scientific community regarding our project.

Finally, we learned more about intellectual property by discussing it with Philippe Lucas from the INPI (National Institute of Industrial Property).

### Week of July 24th to 30th

We attempted, for the first time, to measure the repression of DhdR on the expression of sfGFP in the PURE system. We also determined the concentration of the produced nanobodies.

In addition, we booked the train and the accommodation for our trip to the Jamboree.

Finally, we began filming our project promotion video!

![](https://static.igem.wiki/teams/4768/wiki/notebook/video-2.png)

### Week of July 31th to August 6th

This week was different because we didn’t have access to our lab. As a result, we spent the week writing our wiki and strategizing for entrepreneurship. We enjoyed “sweet” breaks to recharge our energy.

![](https://static.igem.wiki/teams/4768/wiki/notebook/cookie.jpg)

### Week of August 7th to 13th

We finally received our t-shirts!

A perfect timing since a journalist came to our laboratory to write a report about us.
Find the link to the article on [our education and communication page](https://2023.igem.wiki/toulouse-insa-ups/education).

![](https://static.igem.wiki/teams/4768/wiki/notebook/tee-shirt.jpg)

Experimentally, we restarted the cloning process for the T7 constructs using the pET21a (+) instead of the p05-79 and p05-39 plasmids.
We also conducted tests using various concentrations of DhdR on the G-block containing the GFP.

### Week of August 14th to 20th

Experimentally, we finally succeeded in cloning the Pertuzumab antibody linked to the Nterm of the split T7. We attempted to produce this complex in the Puresystem.

Simultaneously, we were in contact with the student responsible for creating the video animation, Oscar Baron. Additionally, we also received the first draft of the video's musical composition from Léo Vendeville.

### Week of August 21th to 27th

We shot several scenes for our video including those captured with a drone.

Many meetings were held to discuss our entrepreneurship and HP strategies, as many people's holidays were coming to an end. We had the opportunity to make progress on our educational workshop.

We also had our first meeting with the Patras-Medicine iGEM team to discuss how we could collaborate together. It was so nice meeting another team and presenting our respective project!

![](https://static.igem.wiki/teams/4768/wiki/notebook/patras.png)

### Week of August 28th to September 3rd

Experimentally, we successfully prepared a lipid film hydration for the first time using lipid-coated beads. We also cloned the SP6 promoter on the rapamycin construction. Finally, we conducted tests to detect the pro-drug and drug using HPLC.

For our promotional video, we recorded the voiceover and completed the editing.

### Week of September 4th to 10th

For the first time, we tried to visualize liposomes expressing sfGFP by fluorescence microscopy. We also succeeded in expressing the anti-rapamycin T7 RNA split polymerase in the PURE system. We planned the remaining experiments to be conducted by the end of the internship by creating diagrams.

![](https://static.igem.wiki/teams/4768/wiki/notebook/sch-ma-12.jpg)

### Week of September 11th to 17th

Experimentally, we conducted toxicity tests with 5-FU and Tegafur on cancer cells. We also assessed the impact of Tegafur on the synthesis of sfGFP in the PURE system. Additionally, we attempted to demonstrate the activity of the anti-rapamycin T7 split in the PURE system.

We started creating the storyboard for our presentation video and put a lot of effort into writing the content for our wiki.

Finally, we tested our game with some friends to further refine the concepts and format.
You can find more information on the game in our  [education and communication page](https://2023.igem.wiki/toulouse-insa-ups/education).

![](https://static.igem.wiki/teams/4768/wiki/notebook/jsp.jpg)

### Week of September 18th to 24th

We visualized the anchoring of liposomes decorated with anti-HER2 nanobodies to Caco-2 cancer cells! To achieve this, we utilized the facilities at LITC (Light Imaging Toulouse CBI) at the CBI (Centre de Biologie Intégrative).

We also decided to compete for the best measurement, best model and best entrepreneurship prizes.

Additionally, we conducted our synthetic biology workshop at a junior high school, where the students were receptive to our explanations and appreciated the experiments!

![](https://static.igem.wiki/teams/4768/wiki/notebook/o-talent.jpg)

### Week of September 25th to October 1st

This week concluded our time in the lab, indicating the end of our internship. As a result, we carried out our final experiments, which included toxicity tests of 5-FU on cancer cells and producing liposomes. Notably, some of these liposomes demonstrated the ability to express the sfgfp gene after incubation in a culture medium with cancer cells!

![Last day of lab](https://static.igem.wiki/teams/4768/wiki/notebook/fin-stage.jpg "Last day of lab")***Last day of lab***

Thanks to our collaboration with Etincelles association, we were able to meet with patients and host our workshop called “Biology to better understand one’s illness”. This workshop proved to be invaluable for both the patients and ourselves.

![](https://static.igem.wiki/teams/4768/wiki/notebook/atelier.jpg)

### Week of October 2nd to 8th

This week marks the beginning of the school year, which meant we had less time to dedicate to our project. It was a demanding week as we needed to finish our wiki and complete the registry.

In parallel, we filmed our presentation video.

![](https://static.igem.wiki/teams/4768/wiki/notebook/tournage.jpg)

### Week of October 9th to 15th

We checked one last time every detail of the wiki and the registry to be ready for the wiki freeze and started to prepare the final presentation. Thank you for reading the entire Notebook. As a reward, here is one of our most recent photos of us enjoying a meal (for a change)!

![](https://static.igem.wiki/teams/4768/wiki/notebook/repas-fin.jpg)

[![](https://static.igem.wiki/teams/4768/wiki/description/previous-button.png)](https://2023.igem.wiki/toulouse-insa-ups/acknowledgement)

[![](https://static.igem.wiki/teams/4768/wiki/description/up-button-removebg-preview.png)](#top)

[![](https://static.igem.wiki/teams/4768/wiki/description/next-button.png)](https://2023.igem.wiki/toulouse-insa-ups/contribution)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

![Menu aside](https://static.igem.wiki/teams/4197/wiki/description/sort-descending.svg)

* Design and cell-free expression of a two-partite RNA polymerase-linked antibody
  + Cycle I: Constructing a HER2 biosensor with the split T7 RNA polymerase
  + Cycle II: Constructing a rapamycin biosensor based on the split T7 RNA polymerase

* Functional 2-hydroxyglutarate biosensor
  + Cycle III: Implementation of a biosensor capable of detecting physiological concentrations of 2-hydroxyglutarate

## Engineering success

Synthetic biology aims at creating and assembling novel biological components, mechanisms, and structures, along with modifying pre-existing natural biological systems for practical use (Nature, 2021). Just like in other branches of engineering, users of synthetic biology adhere to a cyclical approach known as the DBTL cycle (Design, Build, Test, Learn), which is essential for the effective development of novel or improved biological systems.

In CALIPSO, we made use of synthetic biology to engineer next-generation liposomes capable of specifically producing and delivering an anticancer drug next to cancer cells. Building the biosensing module has been at the core of our engineering approach, which we will present on this page. You can find the detailed design of CALIPSO on the [design page](https://2023.igem.wiki/toulouse-insa-ups/design), and the detailed experimental results on the [Module 2: Biosensing systems page.](https://2023.igem.wiki/toulouse-insa-ups/module2)

Design and cell-free expression of a two-partite RNA polymerase-linked antibody

## Cycle I: Constructing a HER2 biosensor with the split T7 RNA polymerase

We decided to engineer a heterodimer consisting of the split-T7 RNA polymerase (two subunits), coupled to anti-HER2 antibodies via a single transmembrane domain. The binding of the two antibodies to distinct epitopes of HER2 not only provides a means to anchor the liposome to the surface of cancer cells, it also enables complementation of the two split T7 RNA polymerase subunits that are facing the opposite side of the membrane. The functionally assembled T7 RNA polymerase can then trigger the expression of the thymidine phosphorylase gene internally.

####

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/design.png)

### **DESIGN**

When we decided to use the split-T7 RNA polymerase as a biosensor, the main challenge appeared to design a chimeric heterodimer harboring two functionalities: an HER2-binding element and a transcriptional activator. Importantly, the two anti-HER2 antibodies had to recognize different epitopes of the receptor, and binding to the target should enable complementation of a functional RNA polymerase, which imposes strong design constraints.

We opted to construct two plasmids, each containing a different antibody against HER2 fused to one of the two fragments of the split-T7 RNA polymerase. Using soluble linkers, Trastuzumab was linked to the C-terminal part of the polymerase (**Trastuzumab-SL-T7Cterm**), corresponding to the part [BBa\_K4768006](http://parts.igem.org/Part%3ABBa_K4768006), while Pertuzumab was linked to the N-terminal part (**Pertuzumab-SL-T7Nterm**), corresponding to the part [BBa\_K4768005](http://parts.igem.org/Part%3ABBa_K4768005).

![](https://static.igem.wiki/teams/4768/wiki/pertu-trastu/sl-trastu.png)***part BBa\_K4768006***

![](https://static.igem.wiki/teams/4768/wiki/pertu-trastu/sl-pertu.png)***part BBa\_K4768005***

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/test.png)

### **BUILD & TEST**

We first tried to construct them by In-Fusion molecular cloning using p0539\_Trastuzumab-SL-T7Cterm and p0579\_Pertuzumab-SL-T7Nterm with native p0539 and p0579 plasmids, and the two antibody sequences ordered from IDT.

* - For **p0539\_Trastuzumab-SL-T7Cterm**, two inserts had to be cloned into plasmid p0539 by In-Fusion to obtain Trastuzumab linked to the C-term fragment of the T7 polymerase. A PCR was first performed to amplify the two gBlocks and linearize the plasmid p0539. The two amplified gBlocks were then cloned by In-fusion for simultaneous insertion into the linearised plasmid. After transformation and colony screening, no recombinant clones were obtained.
* - For **p0579\_Pertuzumab-SL-T7Nterm**, PCR was performed to amplify the gBlock and linearise the native p0579 plasmid. In-Fusion was then carried out to assemble the insert and the linearized plasmid. After transformation and colony screening, no recombinant clones were obtained.

For both constructs, the In-Fusion reaction was attempted again, this time by considering the following aspects:

* - For **Trastuzumab-SL-T7Cterm**, we hypothesized that the primers used for the PCR screening did not work due to unspecified hybridation.
* - For **Pertuzumab-SL-Nterm**, as the homology regions respected all recommendations from Takara, we hypothesized that the size of the linearized vector was relatively small in comparison with that of the insert (respectively 2391 bp and 2087 bp). This could have led to a poor assembly efficiency.

Therefore, we switched the plasmid vector used for In-Fusion to pET21:

* - For **pET21\_Trastuzumab-SL-T7Cterm**, the In-Fusion step with the two gBlocks failed again.
* - For **pET21\_Pertuzumab-SL-NtermT7**, the In-Fusion assembly was successful.

New sets of primers were ordered to allow the In-Fusion assembly with pET21 for both constructs.

For production in PURE system, we do not necessarily need a plasmid. Linear DNA can be used as a template for gene expression. Hence, we directly amplified the gBlock for **Pertuzumab-SL-T7Nterm** and used the PCR product as a template for expression in PURE system. Protein gel analysis suggested that the full-length protein was successfully produced.

![](https://static.igem.wiki/teams/4768/wiki/module-2/pertu-image-1.png)***Figure 1: SDS-PAGE (10% polyacrylamide gel) of Pertuzumab-linker-T7Nterm reaction tube and visualization by Instant Blue coloration. The expected band is visible at 69 kDa.** PUREfrex 2.0 was used. SP6 RNAP was added in the negative control (T-) and Reaction.*

As an alternative to In-Fusion, we attempted to assemble the two inserts for Trastuzumab-Cterm by overlap-PCR.  **No complete PCR product was obtained.**

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/learn.png)

### **LEARN**

We conclude from these experiments that, despite the structural complexity of such chimeric proteins, it was possible to produce one of the two two-partite RNA polymerase-linked antibody proteins with PURE system. However, we repeatedly failed to generate the second construct, which prevented us from running functional assays. We therefore had to change our strategy to demonstrate the feasibility of using the split-T7 RNA polymerase for biosensing with PURE system.

## Cycle II: Constructing a rapamycin biosensor based on the split T7 RNA polymerase

### **DESIGN**

Inspired by a previous study, where the split-T7 RNA polymerase was fused to anti-rapamycin antibodies for rapamycin-induced gene expression ([Bryan C. Dickinson *et. al*](https://www.nature.com/articles/nchembio.2299)), we decided to modify the reported constructs for compatibility with PURE system.

**We designed two new parts** with FKBP and FRB anti-rapamycin antibodies, respectively linked to the C-terminal (FKBP-SL-T7)Cterm and N-terminal (T7Nterm-SL-FRB) parts of the split-T7 RNA polymerase. Both constructs have optimized regulatory sequences for expression in PURE system. Moreover, the two genes are under control of an SP6 promoter to allow for orthogonal transcriptional regulation of the biosensing and reporting genes. We ordered the corresponding transcription cassettes as gBlocks. ***See the parts [BBa\_K4768009](http://parts.igem.org/Part%3ABBa_K4768009) and [BBa\_K4768010](http://parts.igem.org/Part%3ABBa_K4768010).***

![](https://static.igem.wiki/teams/4768/wiki/pertu-trastu/sl-frb.png)***part BBa\_K4768009***

![](https://static.igem.wiki/teams/4768/wiki/pertu-trastu/sl-fkbp.png)***part BBa\_K4768010***

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/design.png)

### **BUILD & TEST**

We performed a two-step PCR on the two gBlocks, and used the products as templates for expressing the two recombinant proteins in PURE system. To enable functional expression of disulfide bond-containing proteins, as it is so for the majority of antibodies, we chose to use the PURE*frex*2.1 kit. Co-translational labeling with Green-Lys, followed by SDS-PAGE analysis indicated successful biosynthesis of  **FKBP-SL-T7Cterm and T7Nterm-SL-FRB**. Activity assays using a T7-promoter-based reporter gene suggested that, in the presence of the chaperone GroE during transcription-translation, our biosensor is responsive to rapamycin (see **[Module 2 : Biosensing systems](https://2023.igem.wiki/toulouse-insa-ups/module2)**).

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/fig3-eng-succ.png)***Figure 2: SDS-page migration of FKBP-SL-T7Cterm and T7Nterm-SL-FRB on 13% acrylamide gel, stain-free and Pro-Q Emerald green revelations merged together.** DhfR is visible at 18 kDa (lane 2), FKBP-SL-T7Cterm at 91 kDa (lane 3) and T7Nterm-SL-FRB at 32 kDa (lane 4).*

![](https://static.igem.wiki/teams/4768/wiki/module-2/rapamycine-5.png)***Figure 3: Normalized fluorescence intensity of the anti-rapamycin biosensor when the biosensor was produced in presence of GroE.** PURE*frex* 2.1 custom kit was used. Fluorescence was measured at 513 nm. The fluorescence with rapamycin was normalized using the sfGFP signal for a native (full-length) T7 RNAP and with rapamycin. The fluorescence without rapamycin was normalized using the sfGFP signal for a native T7 RNAP and without rapamycin.*

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/test.png)

### **LEARN**

We conclude that it is possible to produce a split-T7 RNA polymerase linked to an antibody for ligand-induced protein expression **in PURE system**. However, the inherent limitation of the rapamycin biosensor is that it does not respond to cancer biomarkers and can only serve as a proof-of-concept system. Therefore, we next focused on the development of a biosensor that responds to an oncometabolite that is found at higher concentrations in the microenvironment of cancer cells.

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/learn.png)

Functional 2-hydroxyglutarate biosensor

## Cycle III: Implementation of a biosensor capable of detecting physiological concentrations of 2-hydroxyglutarate

##### Driven by the objective to trigger the biosynthesis of the thymidine phosphorylase only in the tumor’s vicinity, we engineered a biosensing strategy based upon the detection of the oncometabolite 2-hydroxyglutarate (2-HG), which is recognized by the transcriptional repressor DhdR from *Achromobacter denitrificans*.

####

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/design-green.png)

### **DESIGN**

We designed two plasmids: one for the expression and purification of DhdR, the other containing the DhdR binding site (*dhdO*) upstream of the gene encoding for the reporter sfGFP. The plasmid harboring the gene encoding for the Thymidine phosphorylase (*tymp*) in place of *sfGFP* was also prepared but is not described on this page **(see [Cloning results](https://2023.igem.wiki/toulouse-insa-ups/cloning-results), [Module 3: Anticancer drug in situ production](https://2023.igem.wiki/toulouse-insa-ups/module3) and see the parts [BBa\_K4768000](http://parts.igem.org/Part%3ABBa_K4768000) and [BBa\_K4768001](http://parts.igem.org/Part%3ABBa_K4768001)).**

![](https://static.igem.wiki/teams/4768/wiki/parts/part-000.png)***part BBa\_K4768000***

![](https://static.igem.wiki/teams/4768/wiki/parts/part-001.png)***part BBa\_K4768001***

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/build-green.png)

### **BUILD**

DhdR was produced and purified after transformation of *E. coli* BL21 strain. Plasmids containing *sfgfp* under a T7-*dhdO* inducible promoter were successfully cloned and minipreped from Stellar competent cells.

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/test-green.png)

### **TEST**

* 1. We first proved that DhdR is capable of binding to *dhdO*, which inhibits expression of sfGFP in PURE system.
* 2. We determined the minimal concentration of DhdR required for strong repression. The tested concentrations were derived from the kinetical model, which was recalibrated afterward.
* 3. We showed that 2-HG de-represses transcription of DhdR-bound DNA in a concentration-dependent manner, and that our biosensor is sensitive to physiological concentrations of 2-HG.

![](https://static.igem.wiki/teams/4768/wiki/module-2/dhdr-results-4.jpg)***Figure 4: Effect of different concentrations of 2-HG on DhdR repression.** PUREfrex2.1 was used. The intensity value of sfGFP without DhdR and with 10 µM 2-HG was used for normalization. Each concentration was corrected taking into account the inactivation effect of 2-HG on PURE system.*
* 4. We finally sought to demonstrate that liposomes loaded with the 2-HG biosensor could interact with 2-HG-producing cancer-cells cultured in the laboratory. Specifically, we wanted to know if our liposomes could anchor to tumor cells and express sfGFP at the same time. Liposomes functionalized with anti-HER2 nanobodies and folate molecules for specific anchoring were prepared. Preliminary results showed colocalization of GFP-producing liposomes with cultured Caco2 cells ([Best Measurement page](https://2023.igem.wiki/toulouse-insa-ups/measurement)).

![](https://static.igem.wiki/teams/4768/wiki/registry/part-dhdr-fig-9.png)***Figure 5: Fluorescence microscopy images of anti-HER2-nb- and folate-decorated liposomes on top of Caco2 cells (sample 2). a) Imaging of Caco2 cells in brightfield. b) Imaging of a liposome localized with a fluorescent membrane dye. c) Imaging of the same liposome in the GFP channel.***

![](https://static.igem.wiki/teams/4768/wiki/engineering-success/learn-green.png)

### **LEARN**

This engineering cycle allowed us to create a functional 2-HG biosensor based on the DhdR transcriptional repressor in PURE system. This biosensor was capable of responding to physiological concentrations of the oncometabolite in bulk reactions and could be loaded in liposomes targeting cancer cells. In the course of these experiments, we also showed that repression was stronger when using a linear DNA template vs. a plasmid and that 2-HG can partly inhibit gene expression with PURE system.

Conclusion

##### These three engineering cycles allowed us to design, build and test two different biosensing systems. We learned that it is possible to produce a split-T7 RNA polymerase linked to an antibody for ligand-induced protein expression in PURE system. We obtained promising results concerning the functionality of the two-partite RNA polymerase-linked antibody anti-rapamycin we produced in PURE system. The next challenge is thus to produce a biosensing system based on the split-T7 RNA polymerase capable of responding to cancer biomarkers. The final cycle allowed us to obtain a 2-HG biosensor sensitive to physiological concentrations of 2-HG. We went as far as testing our 2-hydroxyglutarate biosensor in liposomes anchored to cancer cells. Although further experiments need to be performed to validate the full spectrum of the liposomes' activity, the outcome of this engineering process constitutes a major step towards the development of intelligent anticancer liposomes.

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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).

## Inspirations

---

* [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)

{% endblock %}