Team:Toulouse-INSA-UPS/Notebook
NOTEBOOK
INTRODUCTION
LABBOOK
To achieve our lab work, we splited into different teams, each working on different aspects of the experimental procedure. Three teams were then formed :
- Team coli: This Team was managed by Younes and included Marion. Their goal was to achieve the production an characterization of Cerberus and Sirius in Escherichia coli
- Team Pichia: This team was ruled by Angeline and included Gaëlle. Their mission was to reproduce the experiments planned on Escherichia coli in Pichia pastoris
- Team Biotin: This team was ruled by Amandine and included Jean and Julien. Their objective was to construct and test our in vivo biotinylation system.
- Team Callulose: This team was managed by Cal and got helped by Younes. His mission was to launch and optimize our bacterial cellulose production.
LABBOOK: JUNE
18/06 -> 22/06 :
To the first week in the lab, we recovered and amplify our differents plasmids witch will be used ( pPICz, pETDuet, pET28, pGAP) but also our bacterial strains E.coli BL21 DE3 and Tuner.
25/06 -> 29/06 :
- Team coli: We started with a PCR on the the gblocks cerberus pastoris . In order to clone this gblocks we prepared the pGAP plasmid to do this, we had amplify it in E.coli stellard cells before a Midiprep. Another aspect was to check the pREAV witch will allow the incorporation of a unnatural amino acid on our Cerberus.
- Team Pichia: Unfortunately, pPIC, pGAP and petDuet were good but our pPIC did not correspond to our expectations so we had to find another one
LABBOOK: JULY
02/07 -> 06/07 :
- Team Biotin: This week, we have done BirA PCR and we had digest the pPIC after his amplification in E.coli stellard cells. This allowed us to do our first cloning with BirA in the pETDuet. We have check clones by digestion and they were OK. It was necessary to sequenc this clones before doing the the next cloning in pETDuet. We recovered an other pPIC and we checked it.
- Team coli: This week, it was the first In fusion cloning with our cerberus in the pET28. We used two differents construction to Cerberus, one with a monomeric streptavidin and another with tetrameric streptavidin . Next we checked the construct by digestion before sending it for sequencing. In the same time we checked our plasmid pEVOL-azF.witch allow the unnatural amino acid incorporation in E.coli.
- Team pichia: This week, we did the Infusion cloning of cerberus strepta in pGAP with the material prepared the week before. So we had to check some clones by digestion before the sequencing. Fortunately they were good and we sent them to the sequencing. It was also week that we have received the GS200 strains cell.
09/07 -> 13/07 :
- Team BiotinOn monday we sent our pETDuet BirA clones obtained last week to the sequencing and we were happy in the end of the week because our clones were validated by sequencing. In the same we continued the PCR with the parts that will serve us to functionalise our cerberus after biotinylation by BirA.
- Team coliThis week, we received the sequencing results for the cerberus and it was still a successfully cloning to Toulouse iGEM team. We cloned sirus (CBM3-RFP) in the pET28 with the Infusion kit. To do this, firstly you did PCR on gBlocks CBM3 in one hand and RFP in other hand, secondly we used the Infusion kit. After verification, Sirius is OK.
- Team pichiaWe have continued to check the pREAV because the plasmid map didnât correspond with our digestion results. We will have to recover another pREAV or another map.
16/07 -> 20/07 :
- Team BiotinThis week, we focused on the Pastoris cloning. In one hand we prepared the pGAP to clone ( amplification, purification and digestion ) mRFP1, ,BFP, and the Sygonadine and we did the three cloning. In other hand, we continued to try to amplify and purify pPIC but unfortunately it necessary to try again because the Midiprep didnât successful.
- Team coliThis week we made or own competent cells using BL21 and tuner strains
- Team pichiaThis week, we did Infusion cloning in the pGAP but this time with the cerberus mSA2. After transformation, culture and miniprep, we checked clones by digestion. The cloning was still a success.
23/07 -> 27/07 :
- Team BiotinThis week, before another midiprep we checked the pPIC ( with a miniprep ) but we still had a problem with the pPIC, after many verification it wasnât the good but we analysed another tubes and we think we thought the right. We amplified it and prepareed it by midiprep.
- Team coliThis week we did the first production essay to our sirius and we were very happy because we obtained a red culture medium.
- Team pichiaThis week, the pichia team helped the other team because this team was ahead and she had to wait for Yeast growth.
30/07 -> 03/08 :
- Team BiotinThis week take back to the pETDuet-BirA to cloned our functionalising proteines (mRFP1 and BFP) in the plasmid. To this we amplified the pETDuet-BirA and after Midiprep and digestion we cloned mRFP1 and BFP with the Infusion kit. After digestion in one hand we we sent clones to the sequencing and in other hand we started production essay in E.Coli BL21.
- Team coliThis week we did the first Sirius purification after production in E. coli BL21. To do this we used a IMAC column and dialysis. In order to proved the CBM3 fixation to the cellulose we prepared the regenerated amorphous cellulose.
LABBOOK: AUGUST
06/08 -> 10/08 :
- Team Biotin: This week, we continued the production essay of biotinylated proteins with E.coli. Furthermore, We cloned BirA in the pPIC plasmid and we checked by digestion and sequencing. After that we cloned RFP, Sygonadine and BFP in this plasmid to produce this proteins in Pichia Pastoris.
- Team coli: In order to show the efficiency of the second Cerberus head, we needed a Cerberus witch have just his CBM3 head and the streptavidin head. Itâs why, we did a directed mutagenesis on the stop codon to create orthos. This mutagenesis was done by PCR on the Cerberus gblock.
13/08 -> 17/08 :
- Team Biotin: This week, we continued the production essay of biotinylated proteins with E.coli, to do this you tested differents solution, with and without biotin in culture medium.
20/08 -> 24/08 :
- Team coli: This week was a particularly busy week, we did the Orthos production and purification but also the same thing to sirius and mRFP1. This allowed us to perform a cellulose pull down assay with sirius and mRFP1 as negative control. In addition , we did the production of Cerberus. To do this we did co transformation of BL21 cells with pET28 Cerberus and pEVOL AzF. The cell culture medium was complemented with with AzF unnatural amino acid to allow his incorporation. Cerberus was purify by a cellulose pool down essay.
- Team pSB1C3: This week, we started the pSB1C3 cloning, to do this, we did PCR on our gblocks. Unfortunately, we didnât succeed to amplify all gblocks. In other hand, we checked our interlab pSB1C3 but it does not correspond with our expectations.We need to find another.
27/08 -> 31/08 :
- Team coliIn order to demonstrate the efficacy of the third Cerberus head we did a click assay with fluorescein DBCO. The aim was to validate the Azf head of the Cerberus.
LABBOOK: SEPTEMBER
03/09 -> 07/09 :
- Team coli: In order to demonstrate the efficacy of the third Cerberus head we was trying to do a click essay with paramagnetic balls. The aim was to see the displacement of the cellulose when we added our cerberus with paramagnetics beads.
- Team pSB1C3: This week, we made the pSB1C3 cloning again, but we used another pSB1C3 which correspond at our expectations. We did PCR on our gblocks but this time we changed the primers and we used the enzymatic technique. At the end of the week we send our differents pSB1C3 to the sequencing.
10/09 -> 14/09 :
- Team Biotin: In order to purify the biotinylated compounds we made a pull down essay with orthos to measure the BFP fluorescence with and without Orthos in the cellulose.
17/09 -> 21/09 :
- Team Biotin: Final assay and we proved the fixation of the biotinylated BFP with our cerberus.
24/09 -> 28/09 :
- Team coli: This week we did our last manipulation to show that we can fix BFP-DBCO to the head streptavidin
NOTEBOOK
NOTEBOOK: FEBRUARY
February 1st, 2018: Kick-off meeting
Gathering the team members, distribute the tasks and of course get to know each othersâŚ
The iGEMâs adventure begins!
February 8th-22nd, 2018: Brainstorming
At the beginning, more than 50 ideas were proposed. The most original ideas were selected during our brainstorming sessions.
At the end of the month, still 11 subjects need more investigations.
February 21st, 2018: Escape game conference
We attend a conference organized by the Catalyseur on the creation of a teaching escape game. We met two professors in high schools option âSciences and technologies laboratoryâ.
NOTEBOOK: MARCH
March 1st-15t, 2018: Brainstorming
Only 5 subjects still in course!
March 10th, 2018: Grimoire
At this event we introduced our card game Microbioworld.
March 20th, 2018: CRISPR/CAS meeting
We meet a CRISPR/CAS expert in the LMGM lab who teaches us a lot about the new possibilities of the system.
NOTEBOOK: APRIL
April 5th, 2018: Final choice of our project
After a long period of brainstorming, we have decided to work on moleculesâ binding on cellulose. Now, we had to think of our projectâs name.
April 12th, 2018: Tour des Sciences
We still present the Microbioworld game during the âTour des sciencesâ.
NOTEBOOK: MAY
May , 2018:
We work on the design of the project with the help of our supervisors.
May 31th, 2018:
We met Philippe Serp and his team to manipulate our graphene on his lab.
NOTEBOOK: JUNE
June 18th, 2018: First day in the lab
We were all very impatient, and finally it has come! Itâs the first lab day.
June 19th, 2018: Fondation INSA Toulouse meeting
Looking for funding, we present our project to the INSA Toulouse fondation.
June 23rd-24th, 2018: Collaboration with Montpellierâs team
We start a collaboration with the Montpellier team to help them in some areas like the wiki. To do this we go to Montpelier.
June 25th, 2018: TWB meet
We went to TWB, one of our main funders.
June 26th, 2018: NUS meeting
We organizing a skype with the NUS team to discuss about a possible collaboration.
June 27th, 2018: Meet with STL teachers
As part of our discussions with the ministry of education, high school teachers visit us.
June 27th, 2018
Entrep: We met Guillaume Boissonat, who co-funded the startup pili.bio. He advises us when creating our the business model.
June 29th, 2018: Primary school intervention
We intervened in a school in order to present Microbiolworld, the research profession, and more generally introduce the world of microbes.
NOTEBOOK: JULY
July 5th, 2018: CALMIPâs meet and visit
We visit CALMIP a high-throughput calcul center which allowed us to make complex calculations for our modeling.
July 6th, 2018: Official announcement of our project
We officially present our project on social media!
July 7th, 2018: 4th Annual Parisian Meet-up
We take part in the annual Parisian Meet-up organized by Pasteur iGEM team.
July 7th-12th, 2018: EuroScience Open Forum (ESOF)
We participate to the EuroScience Open Forum in Toulouse to introduce igem and synthetic biology for the public.
July 19th, 2018: First publications in media
Toulouse media speak about us, we are very happy of this recognition!
June 20th-22nd, 2018: European Meet-up in Munich
Two members of the team travel to Munich to represent our team.
June 30th, 2018: Incubator meeting
We met Yohann Bouvier who is the manager of a startup incubator. He advices us regarding our entrepreneurship approach.
NOTEBOOK: AUGUST
August 1st, 2018: Laval skype
We take contact by skype with the Laval iGEM team.
August 17th, 2018: Shooting photo for the wiki
We put out our nicest clothes to make the pictures used in our wiki.
August 25th-26th, 2018: Collaboration with Bordeauxâs team
We go to Bordeaux to share with them our bacterial cellulose.
August 29th, 2018: First work group with Le Catalyseur
Le Catalyseur is a business incubator, they helped us to write our business plan. This first work group allowed us to structure our company.
August 31st, 2018: Second workshop with Le Catalyseur
During this second meeting, we have worked on the different risk factors to enhance our business.
NOTEBOOK: SEPTEMBER
September 4th, 2018
Catalyseur help us to create a business CANVAS.
September 5th, 2018:
Catalyseur help us to define our strengths, weakness as a business.
NOTEBOOK: OCTOBER
October 3rd, 2018:
We do a general repetition in an international high school near Toulouse.
October 5th, 2018:
Another meeting with the catalyst to talk about industrial property.
No dogs were harmed over the course of this iGEM project.
The whole Toulouse INSA-UPS team wants to thank our sponsors, especially:
And many more. For futher information about our sponsors, please consult our Sponsors page.
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