iGEM 2017 — Team INSA-UPS_France

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# Team:INSA-UPS France/Notebook

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Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Team

* [Members](https://2017.igem.org/Team%3AINSA-UPS_France/Team)
* [Supports](https://2017.igem.org/Team%3AINSA-UPS_France/Supports)
* [Attributions](https://2017.igem.org/Team%3AINSA-UPS_France/Attributions)
* [Collaborations](https://2017.igem.org/Team%3AINSA-UPS_France/Collaborations)

Strategy

* [Description](https://2017.igem.org/Team%3AINSA-UPS_France/Description)
* [Design](https://2017.igem.org/Team%3AINSA-UPS_France/Design)
* [Parts](https://2017.igem.org/Team%3AINSA-UPS_France/Parts)

Model

* [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Model)
* [Simulation](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Simulation)
* [Analysis](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Analysis)
* [Interface](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Interface)

Realisations

* [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments)
* [Notebook](https://2017.igem.org/Team%3AINSA-UPS_France/Notebook)
* [Clonings](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings)
* [Results](https://2017.igem.org/Team%3AINSA-UPS_France/Results)
* [Contribution](https://2017.igem.org/Team%3AINSA-UPS_France/Contribution)
* [Protocols](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols)
* [Safety](https://2017.igem.org/Team%3AINSA-UPS_France/Safety)

Human Practices

* [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Human_Practices)
* [Silver](https://2017.igem.org/Team%3AINSA-UPS_France/HP/Silver)
* [Gold](https://2017.igem.org/Team%3AINSA-UPS_France/HP/Gold_Integrated)
* [Public Engagement](https://2017.igem.org/Team%3AINSA-UPS_France/Engagement)
* [Microbioworld](https://2017.igem.org/Team%3AINSA-UPS_France/Microbioworld)

Application

* [Applied Design](https://2017.igem.org/Team%3AINSA-UPS_France/Applied_Design)
* [Testimonies](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Testimonies)
* [Entrepreneurship](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship)
* [Device Conception](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Device)

# Notebook

![](https://static.igem.org/mediawiki/2017/5/53/T--INSA-UPS_France--Labbook.png)

Here is a summary of what we did for our project, week by week.
You can go on our [Human Practices labbook](https://2017.igem.org/Team%3AINSA-UPS_France/HP/Silver) page to know more about how each event affected us to take a decision about the future of our project.

Brainstorming meeting

Team day

Meetup with other iGEM teams

Public Engagement event

Integrated Human Practices / Entrepreneurship major step

Experiments

### January

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| 22 | 23 | 24 | 25 | 26 | 27 | 28 |
| 29 | 30 | 31 | 1 | 2 | 3 | 4 |

### February

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 29 | 30 | 31 | 1 | 2 | 3 | 4 |
| 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 12 | 13 | 14 | 15 | 16 | 17 | 18 |
| 19 | 20 | 21 | 22 | 23 | 24 | 25 |
| 26 | 27 | 28 | 1 | 2 | 3 | 4 |

### March

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 26 | 27 | 28 | 1 | 2 | 3 | 4 |
| 5 | 6 | 7 | 8 | 9 | 10 | 11 |
| 12 | 13 | 14 | 15 | 16 | 17 | 18 |
| 19 | 20 | 21 | 22 | 23 | 24 | 25 |
| 26 | 27 | 28 | 29 | 30 | 31 | 1 |

### April

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 26 | 27 | 28 | 29 | 30 | 31 | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| 30 | 1 | 2 | 3 | 4 | 5 | 6 |

### May

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 30 | 1 | 2 | 3 | 4 | 5 | 6 |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 | 1 | 2 | 3 |

### June

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 28 | 29 | 30 | 31 | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| 25 | 26 | 27 | 28 | 29 | 30 | 1 |

### July

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 25 | 26 | 27 | 28 | 29 | 30 | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| 30 | 31 | 1 | 2 | 3 | 4 | 5 |

### August

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 30 | 31 | 1 | 2 | 3 | 4 | 5 |
| 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 13 | 14 | 15 | 16 | 17 | 18 | 19 |
| 20 | 21 | 22 | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 | 31 | 1 | 2 |

### September

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 27 | 28 | 29 | 30 | 31 | 1 | 2 |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |

### October

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| 22 | 23 | 24 | 25 | 26 | 27 | 28 |
| 29 | 30 | 31 | 1 | 2 | 3 | 4 |

# Week 1

Our team was being recruited. Nothing happened this week!

# Week 2

Our team was being recruited. Nothing happened this week!

# Week 3

Our team was being recruited. Nothing happened this week!

# Week 4

## 01-25: Kick Off Meeting

Gathering the team members and distribute the tasks... The adventure begins!

![](https://static.igem.org/mediawiki/2017/5/57/T--INSA-UPS_France--Notebook_Week-4.jpg)

# Week 5

## 01-30: 2th brainstorming: on the track of crocodile antimicrobial peptides...

We’ve took charge of differents accounts of the association (facebook, twitter, email, bank account, google drive…), and made a listing of 45 potential subjects for the competition.

# Week 6

## 02-06: 3th brainstorming meeting

We’ve selected 17 subjects that we considered the most originals, the most feasible, and the most interesting. We’ve imagined to make a dressing against nosocomial infections containing bacteria that produces antimicrobial peptides from crocodile. We’ve read that crocodile peptides are not toxic for humans at microbicide quantity.

# Week 7

## 02-16: 4th brainstorming

Only 7 subjects left! To make a safe device, we’ve thought of switch on the dressing by using spores that can be activated with the temperature.

## 02-13 to 02-17: Training course for INSA students at LISBP with our instructor Brice Enjalbert

# Week 8

## 02-23: 5th brainstorming

We made a list of our public engagement events. For our subject, we’ve focused our research on the originality, and we’ve noted that a similar dressing has already been made by an iGEM team before. So we imagined the use of anticoagulant molecules in the dressing. We’ve thought of a bandage for pets.

# Week 9

## 03-02: 6th brainstorming

We’ve analysed the 7 subjects, and we’ve noted some technical issues. For the “crocodile peptides project”, we’ve raised several questions : what organisme should we use to produce antimicrobial peptides? We need to find an organism which is not susceptible to their antimicrobial activity. We need to use a special membrane to contain GMOs inside the dressing and not directly in contact to the skin.

# Week 10

## 03-05: Team building at the restaurant

Our team met for the first time outside the INSA buildings and shared a delicious korean meal after brainstorming the whole afternoon!

![](https://static.igem.org/mediawiki/2017/6/60/T--INSA-UPS_France--Notebook_Week-9.jpg)

## 03-09: 7th brainstorming meeting

We’ve voted for our 4 favourite subjects : “crocodile antimicrobial peptides”, “bioluminescent living board”, “expiration pastille”, and “colourful cellular cycle”.

# Week 11

## 03-12: Brainstorming in a café

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Notebook_Week-10.jpg)

## 03-14: 8th brainstorming

The problem of originality persists for the application: what about a suture thread? For the peptide-producer microorganism, a yeast could be a good solution because the antimicrobial activity of our peptide is bacterial-specific. For the activation of the peptide production, we’ve thought about lyophilisating the engineered yeast.

# Week 12

## 03-21: 9th brainstorming

In view of what has already been done in iGEM for  detection of quorum sensing molecules, it seems that we’ll need a bacteria to detect other quorum sensing molecules because it’s difficult to express a prokaryotic membrane-receptor into a yeast. So we’ve imagined a system with a prokaryote that detect a quorum sensing signal from a pathogenic bacteria, and that triggers peptide production by a yeast.

# Week 13

## 03-26: Brainstorming in a café

![](https://static.igem.org/mediawiki/2017/f/fa/T--INSA-UPS_France--Notebook_Week-12.jpg)

## 03-30: 10th brainstorming

A second device is born: a shaker containing our too organisms (prokaryote-detector and yeast-peptide-producer) to detect and kill *Vibrio cholerae* in the water. We had a idea for our public engagement project: creating a pedagogic card game about microbiology. *MicrobioWorld* conception is in process...

# Week 14

## 04-06: 11th brainstorming

Dropping of the first device because ALS detection of one single Staphylococcus aureus is going to be difficult. In contrary, *Vibrio cholerae* produces a high quantity of CAI-1, a specific quorum sensing molecule, at pathogenic quantity.

## 04-07: choice of the subject

We’ve finally choose to work on cholera water treatment using a prokaryotic-eukaryotic communication.

# Week 15

## 04-13: 12th brainstorming

Actual solution to fight against cholera are not sufficient to prevent great epidemics, we need to contact NGOs to benefit from their experience of the field of a cholera epidemy. For the proof of concept, we need to find an effective cholera detector, and a good peptide producer. The detector bacteria could be *E. coli*, or maybe *V. harveyi*, that already possess the quorum sensing receptor of CAI-1, and all the proteins to do the phosphorylation cascade in response of CAI-1.

## 04-10 to 04-14 Training course for the UPS students at LBME with our instructors Anthony Henras and Yves Romeo

# Week 16

## 04-19: Meeting with Marc Lemonnier, CEO of Antabio

We met Marc Lemonnier, founding CEO of ANTABIO: a start-up that develops novel therapies to treat drug-resistant infections by the most critical Gram-negative pathogens. This was our first meeting within the Entrepreneurship part of the project. The aim was to learn about the approach to follow to start a business and to have a first external opinion regarding the integration of our biological system into the market. You can read the result of [his testimony here.](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Testimonies#a1)

## 04-20: 13th brainstorming

We’ve found a company called “Sunwaterlife” that work on water treatment by filtrating water in Toulouse. We’ve planned to meet them to take advices from their experience, and ask if they could help us to build our device to contain GMMs into a membrane to avoid their dispersion into the environment. For the peptide-producer yeast, we’ve selected *Pichia pastoris*, famous for it’s capacity of fast production of proteins. To make a communication between our two organisms, we’ve thought of using diacetyl: our detector organism produces diacetyl in response of CAI-1 detection, that triggers AMP by *P. pastoris*.

# Week 17

## 04-23: First drafts of Sobki, our mascot!

![](https://static.igem.org/mediawiki/2017/0/02/T--INSA-UPS_France--Notebook_Week-16.jpg)

# 04-25:

Following our appointment with Marc Lemonnier, we began to think about a device so that our system could treat the water in a tank for villagers or the water of a flask for the tourists. The challenge is to confine the microorganisms so that they do not go into the water.

## 04-26: 14th brainstorming

For safety reasons we can’t use *V. cholerae* for our proof of concept, and we can’t use any gene from that bacteria. As *V. harveyi* is genetically close to *V. cholerae* but not pathogenic for human, we can use it’s quorum sensing molecule, the C8-CAI-1 that looks very much as the CAI-1, to mimic *V. cholerae*. Here, the idea is to engineer a bacteria to be a “cholerae-like bacteria” that produces a lot quorum sensing molecules to be detected by our detector *V. harveyi*.

## 04-27:

Téo and Margaux went to a conference about *V. cholerae* to learn more about genetics of that bacteria, and the main differences between *V. cholerae* and *V. harveyi*.

# Week 18

## 05-02: 15th brainstorming

We need to evaluate the volume of water to treat. In the case of our device contain 1L water, it’s for only 1 person but we can imagine to treat a high volume for an entire village. We’ve prepared a list of questions to ask to NGOs to make a user friendly device.

## 05-07:

We’ve met 2 game designers and graphists students, Vincent and Julien, to help us in the conception of our card game about microbiology! They will make the graphism designs of our card game.

# Week 19

## 05-11: 16th brainstorming

For the detection of diacetyl, we found the receptor Odr10, that activate the promoter pFUS for peptide production. For diacetyl production, the gene ALS is known to enhance diacetyl production, so we’ll need to express ALS gene in *V. harveyi* only when C8-CAI-1 is detected. This is complicated because the receptor of C8-CAI-1 induces a repression of the promoter pqrr4. We need to find a inhibitor regulation in our system.

## 05-13: Paul & Margaux officialy joined our team!

![](https://static.igem.org/mediawiki/2017/6/6a/T--INSA-UPS_France--Notebook_Week-19_1.jpg)

# Week 20

## 05-17: 17th brainstorming

We planed to do NMR analysis to detect C8-CAI-1 and diacetyl for the proof of concept. We’ve asked to Bassler’s lab to give us 2 *V. harveyi* strains: a WT one, and the JMH626 strain deleted from the gene CqsA, which can’t produce C8-CAI-1 by itself. We’ll need that strain to detect C8-CAI-1 from our engineered “*E. coli* cholera-like”. Purified diacetyl has been commanded to do induction experiments on *P. pastoris*. We find the Tet on/ Tet off repression system for diacetyl production in response to C8-CAI-1.

## 05-18: iGEM distribution kit received

![](https://static.igem.org/mediawiki/2017/7/70/T--INSA-UPS_France--Notebook_Week-20.jpg)

## 05-21 Sobki is born!

![]()

# Week 21

We made a survey about cholera disease and about our project to know if people are aware of cholera epidemics in Africa and the existing solutions, and to know if people would be ready to use a device containing GMMs to treat the water they drink.

## 05-25: Parts design day at Brice's

![](https://static.igem.org/mediawiki/2017/f/f0/T--INSA-UPS_France--Notebook_Week-21_2.jpg)

# Week 22

## 05-28: Official announcement of our subject

We officialy the subject of our project on social media!

## 05-30 and 31: Exposciences

We took part to “Exposciences” which is a scientific festival. During this event we made children extract banana DNA thanks to simple ingredients that they can find in their kitchen. We also discussed with them about microorganisms thanks to paper fortune teller and this support led them to be curious on the microscopic world around them.

![](https://static.igem.org/mediawiki/2017/f/fb/T--INSA-UPS_France--Engagement-Exposciences_1.jpg)

## 06-01: Interventions in schools

The main goals of these interventions were the discovery of biology and research at school with two workshops: Microorganisms and their environment and growth of microorganisms on a Petri dish.

![](https://static.igem.org/mediawiki/2017/6/61/T--INSA-UPS_France--Notebook_Week-22.jpg)

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Design and orders

* Design of the gBlocks and launching of the synthesis order for IDT
* Design of the PCR oligos

## Design and orders

* Design of the gBlocks and launching of the synthesis order for IDT
* Design of the PCR oligos

# Week 23

## 06-06: Meeting with the CEO of Sunwaterlife

We presented our project to Christophe Campéri-Ginestet who seems to be interested, especially by the detection function of *V. cholerae*. He would like to adapt it to the Sunwaterlife filters in order to have a direct colored response according to the water contamination. In addition, he would be willing to invest in our project in order to apply for JEI status (or “Young Innovative Enterprises” in english), a very interesting status for start-ups allowing them to have financial advantages. Besides, a collaboration is discussed so that Sunwaterlife provide us with their membranes, allowing us to confine our microorganisms within a device. He told us that he might be able to put us in touch with a UNICEF person in charge of cholera epidemics.

You can read the result of [his testimony here.](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Testimonies#a4)

## 06-08: 18th brainstorming

We’ve divided the subject in 3 modules. The “Quorum” module, with *E. coli* engineered to produce C8-CAI-1; the “Detection” module, with V. harveyi engineered to detect C8-CAI-1 and produce diacetyl in response; and the “Killing” module with *P. pastoris* engineered to detect diacetyl and produce crocodile AMP in response.

## 06-09: Intervention in school

The main goals of these interventions were the discovery of biology and research at school with two workshops: Microorganisms and their environment and growth of microorganisms on a Petri dish.

![](https://static.igem.org/mediawiki/2017/6/60/T--INSA-UPS_France--Engagement_school1.JPG)

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Taking care of *V. harveyi*

* Reception and storage of *V. harveyi* BB120 (WT) and *V. harveyi* JMH626 (ΔcqsA ΔluxQ ΔluxN) CmR at -80 °C.
* Investigation of *V. harveyi* antibiotics resistances.

# Week 24

## 06-13: Model

A first version of the SBGN (Systems Biology Graphical Notation) summarizing our synthetic system have been drawn.

## 06-14: Scope statement start

A scope statement was created for a device that treats the water of a tank and another for a device that treats the water in a flask.

## 06-15: 19th brainstorming

We’ve received our IDT parts! We finally choose the name of our project: Croc’n cholera.

## 06-16: Skype with Greece iGEM team

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Amplifying pSB1C3

* Reception of IDT gBlocks and storage
* Preparative work with the cloning vectors : Amplification and stock of pSB1C3

## Taking charge of the lab and of *V. harveyi*

* Growth kinetic assay in order to determine the adequate NaCl concentration for *V. harveyi* growth.
* Reception of IDT gBlocks and storage
* Transformation of DH5α competent cells with pYFP, pDsRed, pPIZα, pBR322 and amplification of the plasmids. The plasmids are double digested with the appropriate restriction enzymes.

## Cloning

* pPICzα, jmp62-DsRed (pDsRed) and jmp62-YFP (pYFP) were transformed into E.coli DHAα in order to amplify them.
* Bacteria grown were then put into liquid culture to do miniprep.
* Digestion BamHI/EcoRI to check pPICzα gel migration profile
  Digestion XbaI/NheI to check jmp62-DsRed gel migration profile
  Digestion BamHI/NheI to check jmp62-YFP gel migration profile

# Week 25

## 06-19: Meeting with Doctors Without Borders, regional manager

She explained that the detection of cholera in the patient is simple because the symptoms are known. She told us that our system would be more useful treating water tanks in villages far from cholera camps rather than tourists. Indeed, governments often hide epidemics from tourists and tourist places take enough precautions to avoid tourists’ contamination.

You can read the result of [her testimony here.](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Testimonies#a5)

## 06-23: Beginning of the marketing approach

A marketing approach has been initiated to study the strengths and weaknesses of our two potential products and to determine the approach to sell them. Thanks to a SWOT analysis on both products, we realized that the product to treat water in a flask is not adapted to the market. This product is therefore abandoned and we focus on a product that processes a larger volume of water, suitable for villagers in the affected countries.

## 06-22 Skype with NUS iGEM team

## 06-23: 20th brainstorming

Meeting with all the team members and supervisors and final choice of the global device purpose: made for people living in remote areas in Africa, which are far from NGOs camps and more vulnerable to a cholera epidemy.

## 06-23: Model

Our mathematical model based on twelve ODEs was built!

## 06-23: First appearance of Sobki, our mascott!

![](https://static.igem.org/mediawiki/2017/2/2a/T--INSA-UPS_France--Logo.png)

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Plasmids preparation

* Transformation of DH5α competent cells with pSB1C3 and amplification of the plasmids. The plasmids are double digested with the appropriate restriction enzymes.
* Scale up of the preparative digestion, in order to have more DNA matrix.
* Reception of the PCR primer.

## Plasmids preparation

* Transformation of DH5α competent cells with pGAP, pSB1C3 and amplification of the plasmids. The plasmids are double digested with the appropriate restriction enzymes.
* Scale up of the preparative digestion, in order to have more DNA matrix.
* Reception of the PCR primer.

## Cloning

* Preparative digestion of pPICZα with BamHI/BglII to clone our gene followed by a gel extraction to purify the open plasmid.
* Glycerol stocks of *E.coli* DHAα containing pPICZα, pDSRed and pYFP were made after liquid culture.

# Week 26

## 06-26: Skype with the iGEM team of Purdue

## 06-28: Meeting with the CEO of Sunwaterlife, building a collaboration

Really interested in the ability to detect *V. cholerae* in water, Christophe Campéri-Ginestet asked us to provide our bibliographic research and results at the end of the iGEM project. In addition, in exchange for our financial investment in our project, we have to fill out the JEI document which attests to our R&D activity for Sunwaterlife. He also put us in touch with Alama Keita, cholera leader of UNICEF in Niger. Finally, we talked to him about our product to contain our microorganisms. He helped us to design a cylindrical device whose sections would be these membranes to ensure GMMs containment.

You can read the result of [his testimony here.](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Testimonies#a4)

## 06-30: First steps of our ethical matrix

![](https://static.igem.org/mediawiki/2017/6/6b/T--INSA-UPS_France--Notebook_Week-26.jpg)

## 06-29: 21th brainstorming

We choose to made a Prezi for the presentation in Boston, the structure has been validated by instructors and students, and we made a first version of our poster for the meetup in Delft. Our wiki is online next day! Our survey has around 400 answers, so we can start analyses of results.

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Cloning VhCqsA

* Amplification of the IDT gblocks and optimisation of the PCR cycle condition for VhCqsA part
* Digestion of the PCR amplicons and ligation with the appropriate cloning vector.

## Assembly of *V. harveyi* gene circuit : starting the sub-cloning work

* Amplification of the IDT gblocks and optimisation of the PCR cycle condition for VhCqsA part
* Digestion of the PCR amplicons and ligation with the appropriate cloning vector.
* Cloning attempt #1 and #2 to sub-clone  Vh1, Vh2 and  Vh3 in their respective vector.

## Cloning

* PCR of DsRed and YFP with primers containing restriction sites. PCR products for these genes have right size. PCR products are purified.
* PCR of our parts (cOT2 ; leucrocine ; DNY15; Odr10) from IDT GBlocks. All PCRs succeeded.
* cOT2 ; leucrocine ; DNY15 ; Odr10 were digested BamHI/BglII to be inserted in pPICZα.
* Transformation of pPICZα-cOT2 ; pPICZα-leucrocine ; pPICZα-Odr10 and pPICZα-DNY15 in *E. coli* DH5α
* Miniprep of liquid culture transformants
* Digestion BmaHI/BglII to check the gel migration profile.
  → It appears that the plasmid was well digested but the insert does not appear. There is an intense band around 1300bp that might correspond to supercoiled plasmid. To conclude it seems like pPICZα closed itself without the insert: BamHI and BglII are compatibles.
* New primers were ordered replace by PCR the BglII site by KpnI site to remove the possibility for the plasmid to close itself.

# Week 27

## 07-06: 22th brainstorming

We’ve prepared some public engagement events for september : an exhibition at the campus science in Toulouse to showcase ancient iGEM Toulouse projects, and synthetic biology, a practical work in an high school, and we’ve planned to participate to the “European Researcher’s Night” with the LISBP by creating a game called “Possible or Impossible”, to open a dialogue about GMO legislation with people.

## 07-07: Delft European iGEM Meetup

![](https://static.igem.org/mediawiki/2017/e/e1/T--INSA-UPS_France--Collaborations_meetup-2.jpg)

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Cloning VhCqsA

* Optimization of PCR cycle
* Cloning of VhCqsA into pSB1C3 : positives clones grew

## Assembly of *V. harveyi* gene circuit : continuing the sub-cloning work

* Assembly of V.harveyi gene circuit: Analysis of the transformants of V.harveyi module (attempt #1 and #2) by checking of the restriction map.
* New attempt (attempt #3) to sub-clone Vh1, Vh2 and  Vh3 in their respective vector.

## Cloning

* While waiting for the new primers, another try was made using for the ligation a 1:10 ratio (pPICZα:insert).
* Transformants appeared in all plates except for Odr10.
* Miniprep and digestions of transformants to check the gel migration profile.
* We obtained 2 clones pPICZα-DNY15. The gel confirmed that D1 and D2 are clones with the entire plasmid pPICZa A open only in BamHI site with one insert in this site. D1 has the insert in the good direction and D2 has the insert in the opposite direction.
* Clones D1 and D2 were stored in glycerol.
* At the end of the week, PCR with new primers containing KpnI instead of BglII were made for all the parts (cOT2 ; leucrocine ; DNY15 ; Odr10). All the PCR worked perfectly, PCR tubes were pooled. Odr10 has secondary products which need gel extraction.
* Digestion of plasmid and parts with KpnI and BamHI

# Week 28

## 07-13: 23th brainstorming

Citation from Cédric : “we must be humble before molecular biology”. Our card game conception progressing well! We find a name : MicrobioWorld, and the main rules has been determined after several crazy gameplay!

## 07-14: Meeting with Doctors Without Borders, cholera specialist

Fransisco Luquero assured us that the current solutions (bleach, boil water) were not the most suitable to treat water from cholera. Our device could be interesting if it is suitable to treat several liters of water and even if it contains GMM, it could be easily accepted by the population as long as it does not change the color and taste of water.

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Assembly of *V. harveyi* gene circuit : continuing the sub-cloning work

* Assembly of V.harveyi gene circuit: Analysis of the transformants of V.harveyi module (attempt #1 and #2) by checking of the restriction map.
* New attempt (attempt #3) to sub-clone Vh1, Vh2 and  Vh3 in their respective vector.

## Cloning

* Ligation and transformation of pPICZα-cOT2 ; pPICZα-leucrocine ; pPICZα-Odr10 and pPICZα-DNY15.
* Miniprep of transformants and gel migration were made. The ladder was clear but there was no DNA on tranformants colums.
* Nanodrop analysis showed that there was not enough DNA.
  → The hypothesis is that the liquid culture was too long so *E. coli* lost the plasmid.

## Experiments with *P. pastoris*

* We did plates of YPD with increasing concentration of zeocin to select *P. pastoris* clones which integrated the most copies of the construction.
* Preparation of competent yeast (*Pichia pastoris*)
* Digestion of D1 clone (pPICZα-DNY15) with HindIII to integrate the plasmid construction at the AOX1 promotor in *P. pastoris* genome.
* Electroporation of competent yeast with the digested DNA.

# Week 29

## 07-17: Model

Our system of ODEs script and function have been developed in Matlab.

## 07-20: 24th brainstorming

We’ve finished a first version of our presentation, which will be presented during the Parisian Meetup. The Matlab programming is done, the next step is to add values from bibliography.

## 07-20: First meeting with Pierre-Alain Hoffmann

We met Pierre-Alain Hoffmann, Deputy Director of CRITT Bio-Industries for the business plan elaboration. The knowledge in creation and development of companies of Pierre-Alain Hoffmann allowed us to understand the goal of a business plan. He accompanied us in this approach, explaining the points to be detailed in order to better integrate our system in the market.

## 07-21: Parisian meetup

![](https://static.igem.org/mediawiki/2017/8/8b/T--INSA-UPS_France--Collaborations_meetup-1.jpg)

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Expressing VhCqsA (2nd)

* Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
* SDS page fo the protein overexpression of our clones (fail)

## Assembly of *V. harveyi* gene circuit : A new strategy

* Amplification of the IDT gblocks, digestion of the PCR amplicons.
* Amplification and digestion of the sub-cloning vectors.
* Ligation and transformation in *E. coli* STELLAR competent cells (attempt #8).

## Cloning

* This time transformation were made with *E. coli* XL1 Blue which are supposed to be more efficient.
* No DNA after gel migration like the previous week.

## Experiments with *P. pastoris*

* Colony PCR of *P. pastoris* clone which grew on YPD zeocin. → All clones have the gene construction in their genome!￼
* The 11 clones of Pichia were grown on plates with 250 µg/mL, 500 µg/mL and 1000 µg/mL to see which one has the best integration of the construction. All clones grew onto 250, 500 and 1000 µg/mL zeocin.

# Week 30

## 07-23: Skype with the iGEM team of Greece

## 07-27: Meeting with Pierre Monsan at TWB

As a preindustrial demonstrator recognized in France, TWB helps many projects to become companies. We presented them our project and had a positive feedback. It is even one of our biggest sponsors.

You can read the result of [their testimony here.](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Testimonies#a2)

## 07-29: Team building in Pyrenees

![](https://static.igem.org/mediawiki/2017/e/ec/T--INSA-UPS_France--Notebook_Week-30.gif)

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Expressing VhCqsA (3rd)

* Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)

## Assembly of *V. harveyi* gene circuit : The first transformant

* TA cloning (attempt #9): amplification of the IDT gblocks with KAPA2G polymerase , digestion of the PCR amplicons. Ligation with pGEM vector TA cloning vector.
* Analysis of the transformants (attempt #8 and #9)  by checking the restriction map.
* The part 1 and 2 were successfully cloned in pBR322 and pGEM.

## Cloning

* PCR were made again of all parts in order to have enough materials to do ligation.
* This time lot of DNA was digested.
* Several strategies were tried to obtain pPICZα-cOT2 ; pPICZα-leucrocine  and pPICZα-Odr10 with DHAα and XL1 Blue strains.
* At the end of the week we got 2 clones of pPICZα-Odr10 in E. coli DHAα

## Experiments with *P. pastoris*

* The supernatant of *P. pastoris* containing the DNY15 gene was extracted and then concentrated via rotavapor and freeze-drying.
* In a 24 wells plate, kinetic growths of *V. harveyi* were followed with or without *P. pastoris* supernatant to check the bactericide effect of DNY15. → this first try was not perfectly made
* Inhibition test with the same supernatant was made on petri dish. → this first try was not perfectly made

# Week 31

## 08-01: 25th brainstorming

Our collaborations with other iGEM teams are in process! We’ve started an experiment for iGEM Purdue.

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Expressing VhCqsA (aborted) + Cloning of VcCqsA

* Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR aborted : no growth of bacteria

## Step 2 : reconstruction of CqsS Rc by ligating Vh2 to Vh1 in pBR322.

* Storage of the transformants with Vh1 and Vh2 in glycerol at -80°C.
* Amplification, digestion of pBR322-Vh1,  pGEM-Vh2 and ligation in *E. coli* TOP10 competent cells (attempt A)
* Transformation of Vh3 (attempt #10) in *E. coli* DH5α competent cells
* Analysis of the transformants (attempt #A and #10)  by checking the restriction map.

## Cloning

* Different strategies were made again to obtain pPICZα-cOT2 and pPICZα-leucrocine in E.coli. → Several clones of pPICZα-cOT2 and pPICZα-leucrocine were obtained!!!
* Liquid culture of DHAα containing pSB1C3 was made to get enough material to do cloning of our parts in it.
* Ligation of YFP in pPICZα-Odr10 and in pPICZα-DNY15 instead of the AMP gene.
* Integration of AMP genes in pSBIC3 (=iGEM parts).

## Experiments with *P. pastoris*￼

* Cytotoxicity test of DNY15 was tried again with kinetic growths of *V. harveyi*.
* Cytotoxicity test of DNY15 was tried again on petri dish: No effect of DNY15 can be seen.
* Genomic integration of pPICZα-cOT2 , pPICZα-leucrocine and pPICZα-Odr10.

# Week 32

## 08-07: Skype with the iGEM team of Boston

## 08-07: Model

All the experimental and literature values have been implemented in our Matlab script, which provides promising results. These first results will be used for preliminary work in design and entrepreneurship.

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Expressing VhCqsA (4th time) + VcCqsA cloning

* Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
* PCR on VcCqsA

## Step 2 CqsS Rc assembly in progress…still no success for Vh3

* Amplification, digestion of pBR322-Vh1  pGEM-Vh2 and ligation in *E. coli* TOP10 competent cells (attempt B and C)
* Transformation of Vh3 (attempt #11 to #13) in *E. coli* TOP10 competent cells using an alternative restriction enzyme :  PstI-HF restriction enzyme is remplaced  by SpeI.

## Cloning

We obtained the YFP gene after the GAP promotor and the FUS1 promotor in *E. coli*.

## Experiments with *P. pastoris*

* pGAP-YFP and pFUS-YFP were transformed in *P. pastoris*.
* *P. pastoris* clones were obtained with pPICZα-Odr10 ; pPICZα-leucrocine and pPICZα-cOT2 but the PCR colony didn’t work. After several trials and controls we concluded that the DNA extraction the way we did it was not enough to do a PCR of the GAP promotor in the pichia genome.
* A kinetic study of *V. harveyi* growth was made using pichia supernatant. → A difference was observed but the experiment needs to be done again.

# Week 33

## 08-16: Skype with the iGEM team of Groningen

## 08-17: “iGEM on ice”

![](https://static.igem.org/mediawiki/2017/7/70/T--INSA-UPS_France--Notebook_Week-33.jpg)

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Cloning of VcCqsA

* From ligation to transformation : transformants grew.

## Step 2 CqsS Rc assembly in progress, preparation of the conjugation

* Analysis of the transformants (attempt #13)  by checking the restriction map : the part 3 is successfully cloned in pSB1C3. The transformant is stored  in glycerol at -80°C.
* Amplification of pBBR1MCS-4 and pBBR1MCS-5 for cloning of Vh3 and for a conjugation assay.
* Ligation of Vh3 in pBBR1MCS-4 and transformation in *E. coli* TOP10 (attempt α).
* Amplification, digestion of pBR322-Vh1  pGEM-Vh2 and ligation in *E. coli* TOP10 competent cells (attempt D to F)

* *P. pastoris* clones were obtained with the YFP reporter behind both promotor: GAP and FUS1.
* Fluorescence test was made but it didn’t work.
* A cytotoxicity test was made on V.harveyi with pichia-AMP supernantant but we had trouble because of volumes used.
* We tried a new DNA extraction protocol to do the PCR colony on *P. pastoris* and it did work.

# Week 34

## 08-24: 26th brainstorming

We finally choose our track! We will be candidate in “information processing”, to highlight the prokaryote-eukaryote communications in our multimicrobial system!

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Confirming VcCqsa clone + Expressing VhCqsA (5th time)

* Transformant were good
* Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)

* Analysis of the transformants (attempt #F)  by checking the restriction map : the part Vh1+Vh2 is successfully cloned in pBR322. The transformant is stored  in glycerol at -80°C.
* Amplification, digestion of pBR322-Vh1+Vh2, pSB1C3-Vh3. Ligation and transformation (attempt #1 and #2) in *E. coli* TOP10 competent cells.
* Ligation of Vh3 in pBBR1MCS-4 and transformation in *E. coli* TOP10 (attempt β and γ)
* Vh3 part validation : diacetyl production assay by NMR detection with *E. coli* BL21.

* Cytotoxicity test was made again but this time on a petri dish. We had a halo for the antibiotic but *P. pastoris’* supernatant didn’t stop *V. harveyi’*s growth.
* We’ve tried to characterize pFUS1 promoter in different media but we got no positive result.

# Week 35

## 08-28: First try for the device modeling

A 3D modeling of the device was initiated on the Solidworks software with the help of a professional in the design of industrial parts. The objective is to model the device in 3D and then to print the prototype.

## 08-31: 27th brainstorming

After much thinking, we are agreed that we want Margaux, Maxant and Margaux to be our speakers during the competition in Boston!

## 08-31: Model

First MCA results! The control exerted by each parameter on the response time can now be analysed.

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Expressing VhCqsA (6th time) + Bioluminescence

* Production of C8-CAI-1 in minimal media, extration on LLE dichloromethane and analysis on NMR (fail)
* Optimisation of *Vibrio harveyi* JMH626 bioluminescent assay

* 2nd  diacetyl production assay by NMR detection with *E. coli* BL21.
* Success of the conjugation assay of pBBR1MCS-4.

* This time, we tested the production of DNY15 in YPD with 40g/L of glucose instead of 30g/L because publications which use pGAP as a promotor to produce AMP sometimes have a higher production with more glucose.
* Cytotoxicity tests using the supernatant from the 40g/L glucose culture media was made. Supernatants were filtered beforehand and half was freeze-dried.  We got a weak halo for the DNY15 production in glucose 40 and supernatant freeze-dried!

# Week 36

## 09-04: Start of our Ulule campaign

## 09-07: 28th brainstorming

## 09-08: Farewell party

![](https://static.igem.org/mediawiki/2017/5/52/T--INSA-UPS_France--Notebook_Week-36.jpg)

## 09-09: Meeting with Marie-Pierre Escudié

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Expressing VhCqsA (7th time)

* Production of C8-CAI-1 in minimal media, and analysis on MS (fail) extration on LLE dichloromethane and analysis on NMR (fail)

* 3rd  Diacetyl production assay by NMR detection with *E. coli* MG1655.
* Amplification of pBBR1MCS-4 and pBBR1MCS-5.

* Because we still had trouble integrating some parts in *P. pastoris’* genome we tried the integration via electroporation and via chemical way at the same time.
* We launched one again *P. pastoris* culture in YPD 40g/L and 50g/L glucose to try producing more DNY15.

# Week 37

## 09-14: 29th brainstorming

We’ve prepared the abstract, and choose a subtitle : “a synthetic microbial consortium”

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Bioluminescence assay

* Trial on the expression of C8-CAI-1 with induction on bioluminescence on liquid media : fail

* 4th diacetyl production assay by NMR detection with *E. coli* MG1655 in M9 media supplemented with xylose and pyruvate.
* Transformation of *E. coli* TOP10 competent cells with GFP from iGEM kit
* Ligation and transformation of pBBR1MCS-4 to RFP from iGEM kit in *E. coli* TOP10 competent cells for conjugation to *V. harveyi*.
* Preparation of the clones for sequencing.

* We prepared sample to sequence the AMP genes we wanted to give to the iGEM registry.
* Culture media from *P. pastoris* culture of last week were freeze-dried. And the cytotoxicity test on petri dish was made again but this time, even the positive control with antibiotic didn’t work.
* *P. pastoris* clones were obtained with pAOX1-leucrocine, pAOX1-cOT2, Odr10-pFUS-RFP.

# Week 38

## 09-21: 30th brainstorming

We’ve finished the experiment time, it’s time to write results for the wiki!

## 09-22: Meeting with UNICEF

He warned us that NGOs would agree to use a GMM system only if efficacy is proven and there is no danger of GMM proliferation. He also gave us an idea of the volume of water a village needs. Moreover, it is also important to make a system with as little waste as possible.

You can read the result of [Alama Keita's testimony here.](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Testimonies#a6)

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

## Bioluminescence assay

* Trial on the expression of C8-CAI-1 with induction on bioluminescence on plate : success

* Results of the 4th Diacetyl production assay by NMR detection: a small peak of diacetyl is visible.
* 5th diacetyl production assay by NMR detection with *E. coli* MG1655 in M9 media supplemented with xylose and pyruvate.
* Results of ligation of RFP in conjugative plasmid: all transformants are red. Two transformants are stored at -80°C in order to be used for conjugation assay.
* Preparative work for the ligation of Vh3 in pBBR1MCS-4 with fresh material

* AMP genes were sent to be sequenced.
* *P. pastoris* producing DNY15 was cultured to do a RT-qPCR experiment to compare it to the WT *P. pastoris*.
* New cytotoxicity tests on petri dish were made but this time it didn’t work because of the paper disk we used. Once again the positive control didn’t work.
* Cultures in BMMY adding methanol every 24h for 120h were made for both pAOX1-leucrocine and pAOX1-cOT2 P.pastoris clones. Half of each supernatant was stored at 4°C and the other half was evaporated to concentrate 10 times.

# Week 39

## 09-27: 31th brainstorming

We’ve started to make rehearsals for the presentation in Boston.

## 09-29: European Researcher's Night

We’ve been to the European Researcher’s Night, a major scientific event that gather researchers and general public in a convivial atmosphere. In 2017, the topic of the event was “Impossible”. In order to follow this thematic, we chose to focus our workshop on the incredible features of biodiversity, how to use it in synthetic biology, and what are legal limits on GMO use in France.

![](https://static.igem.org/mediawiki/2017/0/05/T--INSA-UPS_France--Notebook_Week-39.jpg)

## Experiments

![](https://static.igem.org/mediawiki/2017/4/4e/T--INSA-UPS_France--Experiments_module-coli.png)

![](https://static.igem.org/mediawiki/2017/4/48/T--INSA-UPS_France--Experiments_module-harveyi.png)

![](https://static.igem.org/mediawiki/2017/9/99/T--INSA-UPS_France--Experiments_module-pichia.png)

* Observation by microscopy of *V. harveyi* (rfp) transformant
* Ligation of pBBR1MCS-4 and Vh3 at 16°C overnight, and transformation in competent *E. coli* (Stellars)

* New cytotoxicity tests on petri dishes were made with the leucrocine and cOT2 supernatants. This time with appropriate discs. Positive control did not work again.
* New cytotoxicity tests on petri dishes were made but using pure chloramphenicol. A weak halo was seen. We found out that the *V. harveyi* strain we used to do cytotoxicity test was maybe! the JMH626 one which was chloramphenicol-resistant.
* RT-PCR experiment was made.
* To test the Odr10-RFP system and activate pFUS, nitrogen depletion cultures were done and two sources of nitrogen were tested: sulfate ammonium and glutamine. The fluorescence following was made in a plate reader.
* New cytotoxicity tests on petri dishes were made starting from a fresh *V. harveyi* cuture. Leucrocine, cOT2 and D-NY15 (40g/L and 50g/L glucose) productions were tested. Halos are clearly visible for D-NY15 40g / L not concentrated and concentrated 10 times.
* A 24 well plate was launched by adding leucrocine, cOT2 and D-NY15 supernatant to *V. harveyi* cultures in exponential phase.

# Week 40

## 10-02: Meeting with Pierre-Alain Hoffmann, director of CRITT Bio-industries.

Between these two appointments, we have elaborated the business plan ourselves. Pierre-Alain Hoffmann read it and made a feed-back. He was pleased with our work, ensuring that some start-ups do not start their business with such a business plan.

## 10-05: 32th brainstorming

Rehearsals, and writing of different pages of the wiki.

## 10-04: High school practical work

We prepared an intervention for High School senior students in scientific classes. Our involvement was focused on 3 main goals: implement a learner-centred pedagogical approach in a high school class, make discover biotechnologies through a practical approach, and discuss on “how to be part of science, and which paths to choose after high school?”.

![](https://static.igem.org/mediawiki/2017/8/8a/T--INSA-UPS_France--Notebook_Week-42.jpg)

# Week 41

## 33th brainstorming

Rehearsals, and writing of different pages of the wiki.

## All week: exhibition at Bib'INSA

In order to make former iGEM Toulouse projects understandable for all scientist student (and not only for biologists), we redesigned posters of previous project E. calculus, SubtiTree, ApiColi, Paleotilis, and of our project Croc’n Cholera.

# Week 42

## 10-10: End of the 3D modeling of the device and 3D printing

The device was printed in 3D by the High school Georges Cabanis at Brive-La-Gaillarde.￼

![]()

## 10-11: Model

Some inaccuracies in our mathematical model have been corrected, we have now the final version of all our Matlab files! Results will be used for our entrepreneurship and design work.

## 10-13

We have now our final MCA, and performed a global analysis of our system. We can now definitely conclude about sensitivity and robustness of our model.

## 13-10: Seminar at the campus science Toulouse

After working during several months on synthetic biology for our iGEM project, we needed to organize an event to present our work and the one achieved by the previous iGEM teams. We also wanted to have an exchange of opinions with people who possibly have different values and considerations than us. The intended public was mainly composed of the Paul Sabatier university and INSA Toulouse students, two of the schools we are intending.

# Week 43

## 10-21: Model

An intuitive modeling interface have been built for displaying our model results to non-specialists.

# Week 44

| Realisations pages | | | | | |
| --- | --- | --- | --- | --- | --- |
| [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments) | *Notebook* | [Clonings](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) | [Results](https://2017.igem.org/Team%3AINSA-UPS_France/Results) | [Contribution](https://2017.igem.org/Team%3AINSA-UPS_France/Contribution) | [Protocols](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols) | [Safety](https://2017.igem.org/Team%3AINSA-UPS_France/Safety) |

[![](https://static.igem.org/mediawiki/2017/3/3e/T--INSA-UPS_France--Logo_lisbp.png)](http://www.lisbp.fr/en/index.html)
[![](https://static.igem.org/mediawiki/2017/5/56/T--INSA-UPS_France--Logo_twb.png)](http://www.toulouse-white-biotechnology.com/en/)
[![](https://static.igem.org/mediawiki/2017/6/6d/T--INSA-UPS_France--Logo_fondationinsal.png)](http://fondation.insa-lyon.fr/)
[![](https://static.igem.org/mediawiki/2017/b/b7/T--INSA-UPS_France--Logo_ia2c.png)](https://fr-fr.facebook.com/insaadnconceptclub/)
[![](https://static.igem.org/mediawiki/2017/8/83/T--INSA-UPS_France--Logo_biomerieux.png)](http://www.biomerieux.com/en)
[![](https://static.igem.org/mediawiki/2017/0/09/T--INSA-UPS_France--Logo_sunwaterlife.png)](http://www.sunwaterlife.com/en/)
[![](https://static.igem.org/mediawiki/2017/2/28/T--INSA-UPS_France--Logo_ups.png)](http://www.univ-tlse3.fr/home/home-page-en-379161.kjsp?RH=ACCUEIL)
[![](https://static.igem.org/mediawiki/2017/a/ab/T--INSA-UPS_France--Logo_fsi.png)](http://www.fsi.univ-tlse3.fr/)
[![](https://static.igem.org/mediawiki/2017/c/cf/T--INSA-UPS_France--Logo_ernestsolvay.png)](https://www.kbs-frb.be/)
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[![](https://static.igem.org/mediawiki/2017/9/96/T--INSA-UPS_France--Logo_adisseo.png)](http://www.adisseo.com/en/)
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[![](https://static.igem.org/mediawiki/2017/1/1a/T--INSA-UPS_France--Logo_ambassade.jpg)](https://www.france-science.org/-Homepage-English-.html)
[![](https://static.igem.org/mediawiki/2017/5/51/T--INSA-UPS_France--Logo_LBME.png)](https://www-lbme.biotoul.fr/)
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[![](https://static.igem.org/mediawiki/2017/9/93/T--INSA-UPS_France--Logo_fsie.jpg)](http://en.univ-toulouse.fr/our-strengths)
[![](https://static.igem.org/mediawiki/2017/3/37/T--INSA-UPS_France--Logo_crous.png)](https://www.crous-toulouse.fr/english/)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:INSA-UPS France/Experiments

[![](https://static.igem.org/mediawiki/2017/2/2a/T--INSA-UPS_France--Logo.png)](https://2017.igem.org/Team%3AINSA-UPS_France)

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Team

* [Members](https://2017.igem.org/Team%3AINSA-UPS_France/Team)
* [Supports](https://2017.igem.org/Team%3AINSA-UPS_France/Supports)
* [Attributions](https://2017.igem.org/Team%3AINSA-UPS_France/Attributions)
* [Collaborations](https://2017.igem.org/Team%3AINSA-UPS_France/Collaborations)

Strategy

* [Description](https://2017.igem.org/Team%3AINSA-UPS_France/Description)
* [Design](https://2017.igem.org/Team%3AINSA-UPS_France/Design)
* [Parts](https://2017.igem.org/Team%3AINSA-UPS_France/Parts)

Model

* [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Model)
* [Simulation](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Simulation)
* [Analysis](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Analysis)
* [Interface](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Interface)

Realisations

* [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments)
* [Notebook](https://2017.igem.org/Team%3AINSA-UPS_France/Notebook)
* [Clonings](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings)
* [Results](https://2017.igem.org/Team%3AINSA-UPS_France/Results)
* [Contribution](https://2017.igem.org/Team%3AINSA-UPS_France/Contribution)
* [Protocols](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols)
* [Safety](https://2017.igem.org/Team%3AINSA-UPS_France/Safety)

Human Practices

* [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Human_Practices)
* [Silver](https://2017.igem.org/Team%3AINSA-UPS_France/HP/Silver)
* [Gold](https://2017.igem.org/Team%3AINSA-UPS_France/HP/Gold_Integrated)
* [Public Engagement](https://2017.igem.org/Team%3AINSA-UPS_France/Engagement)
* [Microbioworld](https://2017.igem.org/Team%3AINSA-UPS_France/Microbioworld)

Application

* [Applied Design](https://2017.igem.org/Team%3AINSA-UPS_France/Applied_Design)
* [Testimonies](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Testimonies)
* [Entrepreneurship](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship)
* [Device Conception](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Device)

# Experiments Overview

![](https://static.igem.org/mediawiki/2017/0/08/T--INSA-UPS_France--Experiments_croco.png)

This Realisations section describes all our wet lab work, based on our strategy. We divided this section into the [Clonings page](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) and the [Results page](https://2017.igem.org/Team%3AINSA-UPS_France/Results): the first shows how we integrated the parts of our [Design](https://2017.igem.org/Team%3AINSA-UPS_France/Design) into our chassis, and the second shows the experiments we did to further validate their functionnality. Both are presented for each of our eight modules:

![](https://static.igem.org/mediawiki/2017/6/66/T--INSA-UPS_France--exp_overview.png)

# 1 - Mimicking *Vibrio* sp. presence with an engineered *E. coli*

We engineered here *E. coli* to mimic for safety reason the presence of *Vibrio* species.

* Cloning:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings#cloning1)
* Validation:  [in progress](https://2017.igem.org/Team%3AINSA-UPS_France/Results#result1)

# 2 - *E. coli* producing C8-CAI-1 molecules can be sensed by *V. harveyi*

In this module, we created synthetic communication between our engineered *E. coli* and *V. harveyi*.

* Cloning: No cloning for this module
* Validation:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Results#result2)

# 3 - Modification of *V. harveyi* to detect both C8-CAI-1 and CAI-1

Here, we present our first effective modification of *V. harveyi* and its perspectives for the project.

* Cloning:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings#cloning3)
* Integration in chassis: not done yet
* Validation:  [in progress](https://2017.igem.org/Team%3AINSA-UPS_France/Results#result3)

# 4 - Establishing production of diacetyl to establish communication between prokaryotic and eukaryotic cells

This module is dedicated to the production of diacetyl by bacteria, as a signal relay toward yeast.

* Cloning:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings#cloning4)
* Integration in chassis:  [in progress](https://2017.igem.org/Team%3AINSA-UPS_France/Results#result4)
* Validation:  [in progress](https://2017.igem.org/Team%3AINSA-UPS_France/Results#result4)

# 5 - Diacetyl detection by *Pichia pastoris*

This presents are modification and first assays to engineered *P. pastoris* for diacetyl detection.

* Cloning:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings#cloning5)
* Integration in chassis:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings#cloning5)
* Validation:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Results#result5)

# 6 - *P. pastoris* is able to produce functional antimicrobial peptides

Finally, this module described the efficiency of a yeast-produced crocodile AMP on *V. harveyi*.

* Cloning:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Clonings#cloning6)
* Integration in chassis:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings#cloning6)
* Validation:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Results#result6)

# 7 - Co-cultivate *P. pastoris* and *V. harveyi* is possible

Toward a realistic solution, we tested here how to co-culture our microbial consortium.

* Validation:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Results#result7)

# 8 - Membrane permeability assay

We assessed the capacity of our microorganism-impermeable materials to let AMP go through.

* Validation:  [successful](https://2017.igem.org/Team%3AINSA-UPS_France/Results#result8)

| Realisations pages | | | | | |
| --- | --- | --- | --- | --- | --- |
| *Overview* | [Notebook](https://2017.igem.org/Team%3AINSA-UPS_France/Notebook) | [Clonings](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) | [Results](https://2017.igem.org/Team%3AINSA-UPS_France/Results) | [Contribution](https://2017.igem.org/Team%3AINSA-UPS_France/Contribution) | [Protocols](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols) | [Safety](https://2017.igem.org/Team%3AINSA-UPS_France/Safety) |

[![](https://static.igem.org/mediawiki/2017/3/3e/T--INSA-UPS_France--Logo_lisbp.png)](http://www.lisbp.fr/en/index.html)
[![](https://static.igem.org/mediawiki/2017/5/56/T--INSA-UPS_France--Logo_twb.png)](http://www.toulouse-white-biotechnology.com/en/)
[![](https://static.igem.org/mediawiki/2017/6/6d/T--INSA-UPS_France--Logo_fondationinsal.png)](http://fondation.insa-lyon.fr/)
[![](https://static.igem.org/mediawiki/2017/b/b7/T--INSA-UPS_France--Logo_ia2c.png)](https://fr-fr.facebook.com/insaadnconceptclub/)
[![](https://static.igem.org/mediawiki/2017/8/83/T--INSA-UPS_France--Logo_biomerieux.png)](http://www.biomerieux.com/en)
[![](https://static.igem.org/mediawiki/2017/0/09/T--INSA-UPS_France--Logo_sunwaterlife.png)](http://www.sunwaterlife.com/en/)
[![](https://static.igem.org/mediawiki/2017/2/28/T--INSA-UPS_France--Logo_ups.png)](http://www.univ-tlse3.fr/home/home-page-en-379161.kjsp?RH=ACCUEIL)
[![](https://static.igem.org/mediawiki/2017/a/ab/T--INSA-UPS_France--Logo_fsi.png)](http://www.fsi.univ-tlse3.fr/)
[![](https://static.igem.org/mediawiki/2017/c/cf/T--INSA-UPS_France--Logo_ernestsolvay.png)](https://www.kbs-frb.be/)
[![](https://static.igem.org/mediawiki/2017/6/62/T--INSA-UPS_France--Logo_fondationinsat.png)](http://www.insa-toulouse.fr/fr/institution/fondation-insa-toulouse.html)
[![](https://static.igem.org/mediawiki/2017/9/96/T--INSA-UPS_France--Logo_adisseo.png)](http://www.adisseo.com/en/)
[![](https://static.igem.org/mediawiki/2017/2/2d/T--INSA-UPS_France--Logo_insatoulouse.png)](http://www.insa-toulouse.fr/fr/index.html)
[![](https://static.igem.org/mediawiki/2017/9/91/T--INSA-UPS_France--Logo_veolia.png)](https://www.veolia.com/en)
[![](https://static.igem.org/mediawiki/2017/1/1a/T--INSA-UPS_France--Logo_ambassade.jpg)](https://www.france-science.org/-Homepage-English-.html)
[![](https://static.igem.org/mediawiki/2017/5/51/T--INSA-UPS_France--Logo_LBME.png)](https://www-lbme.biotoul.fr/)
[![](https://static.igem.org/mediawiki/2017/1/16/T--INSA-UPS_France--Logo_metatoul.png)](https://www6.toulouse.inra.fr/metatoul_eng/)
[![](https://static.igem.org/mediawiki/2017/5/5b/T--INSA-UPS_France--Logo_fsdie.png)](http://www.univ-tlse3.fr/associations-%2B/do-you-have-a-project--378066.kjsp?RH=1238417866394)
[![](https://static.igem.org/mediawiki/2017/9/93/T--INSA-UPS_France--Logo_fsie.jpg)](http://en.univ-toulouse.fr/our-strengths)
[![](https://static.igem.org/mediawiki/2017/3/37/T--INSA-UPS_France--Logo_crous.png)](https://www.crous-toulouse.fr/english/)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:INSA-UPS France/Results

[![](https://static.igem.org/mediawiki/2017/2/2a/T--INSA-UPS_France--Logo.png)](https://2017.igem.org/Team%3AINSA-UPS_France)

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Team

* [Members](https://2017.igem.org/Team%3AINSA-UPS_France/Team)
* [Supports](https://2017.igem.org/Team%3AINSA-UPS_France/Supports)
* [Attributions](https://2017.igem.org/Team%3AINSA-UPS_France/Attributions)
* [Collaborations](https://2017.igem.org/Team%3AINSA-UPS_France/Collaborations)

Strategy

* [Description](https://2017.igem.org/Team%3AINSA-UPS_France/Description)
* [Design](https://2017.igem.org/Team%3AINSA-UPS_France/Design)
* [Parts](https://2017.igem.org/Team%3AINSA-UPS_France/Parts)

Model

* [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Model)
* [Simulation](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Simulation)
* [Analysis](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Analysis)
* [Interface](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Interface)

Realisations

* [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments)
* [Notebook](https://2017.igem.org/Team%3AINSA-UPS_France/Notebook)
* [Clonings](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings)
* [Results](https://2017.igem.org/Team%3AINSA-UPS_France/Results)
* [Contribution](https://2017.igem.org/Team%3AINSA-UPS_France/Contribution)
* [Protocols](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols)
* [Safety](https://2017.igem.org/Team%3AINSA-UPS_France/Safety)

Human Practices

* [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Human_Practices)
* [Silver](https://2017.igem.org/Team%3AINSA-UPS_France/HP/Silver)
* [Gold](https://2017.igem.org/Team%3AINSA-UPS_France/HP/Gold_Integrated)
* [Public Engagement](https://2017.igem.org/Team%3AINSA-UPS_France/Engagement)
* [Microbioworld](https://2017.igem.org/Team%3AINSA-UPS_France/Microbioworld)

Application

* [Applied Design](https://2017.igem.org/Team%3AINSA-UPS_France/Applied_Design)
* [Testimonies](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Testimonies)
* [Entrepreneurship](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship)
* [Device Conception](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Device)

# Results

![](https://static.igem.org/mediawiki/2017/0/08/T--INSA-UPS_France--Experiments_croco.png)

[1. Mimicking Vibrio sp. presence](#result1)

[2. *E. coli* producing C8-CAI molecules sensed by *V. harveyi*](#result2)

[3. Modification of *V. harveyi*](#result3)

[4. Production of diacetyl](#result4)

[5. *P. pastoris* is able to detect diacetyl](#result5)

[6. *P. pastoris* is able to produce functional AMP](#result6)

[7. Co-cultivation of *P. pastoris* and *V. harveyi*](#result7)

[8. Permeability assay of the membranes](#result8)

The experimental results described here provide insights of the functionality of our project and clues about how our synthetic consortium will work.

![](https://static.igem.org/mediawiki/2017/6/66/T--INSA-UPS_France--exp_overview.png)

# 1. Mimicking *Vibrio sp*. presence with an engineered *E. coli*

## Background

For safety reasons, we cannot manipulate *V. cholerae*. Therefore to mimic its presence, we engineered *E. coli* to produce a quorum sensing molecule from a non-pathogenic *Vibrio* species i.e. C8-CAI-1 from *V. harveyi* ( [cloning](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) ). C8-CAI-1 is an analogue of *V. cholerae* CAI-1 quorum sensing molecule and is produced by the enzyme CqsA. Besides, it allows to investigate our capacity to create synthetic communication between both engineered *E. coli* and *V. harveyi*.

## Materials and methods

Strain construction can be found here ( [clonings](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) ) and protocols used can be found here  [Protein production and sampling](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot7),  [NMR analysis](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot3) ,  [Cultivation condition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot1)  and  [Medium composition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot0)  ).

## Results and discussion

The figure 1 presents the results obtained with the supernatants of wild type *E. coli* (as a negative control with no C8-CAI-1 produced), wild type *V. harveyi* (as a positive control producing C8-CAI-1), and our engineered *E. coli* strain expressing *cqsA* from *V. harveyi* (i.e. *VhCqsA*,  [Part:BBa\_K2278001](http://parts.igem.org/wiki/index.php/Part%3ABBa_K2278001%20) ). These supernatants were analysed by 1H Nuclear Magnetic Resonance (NMR). Characteristic and observable proton signals of C8-CAI-1 in 1H NMR should appears at approximatively (δ, in ppm) 4.14 as a triplet dedoubled (CH in α of both carbonyl and hydroxyl groups), 2.50-2.36 (CH2 in α of carbonyl group) and 1.92-1.84 (1H of CH2 in β of hydroxyl group) as multiplets.
Unfortunately, the NMR profiles of the three supernatants were similar and no characteristic 1H NMR signal of C8-CAI-1 had been detected neither in wild type *V. harveyi* nor in *E. coli-VhCqsA* strains.
So, NMR approaches failed to confirm the production of C8-CAI-1 molecule in both *E. coli-VhCqsA* and in *V. harveyi* WT strains. This does not mean the tested construction is not functional but more probably that C8-CAI-1 production was below the sensitivity limit of NMR. As a more sensitive alternative, mass spectrometry analyses were also applied on the same samples. However, no conclusive results were obtained (data not shown) and optimization of the method required extra time. We thus cannot exclude that the expression of CqsA is not functional but a more sensitive approach such as *in vivo* strategy should be tested to check the functionality of this part.

![](https://static.igem.org/mediawiki/2017/1/18/T--INSA-UPS_France--Results_2.jpg)

Fig. 1: **Detection of C8-CAI-1 by NMR analysis (500MHz, CDCl3, 298K) in culture supernatant.** Overlaid 1H NMR spectra of freeze-dried supernatants from *E. coli*-pSB1C3 (negative control) in green, *E. coli*-VhCqsA (assay) in red, wild type *V. harveyi* (positive control) in blue. Supernatant have been freeze-dried and re-suspended into deuterated chloroform (CDCl3) before 1H NMR analysis.

## Perspectives

Culture parameters, induction by IPTG or extraction protocol could be optimized to increase C8-CAI-1 production. Alternatively, MS method could be improved to detect the C8-CAI-1.
Alternatively, we can focus on the expression of the *cqsA* gene from *V. cholerae* to create a safe *V. cholerae* effector.

# 2. *E. coli* producing C8-CAI-1 molecules can be sensed by *V. harveyi*

## Background

In response to its quorum sensing molecule, i.e. C8-CAI-1, *V. harveyi* becomes bioluminescent. Here we used it as a sensor to detect the production of C8-CAI-1 by our engineered *E. coli-VhCqsA* strain (i.e.,  [Part:BBa\_K2278001](http://parts.igem.org/wiki/index.php/Part%3ABBa_K2278001) )
.

## Materials and methods

Protocols can be found here:  [Medium composition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot0) ,  [Solid Bioluminescence assay](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot4).

## Results and discussion

We used the JMH626 mutant strain of *V. harveyi*, a strain with deletion of all its quorum sensing production genes, including *cqsA*[1](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285556/). Thus, it is unable to produce its own C8-CAI-1. However, the whole apparatus for response to quorum sensing is still present in this strain. So, the supernatant of the *E. coli-VhCqsA* strain was applied on this strain to check if it was able to trigger luminescence, as a natural property of *V. harveyi* exposed to C8-CAI-1.
Results are presented in Figure 2. In the negative control with *V. harveyi ∆cqsA* supernatant applied to the same strain, a very low basal bioluminescent is observed (likely from promoter leaking). The same very low basal luminescence level was observed with supernatant of wild type *E. coli* strain. As expected, when the supernatant of *V. harveyi* wild type strain was applied to *V. harveyi ∆cqsA*, bioluminescence could be restored. When supernatant of *E. coli-VhCqsA* strain was applied to the *V. harveyi ∆cqsA* strain, bioluminescence was observed with a comparable level to the one observed with wild type *V. harveyi*.
Together, these data demonstrate that C8-CAI-1 is efficiently produced by *E. coli-VhCqsA* and thus validate our  [BBa\_K2278001](http://parts.igem.org/wiki/index.php/Part%3ABBa_K2278001%20) part. More importantly, this result also demonstrates that we successfully created synthetic communication between engineered *E. coli* and *V. harveyi* strains.

![](https://static.igem.org/mediawiki/2017/9/9d/T--INSA-UPS_France--Results_3.jpg)

Fig. 2: **Solid bioluminescence assay**. Two strain of *V. harveyi* were plated (WT and Δ*cqsA*) with the addition of supernatant from *V. harveyi* WT, *V. harveyi* Δ*cqsA* and supernatant from *E. coli* with an empty pSB1C3 vector (*E. coli*) or *E. coli* with pSB1C3-*cqsA* (*E. coli-cqsA*). This picture is a representative picture of the 4 times repeated experiment.

## Perspectives

Next step will be to prove the communication between *E coli* producing *V. cholerae* CAI-1 ( [Part:BBa\_K2278002](http://parts.igem.org/wiki/index.php/Part%3ABBa_K2278002) ) and *V. harveyi* expressing the gene encoding the modified sensor CqsS\* that could sense both C8-CAI-1 and CAI-1 (see next Results module).

# 3. Modification of *V. harveyi* to detect both C8-CAI-1 and CAI-1

## Background

*V. harveyi* has many assets to be a good sensor for *V. cholerae*: it possesses its own pathway of detection for C8-CAI-1, very similar to the *V. cholerae* pathway to detect CAI-1. It has been shown that only a single point mutation on its receptor CqsS allows *V. harveyi* detecting *V. cholerae* CAI-1 molecule[1](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285556/). Because genetic in *V. harveyi* is limited (i.e. classical transformation does not work), we first had to set up a triparental conjugation protocol.

## Materials and methods

Strain construction can be found [here](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) and protocols used can be found there:  [Triparental conjugation](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot5) ,  [Fluorescence microscopy](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot6) ,  [Cultivation condition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot1) ,  [Medium composition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot0).

## Results and discussion

A protocol of triparental conjugation to transfer plasmids to *V. harveyi* was set up from an original conjugation method provided by M. Arlat (LIPM, Toulouse) usually used with *Xanthomonas campestris*. It was adapted to our *V harveyi* strain. It requires an *E. coli* donor with the conjugative plasmid pBBR1MCS-4 + insert (BBa\_J04450), an *E. coli* helper with the helper plasmid pRK2073, and a receiver *V. harveyi*.
Transformants were analyzed by fluorescence microscopy and results are presented in Figure 3. The pictures show that the strain of *V. harveyi* conjugated with the plasmid pBBR1MCS-4 containing the BBa\_J04450 construction is able to express RFP using a promoter and terminator from *E. coli*.
This raised several important conclusions. (i) It clearly demonstrated that *V. harveyi* can be engineered. This is the first use of *V. harveyi* as a genetic chassis in the IGEM competition. (ii) It proved that iGEM registry elements could be used in *V. harveyi* (promoter and terminator for instance). (iii) To the best of our knowledge, this is the first reported use of RFP in *V. harveyi*.

![](https://static.igem.org/mediawiki/2017/8/85/T--INSA-UPS_France--Results_4.jpg)

Fig. 3: **V. harveyi can be modified to produce RFP**. *V. harveyi* JMH626 (panels C & D) and conjugated with pBBR1MCS-4-RFP (panels A & B) in optic microscopy (panel A & C) or fluorescence microscopy (panels B & D). Emission: 530-560nm/Excitation 572-647nm.
On the panel (A), which shows control *V. harveyi* JMH626, no red bioluminescence is observed and the panel (C) prove the presence of cells by optic microscopy. On (B), red bioluminescence is clearly visible. The position of the red stains are correlated to with the location of the *V. harveyi* pBBR1MCS-5-RFP conjugated cells observed by optic microscopy (D).

## Perspectives

Next step will be to integrate the plasmid containing the engineered CqsS\* receptor into *V. harveyi* to create a sensor able to detect both *V. cholerae* CAI-1 and *V. harveyi* C8-CAI-1. Likewise, the Als construction tested in *E. coli* could be now tested in *V. harveyi* (see next module).

# 4. Production of diacetyl to establish communication between prokaryotic and eukaryotic cells

## Background

The quorum sensing signal detected by *V. harveyi* has to be transmitted to the yeast *P. pastoris*. For that, we chose to engineer *V. harveyi* so that it will conditionally produce  [diacetyl](https://2017.igem.org/Team%3AINSA-UPS_France/Design)  as the communication molecule between the bacterium and the yeast. Here, we tested the functionality of our construction containing the *als* gene, encoding the acetolactate synthase that enable diacetyl production (  [Part:BBa\_K2278011](http://parts.igem.org/wiki/index.php/Part%3ABBa_K2278011) ).

## Materials and methods

Strain construction can be found [here](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) and protocols used can be found there:  [Cultivation condition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot1),  [NMR analysis](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot3)  and  [Protein production and sampling](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot7).

## Results and discussion

To check the functionality of the plasmid expressing *als*, we first used *E. coli*. Figure 4 presents the results obtained for the 1H NMR analysis of the supernatant of *E. coli* strains with empty pSB1C3 or pSB1C3-*als*. Diacetyl specific shift was confirmed by spiking experiment. A small peak of diacetyl could be detected in the strain expressing *als* but not in the control. However, the amount seems very low. This could be related to two punctual mutations observed in the *als* sequence of this clone. However, only a single sequencing run was performed for this part, which is insufficient to be affirmative about the reality of these mutations.

![](https://static.igem.org/mediawiki/2017/2/22/T--INSA-UPS_France--Diacetyl_Brice.jpg)

Figure 4: **Validation of diacetyl production by NMR analysis** (800MHz, 10% D2O, 280K). Overlaid 1H NMR spectra of *E. coli* supernatants from *E. coli* pSB1C3 (negative control) in green; *E. coli* pSB1C3 + 0.50 mM diacetyl standard solution (positive control underlying the signature of diacetyl as a chemical shift of δ = 2.34 ppm) in red; two biological replicates of E. coli expressing *als* in blue colors.

## Perspectives

Next step will be to verify the sequence of als and to express the plasmid encoding for the right sequence of Als in *V. harveyi*. Diacetyl production will be then checked again by NMR.

# 5. Diacetyl detection by *Pichia pastoris*:

## Background

To allow a communication between *V. harveyi* and *P. pastoris* mediated by diacetyl, the Odr-10 receptor  [(design)](https://2017.igem.org/Team%3AINSA-UPS_France/Design)  had to be expressed in *P. pastoris*. To check if Odr-10 was functional *in vivo*, we used a pFUS1-RFP reporter system. Indeed when diacetyl binds to Odr-10 a cascade of activation of Ste proteins (endogenous to *P. pastoris*) will lead to the binding of Ste12 on pFUS1 promoter, and the expression of RFP should be activated.

## Materials and methods

Strain construction can be found [here](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) and protocols used can be found there:  [Cultivation condition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot1) ,  [Medium composition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot0)  and  [Plate reader](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot8).

## Results and discussion

We tested the functionality of the complete system Odr-10/pFUS1-RFP by growing the cells on a medium specifically designed to induce the activation of Ste proteins (Johnston et al., 1977). Diacetyl was added at various concentration. Absorbance and fluorescence production by *P. pastoris* strain having integrated the empty plasmid or the plasmid containing Odr-10/pFUS1-RFP system was followed over the time on a microplate reader. A positive control was obtained by growing a *Pichia* strain with a constitutive high expression of RFP (pGAP promoter fused to RFP gene). Results are presented in Figure 5.
In the conditions without diacetyl or with 500 µM of diacetyl, no difference was observed between the control and the strain expressing Odr-10/pFUS1-RFP. However, when diacetyl is present at higher concentration (i.e. 1000 µM), higher expression is
observed than in the negative control.
These data demonstrate the functionality of the complete detection pathway. Incidently, they also show that pGAP (our positive control promoter) is functional in *P. pastoris*, which increase the range of application of this promoter biobrick ( [Part:BBa\_K431009](http://parts.igem.org/wiki/index.php/Part%3ABBa_K431009) ). This was demonstrated again by RT-qPCR in the next Results module. Finally, this result suggests that our engineered *P. pastoris* is ready to communicate with *Vibrio* species if this former one produce diacetyl.

![](https://static.igem.org/mediawiki/2017/7/74/T--INSA-UPS_France--Results_6.jpg)

Figure 5: **Measurement of pFUS1 activity**. *P. pastoris* was grown in CMM media supplemented with glutamine. Negative control (T-) is performed using*P. pastoris* with a genomic integration of pPICZα, *P. pastoris* with a pPICZα-ODR10/pFUS1-RFP genomic integration were grown with 500 µM or 1000 µM of diacetyl. *P. pastoris* with genomic integration of pPICZα-pGAP-RFP (T+) is the positive control. Results are presented as the ratio of RFP fluorescence at 600(+/- 10) nm normalized by absorbance at 595 nm (measure of cell density). Results are from duplicated experiments.

## Perspectives

Next step will be to grow together our *V. harveyi* expressing Als and our *P. pastoris* expressing Odr-10/pFUS-RFP to demonstrate synthetic communication between them.

# 6. *P. pastoris* is able to produce functional antimicrobial peptides

## Background

The final goal of our synthetic consortia was to kill *V. cholerae* with newly described antimicrobial peptides from crocodile. Here, we tested the production by *P. pastoris* of three inovative antimicrobial peptides (AMP), i.e. D-NY15, cOT2 and Leucrocin I (respectively  [Part:BBa\_K2278021](http://parts.igem.org/wiki/index.php/Part%3ABBa_K2278021), [Part:BBa\_K2278023](http://parts.igem.org/wiki/index.php/Part%3ABBa_K2278023)  and  [Part:BBa\_K2278022](http://parts.igem.org/wiki/index.php/Part%3ABBa_K2278022)  )

## Materials and methods

Strain construction can be found [here](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) and protocols used can be found there:  [Cultivation condition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot1) ,  [Medium composition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot0) ,  [Plate reader](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot8),  [Semi quantitative RT-PCR](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot9) and  [On plate toxicity assay](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot10).

## Results and discussion

* **Expression assay**

Because our AMPs have never been produced by *P. pastoris* and their toxicity on this yeast was not assessed, we first validated the expression of the AMPs by RT-qPCR. Figure 6 showed the results obtained for D-NY15 expression in *P. pastoris*. The amount of fluorescence provided by the RT-qPCR with the D-NY15 amorces (curves A1, A2 and A3) is raising after few cycles (8.32 +/- 0.03) whereas the negative control (pPICZα only, curves C1, C2 and C3) starts to be amplified at over 29 cycles (i.e. non-specific amplification). This means that the D-NY15 encoding gene is expressed in *P. pastoris* and non-lethal.

![](https://static.igem.org/mediawiki/2017/0/0e/T--INSA-UPS_France--RTPCR.png)

Fig 6. **RT-qPCR of D-NY15**. RNA were from *P. pastoris* strains having integrated pPICZ or pPICZα-D-NY15. Total RNAs were extracted from transformants and reverse transcriptions were performed using Superscript II reverse transcriptase (Invitrogen). Resulting D-NY15 cDNA were then amplified by quantitative PCR. The curves correspond to *P. pastoris* with integrated pPICZα-D-NY15 (A1, A2, A3), water control (B1 & B2), and *P. pastoris* with integrated pPICZα (C1, C2, C3).

* **Toxicity assay**

To test the toxicity of the three AMPs, disks soaked with the supernatant of *P. Pastoris* with genomic integration of pPICZα, pPICZα-D-NY15, pPICZα-cOT2 or pPICZα-Leucrocin I were put on freshly inoculated solid culture of *V. harveyi*. Results are presented on Figure 7. Inhibition haloes were observed around both the positive control and the patch containing the supernatant from a D-NY15 expressing strain. No growth inhibition were observed around the disks containing the supernatant from COT2 and Leucrocin I expressing strains. These data nicely demonstrated the capacity of the engineered crocodile peptide D-NY15 to inhibit *V. harveyi* growth but also the capacity of *P. pastoris* to produce antimicrobial peptides. This is the first demonstration of AMP production by *P. pastoris* by an IGEM Team and the first use of sequence coming from crocodile in iGEM!

![](https://static.igem.org/mediawiki/2017/9/98/T--INSA-UPS_France--Results_8.JPG)

Figure 7 : **AMP halo assays**. Positive control was performed with chloramphenicol (25 ng/µL), the negative control was performed with the empty plasmid integrated in *P. pastoris*, the assays were performed with the supernatants from *P. pastoris* strains expressing either pPICZα-D-NY15 (D-NY15), pPICZα-Leucrocin I (Leucrocin I) or pPICZα-cOT2 (cOT2).

## Perspectives

Next step will be to prove the inhibitory activity of AMPs in liquid medium containing both modified *P. pastoris* with *V. harveyi*. For cOT2 or Leucrocin I, which cytoxicity was not demonstrated yet, a higher concentration of supernatant could be tested.

# 7. Co-cultivation of *P. pastoris* and *V. harveyi* is possible

## Background

Ultimately our synthetic consortia will be freeze-dried and placed into a plastic bag containing dried cultivation medium. As each microorganism of the consortia has its own cultivation requirements, a difficulty was to find a common media on which *V. harveyi* and *P. pastoris* can grow together.

## Materials and methods

Protocols used can be found here: [Cultivation condition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot1) ,  [Medium composition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot0).

## Results and discussion

* **Selection of the cultivation medium**

*P. pastoris* and *V. harveyi* were grown on six media corresponding to variants of their standard media (LB, LB acetate 2%, LM, LM acetate 2%, YPD, and YPA 1%, see  [Medium composition](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols#prot0) for details).

![](https://static.igem.org/mediawiki/2017/8/82/T--INSA-UPS_France--TableKinetiq.png)

Figure 8: **Table of growth rates (h-1) of V. harveyi and P. pastoris** on six media. Media were inoculated at an initial OD600nm = 0.05. Optical density was measured very hours. Results are issued from a single biological replicate.

As expected, *V. harveyi* and *P. pastoris* were growing well on their respective usual media (LM and YPD ; figure 8), but not as performant on each other medium. Acetate inhibited growth of both microorganism in YPA & LMA but it was better tolerated in LBA. Eventually, LM medium appears as good compromise to ensure a satisfying growth of both strains. This is an interesting result for this project since LM medium is cheap and easy to produce.
All these data have been used to fit our  [model page](https://2017.igem.org/Team%3AINSA-UPS_France/Model/Simulation) in order to help in the design of the [device](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Device).

## Perspectives

Next step will be to challenge the capacity of our strains to recover from freeze-dry since they will be lyophylized in our [device](https://2017.igem.org/Team%3AINSA-UPS_France/Entrepreneurship/Device). Preliminary tries suggested they survived but more accurate measurements will have to be performed.

# 8. Permeability assay of the membranes used for the devices:

## Background

We selected two types of membrane materials, i. e. TPX® X44B 50 PVDF 0.1 µm for making the sachet containing our synthetic biology system composed of *V. harveyi* and *P. pastoris* with nutrient medium. Here we tested the permeability of both to check whether this membrane could contain our synthetic organisms while allowing the antimicrobial peptides and quorum sensing molecules go through from the device compartment to the treated water.

## Results and discussion

We first used the TPX® X44B 50µm material used by  [iGEM Groeningen 2012](https://2012.igem.org/Team%3AGroningen) and  [iGEM Toulouse 2015](https://2015.igem.org/Team%3AToulouse). To assess the porosity of the material, we performed a liquid/liquid transfer assay with bengale pink dye. Bengale pink dye was chosen because of its high molecular mass for a dye (977 g/mol) to get closer to the size of our AMPs. The test was done with a Hele-Shaw measurement cell (two parallel planes spaced by 2 mm) with a camera (EDMUND OPTIC) to monitor the diffusion through the membrane (figure 9). The cell was filled with water and bengale pink was introduced from the top in a cone of membrane (or absorbent paper as a control), in contact with the cell water.

![](https://static.igem.org/mediawiki/2017/5/53/T--INSA-UPS_France--Results_11.png)

Figure 9: **Hele-Shaw measurement cell during the experiment**.

We did not observe diffusion of the dye through the TPX membrane in this experiment (Data not shown). Since we were concerned with the hydrophobicity of the material, we also tried to check if water can go through by putting the material with a drop of water on top of absorbent paper. No moisture on the absorbent paper was observed during the course of the experiment. We conclude that the chemical characteristics of the TPX material does not suit to our project.
Next we tested the material proposed by our partner Sunwaterlife, i.e. a membrane used in their water cleaning system (PVDF 0.1 µm from Orelis Environnement SAS, France). We tested this material with the same Hele-Shaw measurement cell. As shown on figure 10, the dye is able to go through the material. Diffusion was observed in less than 1 minute. This means this material is entirely suitable for our project since the 0.1 µm porosity prevents bacterial diffusion but allows peptides diffusion.

![](https://static.igem.org/mediawiki/2017/9/98/T--INSA-UPS_France--BengalePink.png)

Figure 10: **Bengale pink dye diffusion** through the PVDF membrane 0.1 µm five minutes after the experiment start.

## Perspectives:

Next step will be to test this membrane with our synthetic consortia to check whether bacterial diffusion is prevented while peptides diffusion allowed.

# References:

| Realisations pages | | | | | |
| --- | --- | --- | --- | --- | --- |
| [Overview](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments) | [Notebook](https://2017.igem.org/Team%3AINSA-UPS_France/Notebook) | [Clonings](https://2017.igem.org/Team%3AINSA-UPS_France/Experiments/Clonings) | *Results* | [Contribution](https://2017.igem.org/Team%3AINSA-UPS_France/Contribution) | [Protocols](https://2017.igem.org/Team%3AINSA-UPS_France/Protocols) | [Safety](https://2017.igem.org/Team%3AINSA-UPS_France/Safety) |

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