iGEM 2018 — Team Toulouse-INSA-UPS

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Toulouse-INSA-UPS/Notebook

[![](https://static.igem.org/mediawiki/2018/thumb/f/fb/T--Toulouse-INSA-UPS--All--Yohann--TeamLogo.png/613px-T--Toulouse-INSA-UPS--All--Yohann--TeamLogo.png)](https://2018.igem.org/Team%3AToulouse-INSA-UPS)

* [🏆](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Awards)
* [HOME](https://2018.igem.org/Team%3AToulouse-INSA-UPS)
* TEAM

  [Team Members](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Team)
  [Collaborations](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Collaborations)
  [Sponsors](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Sponsors)
* PROJECT

  [**Description**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Description)
  [**Design**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Design)

  ---

  [Experiments](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Experiments)
  [Notebook](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Notebook)
  [**Results**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Results)
  [**Demonstrate**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Demonstrate)

  ---

  [InterLab](https://2018.igem.org/Team%3AToulouse-INSA-UPS/InterLab)
  [Attributions](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Attributions)
  [**Product Design**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Applied_Design)
* [MODEL](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Model)
* PARTS

  [Parts](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Parts)
  [Basic Parts](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Basic_Part)
* [SAFETY](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Safety)
* HUMAN PRACTICES

  [Human Practices](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Human_Practices)
  [Entrepreneurship](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Entrepreneurship)
  [Education & Engagement](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Public_Engagement)
* [ACHIEVEMENTS](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Achievements)

# NOTEBOOK

---

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

* [LabBook](#NOTEBOOK_Part1)
* [Extra-LabBook](#NOTEBOOK_Part2)

![](https://static.igem.org/mediawiki/2018/1/11/T--Toulouse-INSA-UPS--bannersmall--Brice--Banner.jpg)

* [Sep.](#LabBook_Calendar_Part9)
* [Aug.](#LabBook_Calendar_Part8)
* [Jul.](#LabBook_Calendar_Part7)
* [Jun.](#LabBook_Calendar_Part6)
* [LabBook](#LabBook_Calendar_Part1)

## LABBOOK

---

To achieve our lab work, we split up 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** (and so was dubbed Callulose) and got help from **Younes**. Its mission was to launch and optimize our bacterial cellulose production.

## LABBOOK: JUNE

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 18/06 - 22/06:

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

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 25/06 - 29/06:

* **Team coli**: we started with a PCR on the the gBlock Cerberus-pastoris. In order to clone this gBlock, 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 match our expectations so we had to find another solution.
* **Team callulose**: the first productions of cellulose started in culture tubes.

## LABBOOK: JULY

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 02/07 - 06/07:

* **Team biotin**: this week, we performed a PCR on BirA and we digested the pPIC after its amplification in *E. coli* Stellar cells. This allowed us to do our first cloning with BirA in the pETDuet. We checked the clones by digestion and they were OK. It was necessary to sequence these clones before doing the next cloning in pETDuet. We obtained a different pPIC from a previous year's supplies, 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 constructs by digestion before sending them 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 clone Cerberus-streptavidin though In-fusion into the 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](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 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 PCRs with the parts that will be used to functionalize our Cerberus after biotinylation by BirA.
* **Team coli:** This week, we received the sequencing results for Cerberus and it was again a success for the Toulouse iGEM team. We cloned Sirius (CBM3-RFP) in the pET28 with the In-fusion kit. For 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 match with our digestion results. We needed to find a different pREAV...
* **Team callulose:** we decided to focus the production with *G. hansenii* because it produces more cellulose than *K. rhaeticus*. We also started production in 2L Erlenmeyers.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 16/07 - 20/07:

* **Team biotin:** This week, we focused on the pastoris cloning. We prepared the pGAP vector (amplification, purification and digestion), mRFP1, BFP and scygonadin parts and we cloned all three. 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 DE3 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](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 23/07 - 27/07:

* **Team biotin:** this week, before another midiprep, we checked the pPIC but we still had a mapping problem. After many verifications, it was clearly not correct 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](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 30/07 - 03/08:

* **Team biotin:** this week, we worked on pETDuet-BirA to clone our functionalizing 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 prove 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](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 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](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 13/08 - 17/08:

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

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 20/08 - 24/08:

* **Team coli:** this week was a particularly busy week. We produced and purified Orthos as well as 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 produced Cerberus. To do this, we co-transformed BL21 DE3 cells with pET28 Cerberus and pEVOL AzF. The cell culture medium was complemented with the 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. PCRs were performed on our gBlocks. Unfortunately, we didn’t manage to amplify all the gBlocks.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 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](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 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 movement of cellulose in a presence of a magnet
* **Team pSB1C3:** this week, we tried the pSB1C3 clonings again. 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 for sequencing.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 10/09 - 14/09:

* **Team biotin:** we successfully pulled Orthos down to measure the BFP fluorescence with and without Orthos on cellulose.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 17/09 - 21/09:

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

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 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.

* [Oct.](#OtherBook_Calendar_Part10)
* [Sep.](#OtherBook_Calendar_Part9)
* [Aug.](#OtherBook_Calendar_Part8)
* [Jul.](#OtherBook_Calendar_Part7)
* [Jun.](#OtherBook_Calendar_Part6)
* [May](#OtherBook_Calendar_Part5)
* [Apr.](#OtherBook_Calendar_Part4)
* [Mar.](#OtherBook_Calendar_Part3)
* [Feb.](#OtherBook_Calendar_Part2)
* [Extra-LabBook](#OtherBook_Calendar_Part1)

## Extra-LabBook

---

Here, you can find about some of our activities outside of the lab. More details are provided in the [HP LogBook](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Human_Practices) of the Integrated Human Practices section.

## Extra-LabBOOK: FEBRUARY

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### February 1st, 2018: Kick-off meeting

First meeting of the team, distribution of the tasks and of course we got to know each other.

![](https://static.igem.org/mediawiki/2018/5/54/T--Toulouse-INSA-UPS--NB--Youn--Team.jpg)

The iGEM adventure began!

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### February 8th-22nd, 2018: Brainstorming

At the beginning, more than 50 ideas were suggested. The most original ideas were selected during our brainstorming sessions.

At the end of the month, 11 subjects still needed more investigations.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### February 21st, 2018: Escape game conference

We attended a conference organized by Le Catalyseur on the creation of a teaching escape game. We met two professors in high schools option “Sciences and Technologies of the Laboratory” (STL).

## Extra-LabBOOK: MARCH

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### March 1st-15th, 2018: Brainstorming

Only 5 subjects left!

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### March 10th, 2018: Grimoire

At this event we introduced our card game, Microbioworld.

![](https://static.igem.org/mediawiki/2018/a/aa/T--Toulouse-INSA-UPS--NB--Youn--grim.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### March 20th, 2018: CRISPR/CAS meeting

We met a CRISPR/CAS expert in the LMGM lab who taught us a lot about the new possibilities of the system.

![](https://static.igem.org/mediawiki/2018/2/2b/T--Toulouse-INSA-UPS--NB--Youn--crispr.jpg)

## Extra-LabBOOK: APRIL

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### April 5th, 2018: Final choice of our project

After a long period of brainstorming, we decided to work on the binding of molecules on cellulose. Then, we had to think about the name of our project.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### April 12th, 2018: Tour des Sciences

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

![](https://static.igem.org/mediawiki/2018/7/78/T--Toulouse-INSA-UPS--NB--Youn--tds.jpg)

## Extra-LabBOOK: MAY

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### May 10th, 2018:

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

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### May 31st, 2018:

We met Philippe Serp and his team to talk about experimenting in his lab. We needed them to activate our graphene before using it to functionalise cellulose.

## Extra-LabBOOK: JUNE

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### June 18th, 2018: First day in the lab

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

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### June 19th, 2018: Foundation INSA Toulouse meeting

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

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### June 23rd-24th, 2018: Collaboration with the Montpellier iGEM team

We started a collaboration with the Montpellier team to help them in some areas like the wiki. To do so, we went to Montpellier to talk with them and share our knowledge of the competition.

![](https://static.igem.org/mediawiki/2018/6/60/T--Toulouse-INSA-UPS--NB--Youn--mtp.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### June 25th, 2018: TWB meeting

We met Pierre Monsan, one of the main founders of one of our sponsors, Toulouse White Biotechnology (TWB).

![](https://static.igem.org/mediawiki/2018/a/a6/T--Toulouse-INSA-UPS--NB--Youn--twb.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### June 26th, 2018: NUS meeting

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

![](https://static.igem.org/mediawiki/2018/9/95/T--Toulouse-INSA-UPS--NB--Youn--nusskype.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### June 27th, 2018: Meeting the STL teachers

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

![](https://static.igem.org/mediawiki/2018/1/1f/T--Toulouse-INSA-UPS--NB--Youn--stlvisit.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### June 27th, 2018

For our entrepreneurship approach, 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 us some advice about the possible business model for a Cerberus startup.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### June 29th, 2018: Primary school intervention

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

![](https://static.igem.org/mediawiki/2018/1/19/T--Toulouse-INSA-UPS--NB--Youn--school.jpg)

## Extra-LabBOOK: JULY

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

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

We visited CALMIP, a high-throughput calculations center which allowed us to execute complex algorithms for our modelling.

![](https://static.igem.org/mediawiki/2018/3/3a/T--Toulouse-INSA-UPS--NB--Youn--calmip.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### July 6th, 2018: Official announcement of our project

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

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### July 7th, 2018: 4th Annual parisian meet-up

We took part in the annual parisian meet-up organized by the Pasteur iGEM team.

![Meet up Paris](https://static.igem.org/mediawiki/2018/4/4a/T--Toulouse-INSA-UPS--Collaborations--GB--MeetupParis.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

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

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

![](https://static.igem.org/mediawiki/2018/5/52/T--Toulouse-INSA-UPS--NB--Youn--esof.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### July 19th, 2018: First publications in french newspapers

Newspapers from Toulouse interviewed us about our Cerberus project, we were very happy and grateful for this recognition!

![](https://static.igem.org/mediawiki/2018/c/c7/T--Toulouse-INSA-UPS--NB--Youn--journals.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 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](https://static.igem.org/mediawiki/2018/d/d4/T--Toulouse-INSA-UPS--Collaborations--GB--MeetupMunich.JPG)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 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](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### August 1st, 2018: Laval skype

We contacted the Laval iGEM team in order to create an innovative human practices collaboration.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### August 17th, 2018: Photoshoot for the wiki

We dressed up to take the photos for our wiki.

![](https://static.igem.org/mediawiki/2018/1/1c/T--Toulouse-INSA-UPS--NB--Youn--team.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### August 25th-26th, 2018: Collaboration with Bordeaux iGEM team

We went to Bordeaux to bring them our bacterial cellulose, so they could use it as another carbon source for their Far Waste project.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### 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 session allowed us to structure our company and define our objectives.

![](https://static.igem.org/mediawiki/2018/5/59/T--Toulouse-INSA-UPS--NB--Youn--cat.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### August 31st, 2018: Second workshop with *Le Catalyseur*

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

## Extra-LabBOOK: SEPTEMBER

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### September 4th, 2018

*Le Catalyseur* helped us to create a business model CANVAS.

![](https://static.igem.org/mediawiki/2018/0/00/T--Toulouse-INSA-UPS--NB--Youn--cat2.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### September 5th, 2018:

*Le Catalyseur* helped us to create a SWOT matrix to define our strengths and weaknesses as a business.

## Extra-LabBOOK: OCTOBER

---

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### October 3rd, 2018:

We presented our project in an international high school near Toulouse. This also served as a general repetition for our presentation at the giant Jamboree.

![](https://static.igem.org/mediawiki/2018/3/30/T--Toulouse-INSA-UPS--NB--Youn--ihs.jpg)

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### October 5th, 2018:

We discussed industrial property during another meeting with *Le Catalyseur*.

![icon](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--All--Yohann--StarIcon.png)

#### October 16th, 2018:

We managed to complete all our wiki pages a day earlier, with the precious help of our supervisor Brice. We had to work late into the night, but it was worth it!

![wiki pre-freeze picture](https://static.igem.org/mediawiki/2018/7/73/T--Toulouse-INSA-UPS--HP--Brice--LogBooklast.png)

* [![Up Arrow](https://static.igem.org/mediawiki/2018/1/12/T--Toulouse-INSA-UPS--All--Yohann--Up_Arrow.png)](#TOP)

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

---

The whole **Toulouse INSA-UPS team** wants to thank our sponsors, especially:

![INSA Toulouse](https://static.igem.org/mediawiki/2018/1/1a/T--Toulouse-INSA-UPS--Team--INSAFooter.png)
![UPS](https://static.igem.org/mediawiki/2018/0/02/T--Toulouse-INSA-UPS--Team--UPSFooter.png)
![LISBP](https://static.igem.org/mediawiki/2018/8/8c/T--Toulouse-INSA-UPS--Team--LISBPFooter.png)
![TWB](https://static.igem.org/mediawiki/2018/0/00/T--Toulouse-INSA-UPS--Team--TWBFooter.png)
![Defi](https://static.igem.org/mediawiki/2018/f/f0/T--Toulouse-INSA-UPS--Team--DefiFooter.png)
![Adisseo](https://static.igem.org/mediawiki/2018/0/09/T--Toulouse-INSA-UPS--Team--AdisseoFooter.png)
![Foundation](https://static.igem.org/mediawiki/2018/d/db/T--Toulouse-INSA-UPS--Team--FondationFooter.png)
![ESF](https://static.igem.org/mediawiki/2018/2/2b/T--Toulouse-INSA-UPS--Team--RoiFooter.png)
![Catalyses](https://static.igem.org/mediawiki/2018/5/5d/T--Toulouse-INSA-UPS--Team--Catalyses.png)

And many more. For futher information about our sponsors, please consult **[our Sponsors page](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Sponsors)**.

---

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.*


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Toulouse-INSA-UPS/Experiments

[![](https://static.igem.org/mediawiki/2018/thumb/f/fb/T--Toulouse-INSA-UPS--All--Yohann--TeamLogo.png/613px-T--Toulouse-INSA-UPS--All--Yohann--TeamLogo.png)](https://2018.igem.org/Team%3AToulouse-INSA-UPS)

* [🏆](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Awards)
* [HOME](https://2018.igem.org/Team%3AToulouse-INSA-UPS)
* TEAM

  [Team Members](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Team)
  [Collaborations](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Collaborations)
  [Sponsors](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Sponsors)
* PROJECT

  [**Description**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Description)
  [**Design**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Design)

  ---

  [Experiments](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Experiments)
  [Notebook](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Notebook)
  [**Results**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Results)
  [**Demonstrate**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Demonstrate)

  ---

  [InterLab](https://2018.igem.org/Team%3AToulouse-INSA-UPS/InterLab)
  [Attributions](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Attributions)
  [**Product Design**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Applied_Design)
* [MODEL](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Model)
* PARTS

  [Parts](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Parts)
  [Basic Parts](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Basic_Part)
* [SAFETY](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Safety)
* HUMAN PRACTICES

  [Human Practices](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Human_Practices)
  [Entrepreneurship](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Entrepreneurship)
  [Education & Engagement](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Public_Engagement)
* [ACHIEVEMENTS](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Achievements)

# EXPERIMENTS

---

Here, you will find all the necessary information about the wetlab materials and methods we used in the course of our project.

##### Material

##### Genetic

### *Escherichia coli* vectors

#### pET28

The pET28 vector contains two His Tags in its MCS and is specified for protein expression at high yield. It gives the strain a resistance to Kanamycin and its replication origin is the pBR322 which is a mid - low copy number plasmid (~10). The vector used was a kind gift from IBCG library.

![A generic square placeholder image with rounded corners in a figure.](https://static.igem.org/mediawiki/2018/4/40/T--Toulouse-INSA-UPS--Experiments--Youn--pET28a.PNG)

Figure 1. Map of the pET28 with the MCS Highligthed

#### pETDuet-1

The pETDuet-1 is, like the pET28, specialized in high yield protein production. Its main difference is that it contains two MCS and can therefore express two proteins at the same time under the same expression conditions. It bears ampicilin resistance and is a mid - low copy number plasmid. The plasmid used was a gift from IBCG library.

![A generic square placeholder image with rounded corners in a figure.](https://static.igem.org/mediawiki/2018/d/d1/T--Toulouse-INSA-UPS--Experiments--Youn--pETDuet1.PNG)

Figure 2. Map of the pETDuet-1

### *Pichia pastoris* vectors

#### pPICZ alpha

The pPICZalpha is an integrative plasmid for *Pichia pastoris* protein expression and suitable for *E. coli* replication. Its MCS is suitable for protein expession using the methanol inductible AOX1 Pomoter (PAOX1) and offering the alpha factor secretion signal for protein purification from culture supernatant. It contains a His tag and a Myc Tag for affinity purification stategies.

![A generic square placeholder image with rounded corners in a figure.](https://static.igem.org/mediawiki/2018/8/8e/T--Toulouse-INSA-UPS--Experiments--Youn--pPICZalpha.PNG)

Figure 3. Map of the pPICZ alpha

#### pGAPZalpha

The pGAPZalpha is roughly the same as the pPICZalpha with a different promoter that is the GAP Promoter, a glucose inductible promoter.

![A generic square placeholder image with rounded corners in a figure.](https://static.igem.org/mediawiki/2018/9/97/T--Toulouse-INSA-UPS--Experiments--Youn--pGAPZalpha.PNG)

Figure 4. Map of the pGAPZ alpha

### Primer used

* Cerberus Forward : TAAGAAGGAGATATACCATGGCGGAAGCGGGTATCACC
* Cerberus Reverse : CTCGAGTGCGGCCGCAAGCTTCGGATCGTCCTATGATGGAGG
* Sirius Forward: TAAGAAGGAGATATACCATGAATGCTACGCCAACTAAGGGTGC
* Sirius Reverse: CTCGAGTGCGGCCGCAAGCTTAGCACCGGTGGAGTGACG
* BirA Forward: AAGGAGATATACATATGAAGGATAACACCGTGCCACTGA
* BirA Reverse: CTTTACCAGACTCGATTATTTTTCTGCACTACGCAGGGA
* BFP Forward: ACCACAGCCAGGATCCTATGAGCGAACTGATCAAAGAGAACA
* BFP Reverse: ATGCGGCCGCAAGCTTCTCATGCCATTCAATTTTCTGTGCT
* RFP Forward: AGGAGATATACCATGGCTTCCTCCGAAGACGTTATCAAAG
* RFP Reverse: GTGCGGCCGCAAGCTTAGCACCGGTGGAGTGACG
* Scygonadin Forward: GGCTGAAGCTGAATTCGGCCAGGCACTCAACAAAC
* Scygonadin Reverse: TGGGCCACGTGAATTCTCACTCATGCCATTCAATTTTCTG

##### Microbiological

### *Escherichia coli* strains

**Plasmid amplification strains**

* *Stellar* F-, endA1, supE44, thi-1, recA1, relA1, gyrA96, phoA, Φ80d lacZΔ M15, Δ(lacZYA-argF) U169, Δ(mrr-hsdRMS-mcrBC), ΔmcrA, λ-
* *Top10* F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-

**Protein production strains**

* *BL21(DE3)* B F-*ompT gal dcm lon hsdSB*(*rB-mB-*) λ(DE3[*lacI lacUV5-T7p07 ind1 sam7 nin5*])[*malB+*]K-12(λS)
* *Tuner* F- ompT hsdSB (rB- mB-) gal dcm lacY1(DE3)

### *Pichia pastoris* strains

For protein production we used GS200(ΔARG4, ΔHIS4) Strain for Cerberus production and X33(wt) for Scygonadin

### *Gluconacetobacter hansenii* strain ATCC 53582

##### Chemicals

### Click reagants

* **DBCO-Biotin**, Sigma CAS : 1255942-07-4
* **DBCO-Fluorescein**, Jena Bioscience Cat. No. : CLK-051-1
* **DBCO-Magnetic beads**, Jena Bioscience Cat. No. : CLK-1037-1
* **Fluorescein-Azide**, Jena Bioscience Cat. No. : CLK-80101-5
* **4-L-azidophenylanaline**, Sigma Ref. : 06162

### Graphene Functionalization

* **Graphene**, Sigma Ref. : 900561
* **4-Ethynylaniline**, Sigma Ref. : 481122
* **Isopentyl Nitrite**, Sigma Ref. : 150495
* **N,N-Dimethylformamide**, Sigma Ref. : 227056

### Protein purification

* **Cobalt Affinity Gel**, Sigma Ref. : H8162
* **Protease inhibitor cocktail tablet**, Sigma Ref. : S8830

### Cellulose Binding assay

* **Avicel**, Sigma Ref. : 11365

##### Methods

##### Media & Culture conditions

## Media

---

To prepare each medium, autoclave 20 minutes at 121°C.

### LB Medium

---

##### Cloning & Transformation

## Cloning

---

### 1 . Insert preparation

---

**PCR ClonAMP®**

| Component | Quantity |
| --- | --- |
| Premix ClonAMP® PCR | 25µl |
| Primer 1 | 1.25µl |
| Primer 2 | 1.25µl |
| DNA (0.5 ng/µl) | 4µl |
| Deionized water MQ | 18.5µl |

**PCR ClonAMP program :**

* Cycle 1 : - 98 °C 2min
* Cycle 2 : - 98°C 10s
  - 60°C 5s
  - 72°C 5s/kb
* Cycle 3 : - 72°C 2 min

### 2 . Vector preparation

---

##### 2 . 1 . Miniprep and Midiprep

* ***Miniprep***

This protocol is extracted from the GenElute™ Plasmid Miniprep Kit from Sigma Aldricht.

1. *Harvest cells*
   - High-copy plasmid : 1-3 ml
   - Low-copy plasmid : 1-5 ml
   Pellet 1-5ml of an overnight recombinant *E. coli* culture by centrifuging at **4,500rpm** for **4min**. Discard the supernatant.
2. *Resuspend cells*
   Completely resuspend the bacterial pellet with **200 µl** of the Resuspension Solution. Vortex or pipette up and down to thoroughly mix the cells until homogeneous.
3. *Lyse cells*
   Lyse the resuspended cells by adding **200 µl** of the lysis Solution. Immediately mix the contents by gentle inversion (6-8 times) until the mixture becomes clear and viscous.
   *NB* : Do not allow the lysis reaction to exceed 5 minutes.
4. *Neutralize*
   Add **350 µl** of neutralize solution. Gently invert the tube 4-6 times.
   Pellet the cell debris by centrifuging at **12,000 x g** during **10 minutes**.
5. *Prepare column*
   Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube.
   Add **500 µl** of the Column Preparation Solution to each miniprep column and centrifuge **12,000 x g** during **1min**.
6. *Load cleared lysate*
   Transfer the cleared lysate from step 4 to the column prepared in step 5 and centrifuge at **12,000 x g** for **1 min**. Discard the flow-through liquid.
   Centrifuge again at **12,000 x g** for **1 min**.
7. *Wash column*
   Add **750 µl** of the diluted Wash Solution to the column.
   Centrifuge at **12,000 x g** for **1 min**.
   Discard the flow-through liquid.
   Centrifuge again at **12,000 x g** for **1 min**.
8. *Elute DNA*
   Transfer the column to a fresh collection tube. Add **50 µl** of deionized water (MiliQ).
   Wait **2-3 minutes**.
   Centrifuge **1 minute** at **12,000 x g**.
   The eluate is ready for immediate use of storage at -20°C.

* **Midiprep**

This protocol is extracted from the QIAfilter™ Plasmid Midi Kit (25) from QIAGEN.

High-copy plasmid : 25 ml
Low-copy plasmid : 50-100 ml

1. Harvest bacterial culture after 12-16 h of growth by centriuging at 6000 x g for 15 min at 4°C.
2. Completely resuspend in 4 ml of **Buffer P1**
3. Add 4 ml **Buffer P2** by inverting the sealed tube 4-6 times and incubate at room temperature for up to 5 min. **No more than 5 min !!!**
4. During the incubation, screw the cap onto the outlet nozzle of the QIAfilter Cartridge, and place it in a convenient tube or a QIArack
5. Add 4 ml prechilled **Buffer P3** to the lysate, and mix the solution until it is completely colorless.
6. Pour the lysate into the barrel of the QIAfilter Cartridge. Incubate at room temperature for up to 10min. DO NOT INSERT THE PLUNGER !
7. Equilibrate the QIAGEN-tip by applying 4 ml **Buffer QBT**, and allow the column to empty by gravity flow.
8. Remove the cap from the QIAfilter Cartridge outlet nozzle. Gently insert the plunger into the QIAfilter Cartridge, and filter the cell lysate into the equilibrated QIAGEN-tip. Allow the lysate to enter the resin by gravity flow.
9. Wash the QIAGEN-tip with 20 ml of **Buffer QC**
10. Elute the DNA with 5ml **Buffer QF**.
11. Precipitate DNA by adding 3.5 ml **isopropanol** mix and centrifuge at 4000 rpm for 30min at 4°C. Carefully decant the supernatant.
12. Wash the DNA pelletwith 5 ml **ethanol 70 %** and centrifuge at 4000 rpm for 10 min at 4°C. Carefully decant the supernant.
13. Air-dry the pellet for 20 min and redissolve DNA in 150 µl miliQ water

##### 2 . 3 . Preparative digest gel extraction

---

| Component | Quantity |
| --- | --- |
| DNA plasmid | 5-10µg |
| Cutsmart® buffer | 5µl |
| Enzyme\*\* | 1µl |
| Deionized water MQ | up to 50µl |

​​​​​​​\*\* If you have 2 enzymes 0.5µl of each ones.

#### 3 . Gel extraction purification

---

This protocol is extracted from the NucleoSpin® kit. We would extract both vector and insert with this protocol.

1. Excise DNA fragment and weight it.
2. Solubilize gel slice : for each 100 mg of agarose gel, add 200 µl of Buffer NTI. Incubate sample for 10 min at 50°C, mix by inverting the tube every 2-3 min.
3. Bind DNA : place a column (provided in the kit) into a collection tube (2 ml, also provided) and load up to 700µl sample. Centrifuge for **30s at 11,000g**. Discard flow-through and place the column back into the collection tube.
4. Wash silica membrane : **add 700µl Buffer NT3** to the column. Centrifuge for **30s at 11,000g**. Discard flow-through and place the column back into the collection tube. **Repeat this step** one more time.
5. Dry silica membrane : centrifuge **1 min at 11,000g** to remove Buffer NT3 completely.
6. Elute DNA : place the column into a new 1.5ml Eppendorf. **Add 20 µl of H2O MQ** and incubate at room temperature (18 - 25°C) for 1 min. Centrifuge for **1 min at 11,000g**.

#### 4 . InFusion cloning

---

##### 4 . 1 . Cloning

**Materials**

1. Insert : PCR Fragment purified (ClonAMP®)
2. Vector : Linearized plasmid purified (Cutsmart®)
3. In-Fusion® cloning kit Takara
4. PCR thermocycler

**Protocol**

| Rxn Component | Cloning Rxn |
| --- | --- |
| Purified PCR fragment | 10 - 200 ng\* |
| Linearized plasmid | 50 - 200 ng\* |
| 5X Fusion HD enzyme premix | 2µl |
| Deionized | up to 10 µl |

##### 4 . 2. Transformation into Stellar® competent cells

## Transformation

---

### 1 . *Escherichia coli*

---

##### Protein production & purification

## Protein production

---

### 1. Protein production - *Escherichia coli*

---

**Material**

* LB 10 g/L NaCl (800 ml)
* Your favourite antibiotic
* LB agar plates containing your favourite antibiotic
* IPTG 1M (~1ml)
* Talon Buffer 1x 10mM Imidazole

**Protocol**

* Day one :
  + Transform your plasmid in *E. coli BL21(DE3)* competent cells plate on LB Agar + Your favourite antibiotic and incubate O/N at 37°C
* Day two :
  + Pick 3 colonies and seed then in 9ml LB + Your favourite antibiotic. Incubate O/N at 37°C under 130 rpm shaking.
  + Autoclave 800ml LB 10 g/L NaCl in a 2L baffled flask and preheat it at 37°C O/N.
* Day three:
  + Take a sample of the 800ml culture and measure OD600 as blank.
  + Take 1ml of preculture and measure OD600, seed the preculture in the 800ml LB and take OD.
  + Calculate OD600 assuming that it doubles every 20 minutes and check the OD until it reaches 0.5 ~0.6. Once the exponontial phase is reached, induce with 800µl of 1M IPTG stock solution and incubate at 37°C for 4h or O/N at 16°C.
  + Pellet cells by centrifugate at 5000 x g for 10 minutes and resuspend in 10ml TB 1x 10mM Imidazole if growth was performed at 37°C of 10ml of O/N.
  + Freeze cells for later purification or wait at least 30 minutes to continue the protocol.
  + Sonication : Run on cycle of 1 minute and one of 30 seconds, both at 25 of intensity.
  + Pellet cells by centrifugate at 60,000 x g for 30 minutes and withdraw the supernatant that is the **cell free extract**.
  + Then continue to the protein purification protocol.

### 2. Protein production - *Pichia pastoris*

---

##### Characterization

### Cellulose pull down assay

---

**Material**

* Regenerated Amorphous Cellulose 10 mg/ml
* 50mM Tris HCl pH 8
* Protein sample (concentration about 5mg/ml)
* Ethylene glycol
* RAC previously prepared

**Method**

* Prepare solutions following these amounts :
  + 75µl Tris HCl pH 8
  + 100µl RAC
  + 4µl protein sample (prepare negative control by replacing it with water)
  + 21µl H20 mQ (can aslo be replaced with protein for saturating the RAC)
* Incubate at room temperature for 30 minutes
* Centrifugate at >10,000 x g for 10 minutes
  + Collect supernatant fraction
* Wash with 5 x RAC Volume of Tris HCl
  + Centrifugate at >10,000 x g for 10 minutes
* Eluate protein with 2 x RAC Volume of Ethylene glycol
* Analyse different fractions by SDS PAGE.

### Strain Promoted Alkyne Azide Cycloaddition

**Method**

Add in 100μL a ratio of 1:10 of Azide:DBCO, cover with aluminium foil and incubate O/N under shaking

* [![Up Arrow](https://static.igem.org/mediawiki/2018/1/12/T--Toulouse-INSA-UPS--All--Yohann--Up_Arrow.png)](#TOP)

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

---*

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Toulouse-INSA-UPS/Results

[![](https://static.igem.org/mediawiki/2018/thumb/f/fb/T--Toulouse-INSA-UPS--All--Yohann--TeamLogo.png/613px-T--Toulouse-INSA-UPS--All--Yohann--TeamLogo.png)](https://2018.igem.org/Team%3AToulouse-INSA-UPS)

* [🏆](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Awards)
* [HOME](https://2018.igem.org/Team%3AToulouse-INSA-UPS)
* TEAM

  [Team Members](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Team)
  [Collaborations](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Collaborations)
  [Sponsors](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Sponsors)
* PROJECT

  [**Description**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Description)
  [**Design**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Design)

  ---

  [Experiments](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Experiments)
  [Notebook](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Notebook)
  [**Results**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Results)
  [**Demonstrate**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Demonstrate)

  ---

  [InterLab](https://2018.igem.org/Team%3AToulouse-INSA-UPS/InterLab)
  [Attributions](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Attributions)
  [**Product Design**](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Applied_Design)
* [MODEL](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Model)
* PARTS

  [Parts](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Parts)
  [Basic Parts](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Basic_Part)
* [SAFETY](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Safety)
* HUMAN PRACTICES

  [Human Practices](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Human_Practices)
  [Entrepreneurship](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Entrepreneurship)
  [Education & Engagement](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Public_Engagement)
* [ACHIEVEMENTS](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Achievements)

# RESULTS

---

The whole Cerberus project is based on the construction and validation of a proteic platform to link very different molecules together. This platform is composed of three heads. The first head is a CBM3a domain which specifically binds cellulose. The second head is a streptavidin domain with a strong affinity to biotinylated compounds. The third head is based on the presence of an unnatural amino acid to covalently link any molecule with an alkyne moiety.

To validate the Cerberus platform, we worked head by head, starting from the CBM3a validation, then the streptavidin domain assessment and finally the demonstration of the unnatural amino acid capacity to covalently bound alkynilated molecules. Thus, our first construction has only one head and was named Sirius, after the brightest star of the northern hemisphere, alpha canis majoris. The second construction presents both the CBM3a and streptavidin heads and was nicknamed Orthos, a two-headed mythological dog. The final construction is Cerberus with its three heads.

* [Sirius: CBM3a](#Results_Part1)
* [Orthos: Streptavidin](#Results_Part2)
* [Cerberus: AzF](#Results_Part3)
* [Bacterial Cellulose](#Results_Part4)

![A banner](https://static.igem.org/mediawiki/2018/1/11/T--Toulouse-INSA-UPS--bannersmall--Brice--Banner.jpg)

## Sirius: CBM3a

---

### Background

Sirius is a fusion protein between CBM3a and mRFP1. This design, which consists of fusing CBM3a to a fluorescent moiety, allowed us to investigate the binding capability of the CBM3a domain to cellulose through the four aromatic amino acids of its binding domain. mRFP1 was chosen since it is both a coloured and fluorescent protein.

### Key Achievements

* *Cloning of the part encoding Sirius*
* *Overexpression and purification of Sirius*
* *Fixation of Sirius to cellulose*

### Materials and Methods

The fused CBM3a and mRFP1 sequences were cloned into the pET28 expression vector in fusion with a histidine tag (Figure 1).

![A generic square placeholder image with rounded corners in a figure.](https://static.igem.org/mediawiki/2018/4/4f/T--Toulouse-INSA-UPS--Results--Youn--constructSirius.png)

**Figure 1:** *Scheme of the Sirius construction*

The resulting construct was transformed into *E. coli* strain and expression of the recombinant protein was induced using IPTG. The His-tagged protein was then purified on IMAC resin charged with cobalt and used in cellulose pull down assays. For the experimental details, see [Experiments page](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Experiments).

### Results and Discussion

#### Production and purification of Sirius

Interestingly, *E. coli* turned a deep pink shade after induction, indicating a strong production of Sirius (Figure 2).

![SDS Page](https://static.igem.org/mediawiki/2018/9/96/T--Toulouse-INSA-UPS--Results--Youn--fig2sirius.jpg)

**Figure 2:** *E. coli expressing Sirius*

![SDS Page](https://static.igem.org/mediawiki/2018/c/c3/T--Toulouse-INSA-UPS--Collaborations--GB--SDSPAGE_Analysis.jpg)

**Figure 3:** *SDS-PAGE analysis of Sirius purification. (CFE: cell free extract, FT: flow through, W: washes, E1/40: elution with 40mM imidazole, E1/100: elution with 100 mM imidazole, E2/100: elution with 100 mM imidazole, E1/300: elution with 300 mM imidazole, MW: molecular weight ladder.)*

As shown in Figure 3, we successfully purified the Sirius protein. Indeed, induction with IPTG produced a large amount of a protein at the expected size for Sirius (52 kDa, lane CFE). This protein was then found to be predominant in elution samples (E1/40 and E1/100). We estimated the degree of purity of full-length Sirius at about 72%. In addition to the full-length protein, we observed several extra bands that likely correspond to proteolysis products.

#### Validation

Once produced in , Sirius binding to cellulose was tested using pull down assays. 70 μM of Sirius protein or mRFP1 (without CBM3a) or buffer were incubated with cellulose. After five washes with Tris-HCl buffer, cellulose pellets were recovered and the associated fluorescence was measured.

When mRFP1 was added alone to cellulose, its associated colour mostly remained in the supernatant (Figure 4). When Sirius was associated with cellulose, the pinkish colour was clearly associated to the cellulose pellet. Quantification of the cellulose-associated fluorescence backed up these observations (Figure 5): control experiments showed that only background levels of fluorescence are retained in the cellulose pellet incubated with mRFP1 alone. These experiments have been performed in triplicate and statistical analyses indicate that the high level of fluorescence obtained with Sirius is definitively significant. These results clearly show that the CBM3a domain of Sirius interacts with cellulose, and thus mediates binding of mRFP1 protein to cellulose.

![Fluorescence Retained](https://static.igem.org/mediawiki/2018/6/61/T--Toulouse-INSA-UPS--Results--Youn--CPDSirius.png)

**Figure 4:** *Picture of cellulose pull-down assay with mRFP1 alone (left tube) or with the Sirius construction (right tube)*

![Fluorescence Retained](https://static.igem.org/mediawiki/2018/b/bb/T--Toulouse-INSA-UPS--Collaborations--GB--FluoRetained.jpg)

**Figure 5:** *Binding of Sirius to cellulose. Fluorescence retained in the cellulose pellet after pull down (triplicate test)*

## Orthos: Streptavidin and biotinylated compounds

---

### Background

The next step was to assess the binding capacity of the streptavidin linker. Orthos, named after the guardian of Geryon's cattle, is a fusion protein between CBM3a and a monomeric streptavidin head. The strong affinity of streptavidin for biotin (dissociation constant of 10-13 M) will allow biotinylated organic molecules to be tightly bound to Orthos. We used two types of biotinylated compounds to monitor the ability of our platform to functionalise cellulose: fluorophores (mTagBFP and fluorescein) and antimicrobial peptide (scygonadin).

### Key Achievements

* *Cloning of the part encoding Orthos*
* *Production of Orthos carrying monomeric streptavidin*
* *Production of biotinylated fluorophores* in vitro *and* in vivo
* *Functionalisation of cellulose with biotinylated compounds*

### Materials and Methods

The fusion between monomeric streptavidin and CBM3a was cloned into the pET28 expression vector. The resulting construct was transformed into *E. coli* strain BL21 DE3 and expression of the recombinant protein was induced using IPTG. For Orthos, the histidine tag present on pET28 is not be fused to the CBM3a domain since an amber Stop codon is present between them (Figure 1). The protein was therefore not purified on the IMAC column but purified on Regenerated Amorphous Cellulose (RAC) and used in cellulose pull down assays. For the experimental details, see our [experiments page](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Experiments).

![Fluorescence Retained](https://static.igem.org/mediawiki/2018/7/70/T--Toulouse-INSA-UPS--Results--Youn--constructorthos.png)

**Figure 1:** *Scheme of the Orthos construction*

### Results and Discussion

#### Purification of Orthos

![SDS-PAGE](https://static.igem.org/mediawiki/2018/d/d9/T--Toulouse-INSA-UPS--Collaborations--GB--SDSPAGE_Analysis2.jpg)

**Figure 2:** *SDS-PAGE analysis of purification of Orthos (S: supernatant, W1, W2: washes, E: elution, MW: molecular weight ladder)*

Figure 2 presents the results of the purification of Orthos on RAC. The supernatant (S) contained all proteins produced in *E. coli*, and washes W1 and W2 allowed the elimination of most non-specific interactions with cellulose. The elution step, using ethylene glycol, released the Orthos protein from RAC. The band at about 40 kDa corresponds to Orthos (expected size: 39 kDa) and the band at 15 kDa likely corresponds to a proteolysis fragment of Orthos containing only the CBM3a module.

We estimated that the purification levels of the monomeric Orthos was about 45%, which is sufficient for our validation assays.

#### Validation of monomeric Orthos

##### Validation using *in vivo* biotinylation

In a first set of experiments, we assessed the ability of Orthos to functionalise cellulose with a compound biotinylated *in vivo*. For that purpose, we took advantage of a novel system for *in vivo* protein biotinylation using the biotin ligase BirA. We thus co-expressed BirA and a version of the Blue Fluorescent Protein (BFP) fused to an Avitag in *E. coli*. The Avitag sequence is recognized by BirA for biotin ligation. We added biotin to the culture medium during IPTG induction, in order to produce and purify biotinylated BFP.

We then incubated 3.2 μM of Orthos with 32 μM of *in vivo* biotinylated BFP (Figure 3). For the control experiment BFP (without Orthos), the same quantity of BFP was added to permit a relevant comparison. These samples were then incubated with cellulose. After three washes with Tris-HCl buffer, fluorescence was measured in the cellulose pellets. As shown in Figure 3, fluorescence is twice as intense in Orthos sample than in the control sample containing BFP alone. These experiments have been performed in quadruplicate and a Mann Whitney statistical test indicated that the increased fluorescence signal obtained with Orthos is significant.

![Fluorescence Retained](https://static.igem.org/mediawiki/2018/0/04/T--Toulouse-INSA-UPS--Collaborations--GB--FluoRetainedOrthos.jpg)

**Figure 3:** *Functionalisation of cellulose with Orthos bound to biotinylated BFP. Fluorescence remaining on cellulose fraction after several washes (\*Mann Whitney test p-value 0.01)*

These results demonstrate that Orthos binds efficiently to an *in vivo* biotinylated compound. In addition, they show that Orthos binds to cellulose and thus provide proof of principle that Orthos design allows functionalisation of cellulose with a fluorophore through its streptavidin linker.

##### Validation using *in vitro* chemical biotinylation

In a second set of experiments, we tested the ability of Orthos to functionalise cellulose with *in vitro* biotinylated compounds. Since we had both an azide-functionalised fluorescein (FAM-azide) and biotin-DBCO (an alkyne moiety), we imagined to combine both to produce biotinylated FITC. In addition, it was a nice way to challenge this click chemistry technology. We used the newly emerging technique Cu(I)-free strain-promoted alkyne-azide click chemistry (SPAAC) allowing the coupling *in vitro* of a molecule containing a dibenzocyclooctyne (DBCO) moiety to another molecule bearing an azide function. We used this technique to ligate *in vitro* biotin-DBCO to an azide-functionalised fluorescein (FITC), thus leading to the expected biotinylated FITC.

![Fluorescence Retained](https://static.igem.org/mediawiki/2018/5/52/T--Toulouse-INSA-UPS--Collaborations--angeline--FluoRetainedOrthos2.jpg)

**Figure 4:** *Functionalisation of cellulose with Orthos bound to *in vitro*-biotinylated FITC. Fluorescence remaining on cellulose pellet fraction after several washes (quadruplicate test). (\*Mann Whitney test p-value 0.1)*

Then, 58.0 μM of biotinylated FITC was incubated with 5.8 µM of Orthos protein. The same amount of FITC alone (58.0 μM) was used for the control experiment. These samples were incubated with cellulose and after five washes with Tris-HCl buffer, fluorescence levels were measured in the cellulose pellet fractions (Figure 4). In the presence of Orthos, fluorescence in the cellulose pellet was about twice as high as in control experiments corresponding to the cellulose pellet incubated with FITC alone (without Orthos) or with the reaction buffer only. These experiments were performed in quadruplicate and a Mann Whitney statistical test indicated that the increased fluorescence signal obtained with Orthos is very significant. This result clearly shows that purified Orthos retains the ability to mediate the interaction between cellulose and a compound biotinylated *in vitro*. In addition, this result provides another strong evidence that Orthos allows cellulose to be functionalised using a fluorescent molecule through its streptavidin linker. It also shows that we succeeded in creating biotinylated FITC by click chemistry.

##### Validation using scygonadin

In order to confirm that the streptavidin moiety of Orthos is able to interact with different kind of biotinylated compounds, we also tried to bind it to an antimicrobial peptide, the scygonadin. To biotinylate this compound, we used the *in vivo* protein biotinylation system as the one used for BFP molecule. We co-expressed BirA with a version of scygonadin fused to the Avitag in *Pichia pastoris*.

We then incubated Orthos with *in vivo* biotinylated scygonadin, and few hours later, we added cellulose (under form of a cellulose spot). After two washes with Tris-HCl buffer, cellulose spots with Orthos-scygonadin were deposed on Petri dish pre-inoculated with a *E. coli* layer. We observed small halos of inhibition for some samples after incubation overnight (Figure 5).

![Inhibition halo](https://static.igem.org/mediawiki/2018/5/55/T--Toulouse-INSA-UPS--Collaborations--angeline--Halo.jpg)

**Figure 5:** *Halo of inhibition of Orthos alone (C), scygonadin alone (S) and Orthos+scygonadin (C+S) samples*

We noticed that the inhibition halo was more important for the Orthos-scygonadin sample than the scygonadin alone control. Unfortunately, we repeated this test, but we did not obtain the same result. Therefore, we need to improve our experiment to prove the coupling between Orthos and biotinylated scygonadin. These results are nevertheless encouraging in the perspective of functionalising cellulose using Orthos bound to antibiotic protein.

### Perspectives

---

Our experiments using different kinds of biotinylated compounds provide convincing proofs of concept that Orthos can be conjugated, via its streptavidin head, to biotinylated organic molecules. The resulting molecules interact with cellulose and are therefore potent cellulose functionalising compounds.

We can now envision exploiting the streptavidin linker of Orthos to ligate various compounds that can be biotinylated chemically (*in vitro*) or *in vivo*, via BirA co-expression.

In addition, we incorporated a TEV protease cleavage between the *N*-terminus linker and the streptavidin. This will allow to produce Orthos-bound biotinylated compounds that can be released by TEV cleavage if required, as in the case of odorant molecules for examples for a commercial use.

## Cerberus: AzF

---

### Background

Cerberus, named after the guardian of the gates of the Underworld, is our three-headed protein made of the cellulose-binding molecular platform (CBM3a) fused at the N-terminus to the biotinylated molecule-binding head (monomeric streptavidin) and at the C-terminus to the unnatural amino acid azido-L-phenylalanine (AzF). The incorporation of AzF is achieved through the recognition of the amber stop codon at the C-terminus of CBM3a by an orthogonal AzF-charged tRNA.

![Inhibition halo](https://static.igem.org/mediawiki/2018/8/85/T--Toulouse-INSA-UPS--Results--Youn--constructcerberus.png)

**Figure 1:** *Scheme of the Cerberus construction*

Coupling of molecules to the AzF head can be performed by click chemistry, either through SPAAC or through Cu(I)-catalyzed azide-alkyne click chemistry (CuAAC). To prove the versatility of the system, different compounds such as fluorescent proteins or paramagnetic beads have been clicked onto Cerberus and thus associated with cellulose.

### Key Achievements

* *Cloning of the part encoding Cerberus*
* *Production of Cerberus*
* *Functionalisation of cellulose*

### Materials and Methods

The fusion between the streptavidin linker and the CBM3a domain sequences followed by the amber stop codon was cloned into the pET28 expression vector in fusion with the His-tag. The resulting construct was co-transformed into *E. coli* strain with the pEVOL expression vector. The latter allows to the expression of the amino acyl tRNA synthetase aaRS. In presence of AzF, this enzyme synthesizes the orthogonal AzF-tRNA for in vivo AzF incorporation in place of amber Stop codon. The Cerberus platform was produced by adding IPTG (Cerberus induction), L-arabinose (amino acyl tRNA synthetase production), and AzF to the medium. The His-tagged protein was then purified on IMAC resin charged with cobalt and used in cellulose pull down assays. For the experimental details, see [experiments page](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Experiments).

### Results and Discussion

#### Production and purification of Cerberus

Cerberus production requires a deferred induction. We first induced the synthesis of aarS/tRNA pair with L-arabinose, and added AzF at the same time. One hour later, we induced the production of Cerberus with IPTG.

![SDS-PAGE](https://static.igem.org/mediawiki/2018/7/70/T--Toulouse-INSA-UPS--Collaborations--angeline--SDSPAGE_cerberus.jpg)

**Figure 2:** *SDS-PAGE analysis of the production of Cerberus with monomeric streptavidin (NI: non-induced, I-AzF: induced without AzF, I+AzF: induced with AzF, E1: elution with 40 mM imidazole, E2: elution with 100 mM imidazole, E3: elution with 100 mM imidazole, MW: molecular weight ladder)*

Cerberus production and purification were analysed on SDS gel (Figure 2). Induction in the presence of AzF (Figure 2, lane I+AzF) led to the expression of two major bands at 37 and 39 kDa approximately compared to control conditions, namely the non-induced (lane NI) and induced without AzF (lane I-AzF). We hypothesized that the upper band would correspond to Cerberus (expected molecular weight of Cerberus with monomeric streptavidin: 41 kDa) and the lower bands to the Orthos proteins (Cerberus without the AzF and the His-tag). Surprisingly, both bands were present in the elution fractions which is incoherent since Orthos lacks an His-tag and was supposed to be washed out during purification steps. To confirm our hypothesis, we analysed the same fractions by western blot using antibodies detecting the His-tag (Figure 3).

![Western blot](https://static.igem.org/mediawiki/2018/0/01/T--Toulouse-INSA-UPS--Collaborations--angeline--SDSPAGE_cerberus2.jpg)

**Figure 3:** *Western blot analysis of Cerberus production (NI: non-induced, I-AzF: induced without AzF, I+AzF: induced with AzF, E: elution, MW: molecular weight ladder)*

Anti-His-tag antibodies revealed a band at about 45 kDa in the sample corresponding to IPTG induction in the presence of AzF (Figure 3, lane I+AzF). This band is not present in control samples (Figure 3, lane NI and I-AzF), indicating that it corresponds to the Cerberus protein (theoretical size: 41 kDa). In addition to the full length protein, we observed several extra bands which very likely correspond to proteolysis products since they are detected with the anti-His-tag antibodies. Moreover, the band at 45 kDa is clearly detected in elution samples (Figure 3, lanes E). The band at 37 kDa observed on the SDS PAGE (Figure 2) is not observed on the Western blot. These results show that the experimental setup to produce Cerberus is also likely leading to the production of Orthos when the amber stop codon is not recognized by the AzF-charged orthogonal tRNA. Although Orthos does not contain a His-tag at its C-terminus, the protein seems to be efficiently co-purified with Cerberus. The basis of this observation is unclear but it may suggest that Orthos and Cerberus interact with each other, via their CBM3 or streptavidin moieties.

We estimated that the purification level of Cerberus with monomeric streptavidin in the elution samples was about 62%. These data show that Cerberus was efficiently purified and can be used for pull down assays.

#### Validation

##### Validation using FITC (Fluorescein isothiocyanate) molecules

![Fluorescence Retained](https://static.igem.org/mediawiki/2018/f/f8/T--Toulouse-INSA-UPS--Collaborations--angeline--FluoRetainedCerberus.jpg)

**Figure 4:** *Functionalisation of cellulose with FITC conjugated to Cerberus. Fluorescence remaining in cellulose fraction after several washes (quadruplicate test). (\*Mann Whitney test p-value 0.03)*

To test the potential of Cerberus in functionalising cellulose, we monitored its ability to mediate an interaction between cellulose and a fluorescent compound. To generate a fluorescently labelled Cerberus protein, we performed a SPAAC reaction on 3.2 µM Cerberus protein using 31.9 µM of FITC-DBCO. In control experiment, 31.9 µM of FITC alone was used. These samples were then incubated with cellulose and after five washes with Tris-HCl buffer, fluorescence levels were measured in the cellulose pellet fractions (Figure 9). We observed that the fluorescence levels in the cellulose pellet incubated with Cerberus protein clicked to FITC were about 3 times higher than in the control experiments corresponding to the cellulose pellet incubated with FITC alone. These results show that Cerberus can efficiently be conjugated to fluorescent molecules bearing a DBCO group by click chemistry and that the resulting fluorescent molecules strongly interact with cellulose. Therefore, Cerberus is both a convenient and potent platform to functionalise cellulose.

##### Validation using paramagnetic beads

We further characterize the functionality of Cerberus in a second set of experiments using paramagnetic beads. To bind paramagnetic beads to Cerberus, we performed a click reaction using 3.2 µM of Cerberus protein and 32 µM DBCO-conjugated paramagnetic beads. As a control experiment, 32 µM of paramagnetic beads were used. These samples were then incubated with cellulose, and after five washes with Tris-HCl buffer, we measured the magnetisation of cellulose using a magnet.

[
](https://static.igem.org/mediawiki/2018/1/14/T--Toulouse-INSA-UPS--Collaborations--angeline--ferocell.MOV)

**Figure 5:** *Video of the Cerberus-functionalised paramagnetic cellulose: on the left, control with paramagnetic beads alone; on the right, Cerberus-paramagnetic beads.*

As observed on the video (Figure 5), the cellulose incubated with the Cerberus protein conjugated to paramagnetic beads is quickly and totally attracted by the magnet. In contrast, in the control experiment, no clear movement towards the magnet was observed.

These results show that Cerberus has the ability to interact simultaneously with cellulose and molecules with a DBCO group, confirming that the Cerberus platform allows to functionalise cellulose through its linker containing unnatural amino acid.

#### Towards other functionalisations

To go further in our project, we decided to functionalise two inorganic molecules, graphene and carbon nanotubes (CNT), using a reaction of diazotization. This experiment was performed with the help of the ENSIACET laboratory in Toulouse. To check functionalisation, we analysed samples by Thermogravimetric Analysis (TGA). The concept of TGA consists of measuring the mass change of a sample depending on temperature and time, in a controlled atmosphere. We chose to perform the experiment under nitrogen, N2, an inert gas that avoids mass loss by oxidation, and we studied the mass change from 25 to 1,000 °C (15 °C/min).

![Mass loss](https://static.igem.org/mediawiki/2018/e/ed/T--Toulouse-INSA-UPS--Collaborations--angeline--massloss1.jpg)

**Figure 6:** *Analysis of mass loss for functionalised CNT sample*

![Mass loss](https://static.igem.org/mediawiki/2018/c/cb/T--Toulouse-INSA-UPS--Collaborations--angeline--massloss2.jpg)

**Figure 7:** *Analysis of mass loss for non-functionalised CNT sample*

Figures 6 and 7 present the results of mass loss for non-activated Carbon Nanotube (CNT) and activated CNT samples, respectively. For activated CNT sample (Figure 7), we observed a mass loss of 27.4% around 100 °C corresponding to water loss. A second step of mass loss is observed around 550 °C, with a diminution of 19.8%. For the non-activated sample (Figure 6), we observed two mass losses at 150 and 750 °C corresponding to 1.5% and 3% of mass loss respectively. The significative difference between these two samples allowed us to conclude that the CNT activation has been successful.

![Mass loss](https://static.igem.org/mediawiki/2018/1/1e/T--Toulouse-INSA-UPS--Collaborations--angeline--massloss3.jpg)

**Figure 8:** *Analysis of mass loss for activated graphene sample*

![Mass loss](https://static.igem.org/mediawiki/2018/4/40/T--Toulouse-INSA-UPS--Collaborations--angeline--massloss4.jpg)

**Figure 9:** *Analysis of mass loss for non-activated graphene sample*

Figures 8 and 9 present the results of mass loss for non-activated graphene and activated graphene samples respectively. For activated graphene sample (Figure 9), we observed a mass loss of 31.0% around 100 °C corresponding to water loss. A second step of mass loss is observed around 500 °C, with a diminution of 19.0%. For the non-activated sample (Figure 8), we did not observe a significative mass loss. So we can conclude that the graphene activation has been successful.

Our initial plan was to functionalise cellulose with CNT and graphene in order to change cellulose fibre rigidity and create conductive cellulose, respectively. Unfortunately, we did not have enough time during the course of the iGEM competition to complete these tests. Given the success of the other functionalisation, we have reasonable hopes that these experiments will be successful too.

### Perspectives

Our experiments provide robust proofs of concept that Cerberus can be conjugated, via its unnatural amino acid AzF, to organic or inorganic molecules bearing a DBCO group by click chemistry. The resulting molecules strongly interact with cellulose and are therefore potent cellulose functionalising compounds.

We can now consider fixation of various compounds that can be chemically functionalised. We can also imagine double fixations, for example a fluorophore on the streptavidin head and paramagnetic beads on the AzF linker. This double fixation allows us to envision endless possibilities to functionalise cellulose (see [Demonstrate](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Demonstrate)).

## Bacterial Cellulose production and functionalisation

---

### Background

Our ultimate dream was to be able to produce functionalised cellulose *in vivo*. Some microorganisms naturally produce cellulose. One of these, *Gluconacetobacter hansenii*, can produce cellulose as a biofilm to protect itself. We took advantage of the work of iGEM Imperial team 2014 who set up culture conditions to optimize cellulose production. Our challenge was to assess if our technology is compatible with bacterial cellulose as a first step before producing functionalised cellulose *in vivo*.

### Key Achievements

* *Production of about 100g of dried bacterial cellulose*
* *Functionalisation of bacterial cellulose with Sirius*

### Materials and Methods

*G. hansenii* was grown in Hestrin-Shramm (HS) medium at 30 °C. After one week of culture, bacterial cellulose was extracted and purified. For the experimental details, see our [experiments page](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Experiments).

For the functionalisation, bacterial cellulose was incubated with purified Sirius (CBM3a-mRFP1) or with purified mRFP1 alone as a control. Cellulose was then washed about 10 times with Tris-HCl buffer buffer to remove non-attached proteins.

### Results and Discussion

![Fluorescence Retained](https://static.igem.org/mediawiki/2018/d/d2/T--Toulouse-INSA-UPS--Results--Youn--celluloseUV.jpg)

**Figure 1: a)** *Picture of functionalised cellulose with Sirius (CBM3a fused with mRFP1). Left: control mRFP1 alone; Right: Sirius ;***b)** *Picture under UV of functionalised cellulose with Sirius (CBM3a fused with mRFP1). Left: control mRFP1 alone; Right: Sirius*

Figure 1a presents the results of the functionalisation of our bacterial cellulose with the Sirius fluorescent protein. Only the cellulose sample incubated with Sirius kept the color after several washes with resuspension buffer. Likewise fluorescence remained associated to cellulose only for Sirius (Figure 1b). These results show that CBM3a can interact with the bacterial cellulose and allows fixation of the mRFP1 protein to cellulose.

### Perspectives

Now that we have proved the capacity of CBM3a to interact with bacterial cellulose, we are confident that Orthos and Cerberus proteins should also interact with bacterial cellulose. Now the next step will be to produce functionalised bacterial cellulose *in vivo* . For this, we had planned to produce the Cerberus platform in the yeast *Pichia pastoris* because of its secretion capacity. Indeed, our idea was to co-culture both strains in the same reactor in order to produce functionalised cellulose *in vivo*. This work on *P. pastoris* was performed at the same time as our attempt in *E. coli* but has not been as successful. We managed to produce the Orthos protein in the yeast but we never succeeded in producing Sirius and we never got the plasmid needed to produce the tRNA synthetase necessary for AzF incorporation in *P. pastoris*. In addition, the production of Orthos in the yeast was very low and not sufficient to conduct our assays. So production of functionalised cellulose *in vivo* will require improving the *P. pastoris* part of our project.

* [![Up Arrow](https://static.igem.org/mediawiki/2018/1/12/T--Toulouse-INSA-UPS--All--Yohann--Up_Arrow.png)](#TOP)

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

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The whole **Toulouse INSA-UPS team** wants to thank our sponsors, especially:

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And many more. For futher information about our sponsors, please consult **[our Sponsors page](https://2018.igem.org/Team%3AToulouse-INSA-UPS/Sponsors)**.

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