iGEM 2015 — Team Toulouse

# Team:Toulouse/Notebook

iGEM Toulouse 2015

* [**Home**](https://2015.igem.org/Team%3AToulouse)
  + [Context](https://2015.igem.org/Team%3AToulouse#context)
  + [Achievements](https://2015.igem.org/Team%3AToulouse#achievements)* [**Team**](https://2015.igem.org/Team%3AToulouse/Team)
    + [Presentation](https://2015.igem.org/Team%3AToulouse/Team)
    + [Gallery](https://2015.igem.org/Team%3AToulouse/Gallery)
    + [Fun Facts](https://2015.igem.org/Team%3AToulouse/FunFacts)
  * [**Project**](https://2015.igem.org/Team%3AToulouse/Description)
    + [Project: Description](https://2015.igem.org/Team%3AToulouse/Description)
    + [Design: Attract](https://2015.igem.org/Team%3AToulouse/Description/Attract)
    + [Design: Eradicate](https://2015.igem.org/Team%3AToulouse/Description/Eradicate)
    + [Design: Regulate](https://2015.igem.org/Team%3AToulouse/Description/Regulation)
    + [Device: TrapiColi](https://2015.igem.org/Team%3AToulouse/Design)
  * [**Results**](https://2015.igem.org/Team%3AToulouse/Results)
    + [Attract](https://2015.igem.org/Team%3AToulouse/Results#attract)
    + [Eradicate](https://2015.igem.org/Team%3AToulouse/Results#eradicate)
    + [Regulate](https://2015.igem.org/Team%3AToulouse/Results#regulate)
    + [Containment](https://2015.igem.org/Team%3AToulouse/Results#TPX)
    + [Growth tests](https://2015.igem.org/Team%3AToulouse/Results#biolo)
    + [Cytoxicity](https://2015.igem.org/Team%3AToulouse/Results#tox)
  * [**Parts**](https://2015.igem.org/Team%3AToulouse/Parts)
    + [New BioBricks](https://2015.igem.org/Team%3AToulouse/Parts#biobricks)
    + [Module: Attract](https://2015.igem.org/Team%3AToulouse/Parts#1)
    + [Module: Eradicate](https://2015.igem.org/Team%3AToulouse/Parts#2)
    + [Module: Regulate](https://2015.igem.org/Team%3AToulouse/Parts#3)
  * [**Modeling**](https://2015.igem.org/Team%3AToulouse/Modeling)
    + [Metabolic networks](https://2015.igem.org/Team%3AToulouse/Modeling#part1)
    + [FBA](https://2015.igem.org/Team%3AToulouse/Modeling#part2)
    + [Diffusion](https://2015.igem.org/Team%3AToulouse/Modeling#part5)
    + [Optimisation](https://2015.igem.org/Team%3AToulouse/Modeling#part6)
  * [**Human Practice**](https://2015.igem.org/Team%3AToulouse/Practices)
    + [Meetups & Collaborations](https://2015.igem.org/Team%3AToulouse/Collaborations)
    + [Ethics & IP](https://2015.igem.org/Team%3AToulouse/Practices#Ethics)
    + [Education](https://2015.igem.org/Team%3AToulouse/Practices#Education)
    + [Safety](https://2015.igem.org/Team%3AToulouse/Practices#Safety)
  * [**Wet lab**](https://2015.igem.org/Team%3AToulouse/Notebook)
    + [Notebook](https://2015.igem.org/Team%3AToulouse/Notebook)
    + [Protocols](https://2015.igem.org/Team%3AToulouse/Experiments)
  * [**Attributions**](https://2015.igem.org/Team%3AToulouse/Attributions)
    + [Attributions](https://2015.igem.org/Team%3AToulouse/Attributions)
    + [Supports](https://2015.igem.org/Team%3AToulouse/Supports)

* [- Domestic bees](#domBee)
* [- Collapse of bees](#CCD)
* [*- Varroa destructor*](#varroa)
* [- Outbreak of  *Varroa destructor*](#outB)
* [- Ways to fight *Varroa destructor*](#fight)

### Notebook

![](https://static.igem.org/mediawiki/2015/6/67/TLSE_BG.png)

[### Calendar - 2015](#main2)

* [January](#part1)
* [From February to May](#part2)
* [May](#part3)
* [June](#part4)
* [July](#part5)
* [August](#part6)
* [September](#part7)

---

### January

During this period, we were informed by iGEM toulouse instructors of what this competition is, and what was expected of us.

### From February to May

### 02/02

First team meeting, our instructors informed us on the competition more specifically and how the team is organized. Our first mission was to prepare a presentation for the next week on the following themes:

* Previous subjects. What are the themes explored by iGEM? Give examples of good projects and explain why they won?
* Biobricks? What are they? What are their advantages? What are the materials sent by iGEM? How does the registry work?
* Competition and deadlines. How does the competition works and what are the important deadlines?

Those presentations gave us the opportunity to understand more about the iGEM competition and to get to know each other (since we don’t all come from the same institutions).

![](https://static.igem.org/mediawiki/2015/1/1f/TLSE_notebook_1.png)

Our first team meeting

We also created our email address and a google drive to organise our files online.

### 02/09

Every group of two or three students presented one of the subjects seen above. Every presentation was followed by a Q&A. Thanks to it, everyone understood clearly the topic.

### 02/16

The next step was to present every week, two new project ideas. The presentations were divided in the following parts.

* Context
* Goal
* Originality
* Strategy
* Difficulty
* Modelling
* Device
* Benefit
* Ethics
* Planning
* Why is this project going to win?

### Until May

The following weeks were spent brainstorming. Everyone presented at least two project ideas. Here are few examples of projects reviewed:

* Creation of a quick detection kit of hepatitis
* Degradation of Lignin
* Use of ice nucleation proteins for water desalination
* Creation of a small domestic pollution detector
* ...

### May

### 05/04

Our youngest instructor and team mother Mathilde Béraud went with Benoît Pons to the FSIE committee, and thanks to their intervention, we obtained € 4000 !

### 05/05

Two projects were still in course to be chosen, the use of ice nucleating proteins to purify water and the creation of a
bio trap against the parasitic mite *Varroa destructor*. This last one was chosen to be our project on the grounds that it
was the most feasible. Based on bibliography, the initial idea was to build an engineered bacteria which will have to produce butyrate to attract the parasite. The following weeks were the subject of extensive researches on the topic.

|  |  |
| --- | --- |
| Trouche Blandine | Finance |
| Tanchon Melany | Modelling |
| David Melissa | Events |
| Pons Benoit | Press |
| Etcheberry Thomas | Logistics |
| Le Scornet Alexandre | Registry, Biobricks |
| Pons Marine | Experiments |
| David Anthony | Power Point Presentation |
| Gody Louise | Wiki |
| Chaumont Laetitia | Ethics |

### 05/13

Two members of our team participated in the Toulouse Exposciences. They presented a poster on synthetic biology and the iGEM competition. They also organized banana DNA extraction experiences for children. This successful event was a good opportunity for us to start learning how to design a poster and to communicate about synthetic biology.

### 05/15

We held our first meeting without instructors to discuss the design of the constructions needed to create the engineered bacteria.
Our reflection led us to the conclusion that our construction should respect the bee life cycle. We therefore started searching for beekeepers
that would agree to discuss with us.

### 05/18

We met with Dr Boucher Christian HDR, DR1 INRA retired since 2012 and amateur beekeeper.
The fact that Dr Boucher was both a researcher and a beekeeper made this meeting really beneficial for us.
We learned every important stages in a bee life and how the varroa affected it.
We also learned more about the beekeeping activity and the treatments used to fight against the parasite.
Furthermore we have been able to discuss our early genetic design and how to improve it.

![]( https://static.igem.org/mediawiki/2015/7/72/TLSE_notebook_2.png)

The conclusion of this meeting was that
the bacteria should not produce both attractive and toxic molecules at the same time. These
productions should be cyclic to have the least effect on bees life cycle since formate is also toxic at a lesser extent for bees.

### 05/25

During the whole week we searched for ways to produce our molecules cyclically. We chose to implement
a circadian rhythm cycle which would allow us to produce toxic molecules during the night when bees are a lot less active.
We also convinced Louise Gody to create our logo because she is capable of drawing with talent using a graphical tablet.
We also started to organise our search for financial help. To do so we designed a booklet explaining our project and what iGEM is.

### June

### 06/01

We first started searching for genes that we would like to have in our constructions on the iGEM registry.
After we successfully designed our construct we started thinking about what our device would look like. Since our bacteria needed to perceive red light we decided to place our device at the entry of the hive but outside.

### 06/05

We realised that our the cloning part would be quite long because of the number of genes needed to obtain... We therefore decided to order synthetic sequences for our synthetic pathway constructions.

### 06/06

Laetitia Chaumont, Benoit Pons and Mathilde Beraud went to a laboratory studying varroa(Vacbio EA 4357). Dr. Vetillard allowed us to test the actual effects of different concentrations of butyrate on Varroa. The results were inconclusive. However two parameters had to be improved because butyrate is a very volatile molecule and we conducted experiments in a petri dish thus diffusion complicated the interpretation. Therefore, we decided to set up a more optimised protocol to assess the attraction of varroa by butyrate.

### 06/18

Louise Gody drew our amazing logo!!

![]( https://static.igem.org/mediawiki/2015/f/f8/TLSE_ApiColi_Logo.png)

### 06/22

We finished the design of our constructions and send it to our instructors for checking. In parallel we started training ourselves on basic synthetic biology protocols such as making competent cells, biobrick transformation…

### 06/29

* Modelling started. We attempted to determine which metabolic pathways our molecules are taking part. This gave us the opportunity to have an overview of their roles and effects.
* Everybody trained to master the different protocols of synthetic biology. Our constructs were finally ordered via Biobasic which offered us a discount price.

### July

### 07/01

We designed a new protocol to assess whether butyrate is an attractive molecule for varroas. Thanks to M. Patrick Chekroun who made a T-tube in glass, we were able to define the attraction power of butyrate. [See "Protocols" part](https://2015.igem.org/Team%3AToulouse/Experiments)

### 07/02

Our project was described in a popular newspaper “Metronews”.

### 07/04

Our project made the cover of a popular local newspaper “La Dépêche”.

### 07/06

* Laetitia Chaumont initiated cytotoxicity test. The purpose of those tests are to assess whether our bacteria could withstand various butyrate/formate concentration
* In parallel Melany Tanchon determined the production of butyrate/formate. Those results are directly linked to the undergoing cytotoxicity tests.
* We also initiated the creation of the plans that would be used to 3D print our device. In order to achieve this, Valentin Girin used the software Catia for the modeling of our device.
* Our crowdfunding campaign was launched via the Ulule platform.

![]( https://static.igem.org/mediawiki/2015/3/32/TLSE_Notebook_3.png)

### 07/08

First competent cells obtained with success by Benoît Pons!!

### 07/09

* We Skyped with the iGEM Ankara team which is also working on *Varroa destructor*. We exchanged on our different strategies, and decided to start a collaboration with them. Indeed, we wanted to combine our efforts to fight against varroa and to gather some data about the mite. See  ["Collaborations"](https://2015.igem.org/Team%3AToulouse/Collaborations#ankara)  part for more information.
* We stored two bacterial strains BW25113 and MG1665 in a glycerol solution.

### 07/13

Following the newspaper articles published we discovered a forum of beekeepers discussing about our project on the web. A lot of them were puzzled by our project. We decided to engage the conversation with them and answer all of their questions. They were pleasantly surprised that we took the time to answer all their questions!

### 07/14

We prepared some M9 medium, and also did a cytotoxicity test of formic acid thanks to a plate reader.

### 07/16

We realised two tests: one is a test culture on BW25113 strain under aerobic and micro aerobic conditions. The other permits to determine the cytotoxicity of formic acid.

![]( https://static.igem.org/mediawiki/2015/0/0d/TLSE_Notebook_6.jpg)

48 wells plate, different formic acid concentrations

### 07/17

* Laetitia Chaumont obtained a meeting with Vincent Grégoire Delory, the ethician of Toulouse White Biotechnology (TWB).
* We performed a bacterial culture growth monitoring.
* We did again a cytotoxicity test of formic acid.

### 07/18

* Again a bacterial culture growth monitoring.
* Cytotoxicity test of butyric acid done.

### 07/20

Melissa David, Melany Tanchon and Louise Gody went to Brussels to attend the international Exposciences for the whole week. They presented our project and synthetic biology to people coming from all around the world.

### 07/21 & 07/22

* Bacterial culture growth monitoring (again!!).
* We also realised a cytotoxicity test of butyric acid (again!!).

### 07/23

* We did cloning trainings to be as efficient as possible, Benoît Pons and Marine Pons succeeded the first cloning at the first attempt !!
* Bacterial culture growth monitoring (again!!).
* We also realised a cytotoxicity test of butyric acid (again!!).

### 07/24

* Bacterial culture growth monitoring (again!!).
* We also realised a cytotoxicity test of butyric acid (again!!).

### 07/27

* All the team is now waiting for the synthetic genes and training since the 26th of June. Training with some basic technics such as miniprep, gel extraction, transformation, cloning, cells culture. We are eager to start, but are a bit pessimistic about the next phase of the project. Nonetheless, good mood of the team!!
* Bacterial culture growth monitoring (again!!).
* We also realised a cytotoxicity test of butyric acid (again!!).

### 07/29

Preparation of NMR tubes for the analysis of supernatants.

### 07/30

We did a cytotoxicity test of butyric acid and we were **trained** to be able to use the NMR machine.

![](https://static.igem.org/mediawiki/2015/0/08/TLSE_Protocols_Culture_4.png)

NMR training

### 07/31

An enzymatic test was done on the Biosilta kit with a plate reader.

### August

### 08/03

**Culture tests started !**

### 08/04

* We did a very good glucose dosage.
* An enzymatic test was done on the Biosilta kit with an OD reading, until the stabilization of bacterial growth.

### 08/05

We received from Dr Nico Claasens lab (Wageningen University) the strains containing PCB en Cph8 vectors responsible for the circadian regulation. We could now begin the genetic construction, but we faced a problem with the vectors: receptors genes were cloned into vectors incompatible with our final construction plan, so we had to look for an alternative solution.

### 08/15

* Thomas and Mélany worked together on the attraction module: **production of butyrate**
* Laetitia worked on the eradication module: **production of formate**
* Benoît developed the **regulation module**
* Marine and Anthony worked on the **final construction**
* Alexandre and Mélissa did the different **biobricks**

### 08/17

Preparation of NMR samples.

### 08/19

Enzymatic tests on glucose oxydase.

### 08/20

We received our synthetic genes after more than one month of waiting for them. There is only one month left and we have to work harder to reach project goals. Now the real fight begins, hoping to achieve that we planed.

### 08/22

The synthetic genes, which were cloned into pUC57 vectors, were amplified and then cloned during the weekend. We were excited to experiment something new, and see if all our efforts spent on gene design paid off.

### 08/23

* Miniprep of the clones containing the synthetic genes.
* First miniprep, digestion, gel extraction, cloning and transformation were performed for all the different modules.

### 08/24

* Some cloning worked and others did not. Verification of the clones. Start again for the ones that did not work...

### 08/25

+ The first cloning worked for the final construction: a first step has been completed (the formate genes were cloned into the PL-CI pUC57 plasmid). Unfortunately we lost a team mate in this battle, because Anthony had to leave the project in order to start his courses in Sweden.
+ Tests of formate production were made in Falcon tubes in order to mimic micro aerobic conditions.
+ Culture tests were realized for different concentrations of the Biosilta medium.

### 08/26 & 08/27

The production of formic acid in the culture of the producing strain was followed.

### 08/28

The formate genes (pflA and pflB), the butyrate genes without ccr and the POmpC-LacI box-CI were cloned into pSB1C3. The butyrate genes were cloned into the PL-LacI-ccr pUC57 Plasmid.

### 08/29

+ Blandine took part to iGEM meetup France organized by iGEM Bordeaux Team, where she met different teams, discussing with them, and presenting our project.
+ This same day, we succeeded the cloning of ccr-butyrate together.

### 08/30

Preparation of NMR samples and analysis.

### September

### 09/01

+ **Success** of the PcyA-tesB cloning
+ French institution called DRAAF announced us that they support us with **€ 3000!!**

### 09/04

Anthony met Felix from iGEM Stockholm Team, and discussed about the iGEM experience, all the work done at this moment, his disappointments, … He understood that all iGEM teams were in the same situation: URGENCY.

### 09/07

We succeeded **the first In Fusion cloning step** and the cph8 cloning.

### 09/10

+ Even if the experiments did not work as expected, all the team smiles again when we received our polo shirts and berets that we will wear in Boston: we are ready to fight !
+ Another good news was the success of the first steps of the circadian cloning made by Benoît Pons

![](https://static.igem.org/mediawiki/2015/5/56/TLSE_Notebook_5.jpg)

### 09/12

Blandine, who created our Ulule account (Ulule is a crowdfunding platform) informed us that it was a real success. Indeed, during the crowdfunding period, we obtained **€ 1,780**, with the support of 59 contributors.

### 09/15

This day, we succeeded the cloning of ccr-butyrate (done the 08/29), PL-LacI-ccr-Butyrate genes (done the 08/28) and POmpC-lacI box-CI-Formate genes (done the 08/25) into pSB1C3.

### 09/16

All Biobricks were prepared and sent to be sequenced

### 09/17

One day before THE deadline we sent our Biobrick to iGEM !!

### 09/19

Working past midnight in the lab ? Strong headaches and itching eyes but still on the computer ? Fighting with HTML tags and continuously looking at the clock ? No doubt, this is the final effort before the dreadful wikifreeze !!!

###### iGEM Toulouse 2015

INSA Toulouse
135, Avenue de Rangueil
31400 TOULOUSE
France

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