Team:Toulouse/Notebook

iGEM Toulouse 2015

Notebook

Calendar - 2015

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 send 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).

Our first team meeting

We also created our mail 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 so that 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
Modeling
Device
Benefit
Ethic
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 a few examples of projects reviewed:

Creation of a rapid detection kit for 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 research on the topic.

Moreover, it was time to choose attributions to make the project management easier, below in the table, the main assignments.

Trouche Blandine	Finance
Tanchon Melany	Modeling
David Melissa	Events
Pons Benoit	Press
Etcheberry Thomas	Logistics
Le Scornet Alexandre	Registry, Biobricks
Pons Marine	Experiences
David Anthony	Power Point Presentation
Gody Louise	Wiki
Chaumont Laetitia	Ethic

05/13

Two members of our team participated in the Toulouse Exposcience. 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 reflexion led us to the conclusion that our construction would depend on 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.

In conclusion of this meeting we decided 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 bee’s life cycle since formate is also toxic at a lesser extent for bees.

05/25

During the whole week we searched for means to produce our molecules cyclically. We found a way to produce our molecules on a circadian rhythm which allows us to produce toxic molecules during the night when bees are a lot less active.
Since we agreed on the regulation constructions we started to look for means to build our construction.
We also started to think about our logo and decided that Louise Gody will create our logo since she is capable of drawing using a graphical tablet.
We also started to organize our search for financial help. To do so we designed a booklet explaining our project and what iGEM is.

June

06/01

We started searching for each individual genes by first looking on the iGEM registry.
Knowing that the design of our construct was agreed upon we also 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 where bees need to enter the beehive.

06/05

We realised that our work would be too heavy seeing the number of genes needed to obtain and the cloning work required.. We therefore decided to synthesise the sequence needed to realise our construction.

06/06

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

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

Modeling 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 glass T-tube, we were able to define the attraction power of butyrate. See "Protocols" part

07/02

Our project was in a popular newspaper “Metronews”.

07/04

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

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.

07/08

First competent cells 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 us in order to combine our efforts to fight against varroa and to gather some data about the mite. See "Collaborations" 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 perplexed by our project. We decided to engage the conversation with them and answer all of their questions.

07/14

We prepared precursor molecules for a M9 medium, and also did a cytotoxicité test of formic acid thanks to a plate reader.

07/16

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

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 did a bacterial culture monitoring thanks to an Optical Density (OD) reading and culture plating.
We did again a cytotoxicity test of formic acid.

07/18

We did a bacterial culture monitoring thanks to an Optical Density (OD) reading and culture plating.
We did a cytotoxicity test of butyric acid.

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

We did a bacterial culture monitoring thanks to an Optical Density (OD) reading and culture plating.
We did a cytotoxicity test of butyric acid.

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 !!
We did a bacterial culture monitoring thanks to an Optical Density (OD) reading and culture plating.
We did a cytotoxicity test of butyric acid.

07/24

We did a bacterial culture monitoring thanks to an Optical Density (OD) reading and culture plating.
We did a cytotoxicity test of butyric acid.

07/27

All the team was waiting for the synthetic genes and training since 26th june with some basic technics such as miniprep, gel extraction, transformation, cloning, cells culture. We started to be pessimists about the next phase of the project, but we kept a good mood in the team.
We did a bacterial culture monitoring thanks to an Optical Density (OD) reading and culture plating.
We did a cytotoxicity test of butyric acid.

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.

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

08/15

After the ordering of our synthetic genes, we were waiting for them.
We decided to work in pairs per module to be as effective as possible:

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. There is only one month left and we had to work harder to reach project goals. Now the real fight could begin, hoping to achieve that we planed.

08/22

The synthetic genes, which were cloned into pUC57 vectors, were transformed into E. coli strain in order to realize miniprep 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 bacteria containing synthetical genes, followed by a digestion by NotI restriction enzyme of the plasmid containing the formate genes (pflA and pflB). Then a ligation of puc57 wad done in ordr to obtain the empty vector. Finally, a transformation was realized and the results were good. Indeed, there was no colony in the negative control plate, many colonies on the positive control and one clone for our ligation.
First miniprep, digestion, gel extraction, cloning and transformation were performed for all the different modules and we are eager to see clones on LB plates.

08/24

We had some cloning which worked and others which did not. To check that it really worked for some, we decided to do a miniprep.Indeed, if the insert has been cloned into the vector, the plasmid will be heavier. So we had to do cells culture with some clones.
For the clonings which did not work, we had to do them again.
We checked the only clone obtained the 23/08 and we concluded that it corresponds to puc57.
Recircularized puc57 vector became the vector control for culture tests.

08/25

The first clonings 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 teammate 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 formic culture was followed.

08/28

The formate genes (pflA and pflB), the butyrate genes without ccr and the P_OmpC-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 tubes.

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, because of time lack.

09/07

We succeeded the first In Fusion cloning and the crt-ho1 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 were ready to fight !
Another good new was the success of circadian clonings made by Benoît Pons

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 P_OmpC-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 !!

References