Difference between revisions of "Team:Toulouse-INSA-UPS/Notebook"

Line 97: Line 97:
 
             <h4 class="heavy">18/06 - 22/06:</h4>
 
             <h4 class="heavy">18/06 - 22/06:</h4>
 
<p>
 
<p>
−
For the first week in the lab, we recovered and amplified our plasmids (pPICz, pETDuet, pET28, pGAP) but also our bacterial strains <i>E. coli BL21 DE3</i> and Tuner.
+
For the first week in the lab, we recovered and amplified our plasmids (pPICz, pETDuet, pET28, pGAP) but also our bacterial strains <i>E. coli</i> BL21 DE3 and Tuner.
 
</p>
 
</p>
 
 
Line 147: Line 147:
 
             <h4 class="heavy">02/07 - 06/07:</h4>
 
             <h4 class="heavy">02/07 - 06/07:</h4>
 
<ul>
 
<ul>
−
<li><strong>Team biotin</strong>: this week, we have done BirA PCR and we had digested the pPIC after his amplification in <i>E. coli</i> Stellard cells. This allowed us to do our first cloning with BirA in the pETDuet. We have checked the clones by digestion and they were OK. It was necessary to sequence this clones before doing the next cloning in  pETDuet. We found another pPIC and we checked it.   
+
<li><strong>Team biotin</strong>: this week, we have done BirA PCR and we had digested the pPIC after his amplification in <i>E. coli</i> Stellar cells. This allowed us to do our first cloning with BirA in the pETDuet. We have checked the clones by digestion and they were OK. It was necessary to sequence this clones before doing the next cloning in  pETDuet. We found another pPIC and we checked it.   
 
</li>
 
</li>
−
<li><strong>Team coli</strong>: this week, it was the first In-fusion cloning with our Cerberus in the pET28. We used two differents construction for Cerberus, one with a monomeric streptavidin and another with a  tetrameric streptavidin. Then, we checked the construct by digestion before sending it for sequencing. In the same time we checked our plasmid pEVOL-azF which allow the unnatural amino acid incorporation in <i>E. coli</i>.
+
<li><strong>Team coli</strong>: this week, it was the first In-fusion cloning with our Cerberus in the pET28. We used two differents construction for Cerberus, one with a monomeric streptavidin and another with a  tetrameric streptavidin. Then, we checked the construct by digestion before sending it for sequencing. In the same time we checked our plasmid pEVOL-AzF which allows the unnatural amino acid incorporation in <i>E. coli</i>.
 
</li>
 
</li>
 
<li><strong>Team pastoris</strong>: this week, we did the In-fusion cloning of Cerberus-strepta in pGAP with the material prepared the week before. So we had to check some clones by digestion before sequencing. Fortunately, they were good. It was also this week that we received the <i>Pichia pastoris</i> GS200 strains cell.
 
<li><strong>Team pastoris</strong>: this week, we did the In-fusion cloning of Cerberus-strepta in pGAP with the material prepared the week before. So we had to check some clones by digestion before sequencing. Fortunately, they were good. It was also this week that we received the <i>Pichia pastoris</i> GS200 strains cell.
Line 193: Line 193:
 
             <h4 class="heavy">16/07 - 20/07:</h4>
 
             <h4 class="heavy">16/07 - 20/07:</h4>
 
             <ul>
 
             <ul>
−
<li><strong>Team biotin:</strong> This week, we focused on the pastoris cloning. In one hand, we prepared the pGAP vector (amplification, purification and digestion), mRFP1, BFP and Sygonadine parts and we did the three cloning. We tried to amplify and purify pPIC but unfortunately it was necessary to try again because the Midiprep didn’t succeed.</li>
+
<li><strong>Team biotin:</strong> This week, we focused on the pastoris cloning. In one hand, we prepared the pGAP vector (amplification, purification and digestion), mRFP1, BFP and scygonadin parts and we did the three cloning. We tried to amplify and purify pPIC but unfortunately it was necessary to try again because the Midiprep didn’t succeed.</li>
 
<li><strong>Team coli:</strong> this week, we made or own competent cells using BL21 and tuner strains</li>
 
<li><strong>Team coli:</strong> this week, we made or own competent cells using BL21 and tuner strains</li>
 
<li><strong>Team pastoris:</strong> this week, we did In-fusion cloning in the pGAP but this time with the cerberus mSA2. After transformation, culture and miniprep, we checked clones by digestion. The cloning was a success.</li>
 
<li><strong>Team pastoris:</strong> this week, we did In-fusion cloning in the pGAP but this time with the cerberus mSA2. After transformation, culture and miniprep, we checked clones by digestion. The cloning was a success.</li>
Line 232: Line 232:
 
             <h4 class="heavy">30/07 - 03/08:</h4>
 
             <h4 class="heavy">30/07 - 03/08:</h4>
 
             <ul>
 
             <ul>
−
<li><strong>Team biotin:</strong> this week, we worked on pETDuet-BirA to clone our functionalising proteins (mRFP1 and BFP). We amplified the pETDuet-BirA and after Midiprep and digestion, we cloned mRFP1 and BFP with the In-fusion kit. After digestion, we sent clones to sequencing and started production assay in <i>E. coli</i> BL21.</li>
+
<li><strong>Team biotin:</strong> this week, we worked on pETDuet-BirA to clone our functionalising proteins (mRFP1 and BFP). We amplified the pETDuet-BirA and after Midiprep and digestion, we cloned mRFP1 and BFP with the In-fusion kit. After digestion, we sent clones to sequencing and started production assay in <i>E. coli</i> BL21 DE3.</li>
−
<li><strong>Team coli:</strong> we did the first Sirius purification after production in <i>E. coli</i> BL21. To do this, we used an IMAC column and dialysis. In order to proved the CBM3 fixation to cellulose, we prepared the regenerated amorphous cellulose.</li>
+
<li><strong>Team coli:</strong> we did the first Sirius purification after production in <i>E. coli</i> BL21 DE3. To do this, we used an IMAC column and dialysis. In order to proved the CBM3 fixation to cellulose, we prepared the regenerated amorphous cellulose.</li>
  
 
<li><strong>Team callulose:</strong> We intensified the cellulose production to be able to give 5g of dried cellulose to the Bordeaux iGEM team at the end of the month.</li>
 
<li><strong>Team callulose:</strong> We intensified the cellulose production to be able to give 5g of dried cellulose to the Bordeaux iGEM team at the end of the month.</li>
Line 305: Line 305:
 
             <h4 class="heavy">20/08 - 24/08:</h4>
 
             <h4 class="heavy">20/08 - 24/08:</h4>
 
             <ul>
 
             <ul>
−
<li><strong>Team coli:</strong> this week was a particularly busy week. We did the Orthos production and purification but also the same for Sirius and mRFP1. This allowed us to perform a cellulose pull down assay with Sirius and mRFP1 as a 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 its incorporation. Cerberus was purified by a cellulose pool down assay.</li>
+
<li><strong>Team coli:</strong> this week was a particularly busy week. We did the Orthos production and purification but also the same for Sirius and mRFP1. This allowed us to perform a cellulose pull down assay with Sirius and mRFP1 as a negative control. In addition, we did the production of Cerberus. To do this, we did co-transformation of BL21 DE3 cells with pET28 Cerberus and pEVOL AzF. The cell culture medium was complemented with with AzF unnatural amino acid to allow its incorporation. Cerberus was purified by a cellulose pull-down assay.</li>
 
<li><strong>Team pSB1C3:</strong> we created this new team to start the pSB1C3 clonings. PCR was performed on our gblocks. Unfortunately, we didn’t succeed to amplify all the gblocks.</li>
 
<li><strong>Team pSB1C3:</strong> we created this new team to start the pSB1C3 clonings. PCR was performed on our gblocks. Unfortunately, we didn’t succeed to amplify all the gblocks.</li>
 
</ul>
 
</ul>
Line 323: Line 323:
 
             <h4 class="heavy">27/08 - 31/08:</h4>
 
             <h4 class="heavy">27/08 - 31/08:</h4>
 
             <ul>
 
             <ul>
−
<li><strong>Team coli:</strong> in order to demonstrate the efficiency of the third Cerberus head, we did a click assay with fluorescein DBCO.</li>
+
<li><strong>Team coli:</strong> in order to demonstrate the efficiency of the third Cerberus head, we did a click assay with DBCO-fluorescein.</li>
 
</ul>
 
</ul>
 
         </div>
 
         </div>
Line 376: Line 376:
 
             <h4 class="heavy">10/09 - 14/09: </h4>
 
             <h4 class="heavy">10/09 - 14/09: </h4>
 
             <ul>
 
             <ul>
−
<li><strong>Team biotin:</strong> we successfully made a pull down assay with Orthos to measure the BFP fluorescence with and without Orthos on cellulose.</li>
+
<li><strong>Team biotin:</strong> we successfully made a pull-down assay with Orthos to measure the BFP fluorescence with and without Orthos on cellulose.</li>
 
</ul>
 
</ul>
 
 
Line 394: Line 394:
 
             <h4 class="heavy">17/09 - 21/09:</h4>
 
             <h4 class="heavy">17/09 - 21/09:</h4>
 
             <ul>
 
             <ul>
−
<li><strong>Team biotin:</strong> this was the final assay and we proved the fixation of the biotinylated BFP on cerberus.</li>
+
<li><strong>Team biotin:</strong> this was the final assay and we proved the fixation of the biotinylated BFP on Cerberus.</li>
  
−
<li><strong>Team callulose:</strong> we managed to produce a pink bacterial cellulose.</li>
 
 
 
 
</ul>
 
</ul>
Line 415: Line 414:
 
             <h4 class="heavy">24/09 - 28/09:</h4>
 
             <h4 class="heavy">24/09 - 28/09:</h4>
 
             <ul>
 
             <ul>
−
<li><strong>Team coli</strong>: for our last lab week, we did two manipulations, the first to show that we can fix BFP-DBCO to the head streptavidin and the second to test the specificity of CBM3a to bacterial cellulose. And we successfully validated these two experiments!</li>
+
<li><strong>Team coli & callulose</strong>: for our last lab week, we managed to produce a pink bacterial cellulose by using our purified Sirius protein.</li>
 
</ul>
 
</ul>
 
 

Revision as of 16:30, 16 October 2018

NOTEBOOK

Here, you will find information about the temporal aspects of our project.

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 and characterization of Cerberus and Sirius in Escherichia coli
  • Team pastoris: 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 to test our in vivo biotinylation system.
  • Team callulose: this team was managed by Callum (so the coined Callulose name) and got help from Younes. Its mission was to launch and optimize our bacterial cellulose production.

LABBOOK: JUNE

icon

18/06 - 22/06:

For the first week in the lab, we recovered and amplified our plasmids (pPICz, pETDuet, pET28, pGAP) but also our bacterial strains E. coli BL21 DE3 and Tuner.

icon

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 by amplifying it in E. coli Stellar cells before a Midiprep. Another aspect was to check the pREAV which will allow the incorporation of a unnatural amino acid in our Cerberus.
  • Team pastoris: pPIC, pGAP and pETDuet were good but pPIC did not correspond to our expectations so we had to find another solution.
  • Team callulose: the first productions of cellulose started in culture tubes.

LABBOOK: JULY

icon

02/07 - 06/07:

  • Team biotin: this week, we have done BirA PCR and we had digested the pPIC after his amplification in E. coli Stellar cells. This allowed us to do our first cloning with BirA in the pETDuet. We have checked the clones by digestion and they were OK. It was necessary to sequence this clones before doing the next cloning in pETDuet. We found another 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 for Cerberus, one with a monomeric streptavidin and another with a tetrameric streptavidin. Then, we checked the construct by digestion before sending it for sequencing. In the same time we checked our plasmid pEVOL-AzF which allows the unnatural amino acid incorporation in E. coli.
  • Team pastoris: this week, we did the In-fusion cloning of Cerberus-strepta in pGAP with the material prepared the week before. So we had to check some clones by digestion before sequencing. Fortunately, they were good. It was also this week that we received the Pichia pastoris GS200 strains cell.
icon

09/07 - 13/07:

  • Team biotin: on monday we sent our pETDuet BirA clones obtained last week to sequencing and we were happy that our clones were validated. In the same time, we carried on the PCR with the parts that will serve us to functionalise our Cerberus after biotinylation by BirA.
  • Team coli: This week, we received the sequencing results for Cerberus and it was again a successfully cloning for the Toulouse iGEM team. We cloned Sirius (CBM3-RFP) in the pET28 with the In-fusion kit. To do this, we performed PCR on the gBlocks then we used the In-fusion kit. After verification, Sirius was OK.
  • Team pastoris: we checked the pREAV and found out the plasmid map didn’t correspond with our digestion results. We needed to find another pREAV...
  • Team callulose: we decided to focus the production with G. hansenii because it produces more cellulose than K. rhaeticus. We also started to produce in 2L Erlenmeyers.
icon

16/07 - 20/07:

  • Team biotin: This week, we focused on the pastoris cloning. In one hand, we prepared the pGAP vector (amplification, purification and digestion), mRFP1, BFP and scygonadin parts and we did the three cloning. We tried to amplify and purify pPIC but unfortunately it was necessary to try again because the Midiprep didn’t succeed.
  • Team coli: this week, we made or own competent cells using BL21 and tuner strains
  • Team pastoris: this week, we did In-fusion cloning in the pGAP but this time with the cerberus mSA2. After transformation, culture and miniprep, we checked clones by digestion. The cloning was a success.
icon

23/07 - 27/07:

  • Team biotin: this week, before another midiprep, we checked the pPIC but we still had a mapping problem. After many verification, it was clearly not good but we found another source for this plasmid and it seemed OK. We amplified it by midiprep.
  • Team coli: this week, we did the first production assay for Sirius and we were very happy because we obtained a red culture medium.
  • Team pastoris: this week, the pichia team helped the other teams because it was ahead of the planning and had to wait for yeast growth.
icon

30/07 - 03/08:

  • Team biotin: this week, we worked on pETDuet-BirA to clone our functionalising proteins (mRFP1 and BFP). We amplified the pETDuet-BirA and after Midiprep and digestion, we cloned mRFP1 and BFP with the In-fusion kit. After digestion, we sent clones to sequencing and started production assay in E. coli BL21 DE3.
  • Team coli: we did the first Sirius purification after production in E. coli BL21 DE3. To do this, we used an IMAC column and dialysis. In order to proved the CBM3 fixation to cellulose, we prepared the regenerated amorphous cellulose.
  • Team callulose: We intensified the cellulose production to be able to give 5g of dried cellulose to the Bordeaux iGEM team at the end of the month.

LABBOOK: AUGUST

icon

06/08 - 10/08:

  • Team biotin: this week, we worked on the production assay for biotinylated proteins in E. coli. Furthermore, we cloned BirA in the pPIC plasmid and we checked it by digestion and sequencing. After that, we cloned RFP, scygonadin and BFP in this plasmid to produce the proteins in Pichia pastoris.
  • Team coli: in order to show the efficiency of the second Cerberus head, we needed a Cerberus which has just its CBM3a head and the streptavidin head. We did a directed mutagenesis on the stop codon to create Orthos.
icon

13/08 - 17/08:

  • Team biotin: we produced biotinylated proteins with E. coli, with and without biotin in the culture medium.
icon

20/08 - 24/08:

  • Team coli: this week was a particularly busy week. We did the Orthos production and purification but also the same for Sirius and mRFP1. This allowed us to perform a cellulose pull down assay with Sirius and mRFP1 as a negative control. In addition, we did the production of Cerberus. To do this, we did co-transformation of BL21 DE3 cells with pET28 Cerberus and pEVOL AzF. The cell culture medium was complemented with with AzF unnatural amino acid to allow its incorporation. Cerberus was purified by a cellulose pull-down assay.
  • Team pSB1C3: we created this new team to start the pSB1C3 clonings. PCR was performed on our gblocks. Unfortunately, we didn’t succeed to amplify all the gblocks.
icon

27/08 - 31/08:

  • Team coli: in order to demonstrate the efficiency of the third Cerberus head, we did a click assay with DBCO-fluorescein.

LABBOOK: SEPTEMBER

icon

03/09 - 07/09:

  • Team coli: in order to demonstrate the efficiency of the AzF head, we tried to click paramagnetic beads on it. The aim was to see the displacement of the cellulose when in a presence of a magnet
  • Team pSB1C3: this week, we tried again the pSB1C3 clonings. We used another pSB1C3 and tried a classic cloning with restriction enzymes instead of In-fusion. At the end of the week, we sent our pSB1C3 to sequencing.
icon

10/09 - 14/09:

  • Team biotin: we successfully made a pull-down assay with Orthos to measure the BFP fluorescence with and without Orthos on cellulose.
icon

17/09 - 21/09:

  • Team biotin: this was the final assay and we proved the fixation of the biotinylated BFP on Cerberus.
icon

24/09 - 28/09:

  • Team coli & callulose: for our last lab week, we managed to produce a pink bacterial cellulose by using our purified Sirius protein.

Here, you can find about some of our activities outside of the lab. More details are provided in the HP LogBOOK of the Integrated Human Practices section.

Extra-LabBOOK

Extra-LabBOOK: FEBRUARY

icon

February 1st, 2018: Kick-off meeting

Gathering the team members, distribute the tasks and of course get to know each others…

The iGEM’s adventure begins!

icon

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.

icon

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 “Sciences and technologies laboratory”.

Extra-LabBOOK: MARCH

icon

March 1st-15t, 2018: Brainstorming

Only 5 subjects still in course!

icon

March 10th, 2018: Grimoire

At this event we introduced our card game Microbioworld.

icon

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.

Extra-LabBOOK: APRIL

icon

April 5th, 2018: Final choice of our project

After a long period of brainstorming, we have decided to work on molecules’ binding on cellulose. Now, we had to think of our project’s name.

icon

April 12th, 2018: Tour des Sciences

We presented the Microbioworld game during the ”Tour des sciences”.

Extra-LabBOOK: MAY

icon

May , 2018:

We worked on the design of the project with the help of our supervisors.

icon

May 31th, 2018:

We met Philippe Serp and his team to manipulate our graphene on his lab.

Extra-LabBOOK: JUNE

icon

June 18th, 2018: First day in the lab

We were all very impatient, and finally it has come! It’s the first lab day.

icon

June 19th, 2018: Fondation INSA Toulouse meeting

Looking for funds, we presented our project to the INSA Toulouse fondation.

icon

June 23rd-24th, 2018: Collaboration with iGEM Montpellier’s team

We starte a collaboration with the Montpellier team to help them in some areas like the wiki. To do so, we went to Montpellier.

icon

June 25th, 2018: TWB meeting

We have met Pierre Monsan in Toulouse White Biotechnology (TWB), one of our main funders.

icon

June 26th, 2018: NUS meeting

We organized a skype meeting with the NUS iGEM team to discuss about a possible collaboration.

icon

June 27th, 2018: Meet with STL teachers

As part of our discussions with the ministry of education, high school teachers visited the lab where we worked during summer.

icon

June 27th, 2018

Entrep: We met Guillaume Boissonat, who co-funded the startup pili.bio. He shared with us his own experience as a co-funder, and he gave to us some advice about the possible business model for Cerberus Startup.

icon

June 29th, 2018: Primary school intervention

We intervened in a primary school in order to present Microbiolworld, the research profession, and more generally introduce the world of microbes to children.

Extra-LabBOOK: JULY

icon

July 5th, 2018: CALMIP’s meet and visit

We visited CALMIP a high-throughput calcul center which allowed us to make complex calculations for our modelling.

icon

July 6th, 2018: Official announcement of our project

We officially announced the purpose of our project on social media!

icon

July 7th, 2018: 4th Annual Parisian Meet-up

We took part in the annual Parisian Meet-up organized by Pasteur iGEM team.

Meet up Paris
icon

July 7th-12th, 2018: EuroScience Open Forum (ESOF)

We participated to the EuroScience Open Forum in Toulouse to introduce the iGEM competition and synthetic biology to the public.

icon

July 19th, 2018: First publications in media

Toulouse media spoke about us and Cerberus, we were very happy of this recognition!

icon

July 20th-22nd, 2018: European Meet-up in Munich

Two members of the team travelled to Munich to represent our team at the European meet-up.

Meet up Munich
icon

July 30th, 2018: Incubator meeting

We met Yohann Bouvier who is the manager of a startup incubator, named "le starter". He gave us some advice regarding our entrepreneurship approach.

Extra-LabBOOK: AUGUST

icon

August 1st, 2018: Laval skype

We contacted the laval iGEM team in order to create an innovative human practice collaboration.

icon

August 17th, 2018: Shooting photo for the wiki

We dressed up to take the pictures used in our wiki.

icon

August 25th-26th, 2018: Collaboration with Bordeaux’s team

We went to Bordeaux to bring them our bacterial cellulose. Then, they will use it as another carbon source for there Far Waste project.

icon

August 29th, 2018: First working group with Le Catalyseur

Le Catalyseur is a business incubator, they helped us to write our business plan. This first working group allowed us to structure our company and defined our objectives.

icon

August 31st, 2018: Second workshop with Le Catalyseur

During this second meeting, we identified the critical points and risk factors we need to enhance to conducted Cerberus as a business.

Extra-LabBOOK: SEPTEMBER

icon

September 4th, 2018

Le Catalyseur helped us to create a business model CANVAS.

icon

September 5th, 2018:

Le Catalyseur helped us to create a SWOT matrice to define our strengths and weakness as a business.

Extra-LabBOOK: OCTOBER

icon

October 3rd, 2018:

We have done a general repetition in an international high school near Toulouse.

icon

October 5th, 2018:

Another meeting with Le Catalyseur to talk about industrial property.

No dogs were harmed over the course of this iGEM project.

INSA Toulouse UPS LISBP TWB Defi Adisseo Foundation ESF Catalyses


The content provided on this website is the fruit of the work of the Toulouse INSA-UPS iGEM Team. As a deliverable for the iGEM Competition, it falls under the Creative Commons Attribution 4.0. Thus, all content on this wiki is available under the Creative Commons Attribution 4.0 license (or any later version). For futher information, please consult the official website of Creative Commons.

This website was designed with Bootstrap (4.1.3). Bootstrap is a front-end library of component for html, css and javascript. It relies on both Popper and jQuery. For further information, please consult the official website of Bootstrap.