iGEM 2020 — Team Toulouse_INSA-UPS

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# Team:Toulouse INSA-UPS/Notebook

![](https://static.igem.org/mediawiki/2020/2/21/T--Toulouse_INSA-UPS--2020_newbanner.jpg)

[![](https://static.igem.org/mediawiki/2020/9/98/T--Toulouse_INSA-UPS--2020_IGEMINI_blanc.svg) Toulouse INSA-UPS](https://2020.igem.org/Team%3AToulouse_INSA-UPS)

* [Home](https://2020.igem.org/Team%3AToulouse_INSA-UPS)
* Team

  [Team members](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Team)
  [Attributions](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Collaborations)
  [Partnership](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Partnership)
* Project

  [Description](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Description)
  [Design](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Design)
  [Modeling](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Model)
  [Experiments](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Experiments)
  [Results](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Results)
  [Contribution](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Contribution)
  [Engineering Success](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Engineering)
  [Notebook](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Notebook)
* Implementation

  [Implementation](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Implementation)
  [Sustainable Development Impact](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Sustainable)
  [Supporting Entrepreneurship](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Entrepreneurship)
* [Parts](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Parts)
* Human Practices

  [Overview](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices)
  [Integrated Human Practices](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#Integrated_Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Education)
  [Contribution](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Contribution)
* [Safety](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Safety)
* 🏆

  [Achievements](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Achievements)
  [Awards](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Awards)
  [Poster](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Poster)
  [Promotion video](https://video.igem.org/videos/watch/02a9ba65-6ec1-4be7-b3e7-07cc5a3ff566)
  [Presentation video](https://video.igem.org/videos/watch/0195342d-4c1d-4908-ae87-328fbcbbbc22)

![PF](https://static.igem.org/mediawiki/2020/e/e4/T--Toulouse_INSA-UPS--2020_petite_fusee.png)

## Notebook

Here is an overview over the accomplishments and the challenges we had to manage during our project.

If you want to learn more about our brainstorming stage, from december to march, check out our description page [here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Description#brainstorming).

To know more about our collaborations click [here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Collaborations), and about our two partnerships click [here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Partnership).

December
January
February
March
April
May
June
July
August
September
October

December

Week 48
Week 49
Week 50
Week 51
Week 52
Week 1

January

Week 1
Week 2
Week 3
Week 4
Week 5

February

Week 5
Week 6
Week 7
Week 8
Week 9

March

Week 9
Week 10
Week 11
Week 12
Week 13
Week 14

April

Week 14
Week 15
Week 16
Week 17
Week 18

May

Week 18
Week 19
Week 20
Week 21
Week 22

June

Week 23
Week 24
Week 25
Week 26
Week 27

July

Week 27
Week 28
Week 29
Week 30
Week 31

August

Week 31
Week 32
Week 33
Week 34
Week 35
Week 36

September

Week 36
Week 37
Week 38
Week 39
Week 40

October

Week 40
Week 41
Week 42
Week 43
Week 44

## Week of December 9th to 15th

### Project

We held the first meeting where we met the rest of the team and some of the advisors (Pierre and Eliot were still in Japan for their exchange semester). We also defined some team roles:

* Arno: Wiki leader
* Cécilia: Finances
* Laura: Organization
* Laurène: Communication
* Solène: Human Practices

![](https://static.igem.org/mediawiki/2020/1/12/T--Toulouse_INSA-UPS--2020--PremierJour.jpg)

## Week of December 16th to 22th

### Project

We had our second meeting where we were introduced to the judging handbook. Each of us thought of a possible project before joining the team so we briefly presented them to the rest of the team.

## Week of January 6th to 12th

### Project

We met again after the Christmas break, and continued discussing our possible projects ideas: a tumoral biodetector, production of vitamins, algae treatment, spider silk production…

### Human Practices

![](https://static.igem.org/mediawiki/2020/d/d8/T--Toulouse_INSA-UPS--2020--Mairie.jpg)

We were invited to the “Soirée des talents Toulousains 2019”, an award ceremony for Toulouse inhabitants of the year 2019 organized by Toulouse Métropole. It was the perfect opportunity to start team building, and also to network with local entrepreneurs.

## Week of January 13th to 19th

### Project

We started searching possible ways to fund our project, and continued discussing our possible ideas between us.

## Week of January 20th to 26th

### Project

During this week's meeting, we had the idea for the first time of using electrosynthesis in our project.

Contenu Semaine 5

## Week of February 3rd to 9th

### Project

Out of the eight project ideas we decided to dismiss some of them: toxic red mud treatment, algae treatment, tumoral biodetector.

### Human Practices

We attended TWB start-up day, this event brings together European key players in the biotech start-up business. We also had the chance to meet and chat with Randy Rettberg, the iGEM president.

![](https://static.igem.org/mediawiki/2020/a/a2/T--Toulouse_INSA-UPS--2020--TWBDAY.jpg)

## Week of February 10th to 16th

### Project

As we continued exploring all our ideas, we decided to stop searching about the production of spider silk. We also decided to merge our idea about vitamin production and space to produce nutrients for astronauts.

### Team building

We finally met Eliot and Pierre, who came back from their semester in Japan and we had a lovely team Building dinner with previous iGEMERS (iGEM Toulouse 2018, 2017).

![](https://static.igem.org/mediawiki/2020/0/06/T--Toulouse_INSA-UPS--2020--TeamDinner.jpg)

Semaine 8

## Week of February 24th to 1st of March

### Human Practices

We had our first meeting with Alain Maillet, scientific leader at MEDES - Institute for Space Medicine and Physiology. We discussed about the loss of nutritional value of food over time and also about the available resources in a space station. You can learn more about it [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step0)].

## Week of March the 2nd to 8th of March

### Project

Brainstorming continued. We focused on the production of nutrients in space for astronauts and a possible solution for the sharka virus.

Semaine 11

## Week of March 16th to 22nd

### Covid-19

On the 16th of march our country started a lockdown due to COVID-19, from now on all our meetings will be held remotely.

### Project

After a long discussion with the whole team, we decided that we will work on the production of molecules in space for our project. We will grow two microorganisms in coculture: *Clostridium ljungdahlii PETC* (an acetogen) and *Saccharomyces cerevisiae* (a yeast).

### Human Practices

We interviewed Jean-Marie François, specialist in *Saccharomyces cerevisiae*. For more information click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step5)].

We also Interviewed Benjamin Erable, responsible for research at CNRS, specialist in electroactive biofilms. For more information click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step2)].

![](https://static.igem.org/mediawiki/2020/c/cd/T--Toulouse_INSA-UPS--2020--StayHome.jpg)

## Week of March 23rd to 29th

### Project

We designated Eliot as our modeling leader. We started brainstorming on which molecules we want to produce in space.

## Week of March 30th to 5th of April

### Project

We started searching which are the available resources that we could use in space for our project.

## Week of April the 6th to 12th

### Project

This week, we focused on different vitamins that would be interesting to produce in space, we thought about producing vitamin D, vitamin K, vitamin A, or vitamin E.

### Human Practices

For our Human Practices, we decided to focus on how synthetic biology is essential for long-duration space travel.

## Week of April the 13th to 19th

### Project

We have chosen Pierre as our entrepreneurship leader, and Solène as our Ethics leader. We also decided on our project name: **iGEMINI**. Brainstorming about the molecules we want to produce continued. This week, in addition to working on vitamins, we also searched what flavoring molecules we could synthesize.

### Human Practices

We interviewed Dr. Gilles Truan, the head of the Molecular and Metabolic Engineering (I2M) team and responsible for the Metabolic Engineering axis which includes three TBI teams. You can learn more about this interview [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step3)].

## Week of April 20th to 26th

### Project

We decided on producing provitamin A, and as flavoring molecules we wanted to produce limonene, geraniol and brazzein.

## Week of April 27th to 3rd of May

### Project

Optogenetics systems were chosen to let astronauts select the flavor they prefer. There would be two LEDs colors: red and blue.

## Week of May 4th to 10th

### Project

We started designing our experimental plan for the summer

![](https://static.igem.org/mediawiki/2020/a/a0/T--Toulouse_INSA-UPS--2020--week19.jpg)

## Week of May the 11th to 17th

### Project

We started designing the sequences we want to clone. We also had our second meeting with Alain Maillet where we got the wonderful news that the French National Centre for Space Studies (CNES) wanted to sponsor our project!

![](https://static.igem.org/mediawiki/2020/5/52/T--Toulouse_INSA-UPS--2020--week20.jpg)

### Human Practices

We interviewed Dr. Natalie Ley (Head of the Microbiology Research Unit, coordinator of the Space Life Science program at SCK-CEN) and Dr. Rob Van Houdt (specialist in genetic engineering at Microbiology Research Unitat at SCK-CEN). For more information click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step7)].

![](https://static.igem.org/mediawiki/2020/a/a2/T--Toulouse_INSA-UPS--2020--Natalie.jpg)

## Week of May the 18th to 24th

### Project

We continued designing the sequences we want to clone.

![](https://static.igem.org/mediawiki/2020/3/32/T--Toulouse_INSA-UPS--2020--week21.jpg)

### Human Practices

We had our third meeting with Alain Maillet, this time we discussed the recycling system that is currently in place at the international space station.

## Week of May 25th to 31th

### Project

We kept designing the sequences we wanted to clone.

 ![](https://static.igem.org/mediawiki/2020/e/e4/T--Toulouse_INSA-UPS--2020--week22.jpg)

### Human Practices

We discussed with Marie-Pierre Escudié, our Ethics advisor, to decide what we want to achieve for our integrated human practices and public and education engagement. You can read the full interview [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step6)].

## Week of June 1st to 7th

### Project

We finished our sequences design using Benchling.

 ![](https://static.igem.org/mediawiki/2020/a/a0/T--Toulouse_INSA-UPS--2020--week23.jpg)

### Human Practices

We participated at Exposcience, an exposition for young scientists in Occitanie, where we presented our project and discussed it with middle school students.

![](https://static.igem.org/mediawiki/2020/9/9c/T--Toulouse_INSA-UPS--2020--exposcience.jpg)

### Collaborations and Partnerships

#### AshesiGhana iGEM team

New techniques developed for space could be used on Earth and have a major impact on a world where the population is growing and where resources are diminishing. For these two major reasons, we wondered about the possible use of our production device on earth. We looked for areas in the world where vitamin A deficiency is present and found out that Africa is part of these areas, so we contacted Team iGEM AshesiGhana.
In our first meeting with AshesiGhana iGEM Team we presented both of our projects and talked about potential collaborations. Some ideas came up, for instance, to give a survey to Ghanaian people about the acceptability of our vitamin production device.

## Week of June 8th to 14th

### Project

We were finally able to go to the lab! Five out of the eight team members started, we wore masks all day and made sure we were keeping safe distances between us. We got our first genetic elements from Gilles Truan.
During this first week, we finished designing all the primers that we will need, we ran the order for our DNA sequences with IDT, and started designing our fermentation protocols.

![](https://static.igem.org/mediawiki/2020/e/e4/T--Toulouse_INSA-UPS--2020--firstday.jpg)

![](https://static.igem.org/mediawiki/2020/3/3d/T--Toulouse_INSA-UPS--2020--week24.jpg)

### Entrepreneurship

We had our first online meeting with Le Catalyseur, to start talking about how we could create a start-up from our project.

## Week of June 15th to 21th

![](https://static.igem.org/mediawiki/2020/8/8c/T--Toulouse_INSA-UPS--2020--SocialDistancing.jpg)

### Project

The whole team is back in the lab! As always we followed the rules established by our university: face masks at all times, safe distances between us, and often hands washings.
We did the security training required by TBI.

### Human Practices

We had our first meeting with Arnaud Cockx, Gas-Liquid Reactor Engineering professor at INSA Toulouse, to discuss our bioreactor, read the full interview [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step9)].

### Collaborations and Partnerships

#### AshesiGhana iGEM team

We created our Google Drive work space to write down our ideas about our partnership.

## Week of June 22nd to 28th

### Project

#### Clonings

##### Plasmids

Amplification and verification of all the plasmids that we were given to us.

##### tHmg1-CrtE

Amplification and purification from the plasmid by PCR.

##### CrtYBekI

Size problem with our plasmid amplification.

##### Rose Brazzein

Amplification of one of the fragments from the plasmid by PCR.

![](https://static.igem.org/mediawiki/2020/b/ba/T--Toulouse_INSA-UPS--2020--week26.jpg)

#### Cococulture

First cultures with *Saccharomyces cerevisiae* on acetate and ethanol.

### Collaborations and Partnerships

#### AshesiGhana iGEM team

AshesiGhana team have created our slack work space in order to easily exchange information between our teams. We talked about attaining both silver and gold medals criteria.

## Week of June 29th to 5th of July

### Project

#### Clonnings

##### tHmg1-CrtE

Infusion of the three fragments into pUC19.

##### CrtYBekI

Amplification of the fragment, then cloning into pUC19.

##### Rose/Brazzein

Amplification of fragments.

![](https://static.igem.org/mediawiki/2020/3/3c/T--Toulouse_INSA-UPS--2020--week27.jpg)

#### Cococulture

First culture monitoring yeast with a medium containing 60% acetate and 40% ethanol,

### Collaborations and Partnerships

We participated in the German Meetup on the 4th and 5th of July, where we presented our team and our poster. We got first place in the poster competition and won a 5000€ centrifuge ! It was amazing to be able to meet with other teams and learn about their projects.

![](https://static.igem.org/mediawiki/2020/2/23/T--Toulouse_INSA-UPS--2020--germanmeetup.jpg)

## Week of July 6th to 12th

### Project

#### Clonings

We received all the DNA blocks from IDT, so we can finally start all the constructions that were not started yet.

##### tHmg1-CrtE

Infusion of the other three fragments into pUC19.

##### CrtYBekI

Fail of Last week’s cloning.

##### Rose/Brazzein

Amplification of the fragment by PCR and subcloning in pUC19.

##### Red regulation

Amplification of four fragments by PCR and cloning into pUC19. Negative results.

##### Lemon

Amplification of the fragment by PCR and subcloning in pUC19. Negative results.

![](https://static.igem.org/mediawiki/2020/1/1f/T--Toulouse_INSA-UPS--2020--week28.jpg)

#### Cococulture

Test of different media concentrations for the growth of *S. cerevisiae*.

### Human Practices

We had a meeting with Arnaud Cockx to talk about the modelling of our project.

We interviewed former Astronaut Phillipe Perrin, we talked about nutrition in space. To watch the full interview click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#text11)].

### Collaborations and Partnerships

#### iGEM France

Meeting with all the iGEM teams in France. iGEM Grenoble suggested doing a video with all the teams which sounds very exciting!

#### AshesiGhana iGEM team

This was an update meeting on what we have done. We have created a file « notebook » to follow our meeting through the year. We discussed resources that are available in Ghana that we could use for our Vitamin A production device. We thought to do an experiment for the iGEM Ghana team and advised them to use the sponsorship with IDT and get 20kb free of DNA.

#### Concordia iGEM team

We had our first meeting with the Concordia iGEM team where we presented both of our projects and talked about potential collaborations. Some ideas came up. We then created a Google drive work space as well as a Slack to easily communicate with all the members of each team.

![](https://static.igem.org/mediawiki/2020/0/09/T--Toulouse_INSA-UPS--2020_first-meeting-concordia.png)

### Entrepreneurship

We had a meeting with Le Catalyseur in order to discuss our project potential risks, and their solutions.

## Week of July 13th to 19th

### Project

#### Clonings

##### CrtYBekI

For both parts of the construction: fragments were cloned and transformed into *E. coli* stellar cells. One of the clones has been sequenced with positive results.

##### Rose/Brazzein

For one of the inserts we did a miniprep, but it turned out negative. On the other hand, we sent the other inserts to be sequenced.

##### Lemon

We did the same cloning again and verified the transformation by enzymatic digestion. Sadly, none of the clones have the insert, which means that we have to rethink our design.

##### Red regulation

After retesting our strategy we decided to design a new strategy with more but smaller blocks.

##### Blue regulation

We finally received the plasmids containing some of the fragments.

![](https://static.igem.org/mediawiki/2020/4/4e/T--Toulouse_INSA-UPS--2020--week29.jpg)

### Human Practices

We had a meeting with Alain Maillet at CNES, to discuss the progress of our project.

![](https://static.igem.org/mediawiki/2020/7/7b/T--Toulouse_INSA-UPS--2020--CNESMeeting.jpg)

## Week from the 20th to 26th of July

### Project

#### Clonings

##### Rose/Brazzein

We got our sequencing results: the sequence for the rose is good!

As for the other part of our insert, brazzein, we unfortunately didn’t get any positive results so we redesigned our strategy.

##### Red regulation

We amplified the fragments by PCR.

##### Blue regulation

The fragments were amplified by PCR from the plasmids we received last week.

![](https://static.igem.org/mediawiki/2020/a/a2/T--Toulouse_INSA-UPS--2020--week30.jpg)

### Human Practices

![](https://static.igem.org/mediawiki/2020/a/ad/T--Toulouse_INSA-UPS--2020--France3.jpg)

On Monday, we were interviewed with France 3 Occitanie, a local television channel. We interviewed Karen Dhanraj and Romain Maillet who are specialists about the nutritive aspect of the yeast. The interview report is available [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#text13)].

On Thursday we interviewed Dr. Brigitte Godard, the European astronaut’s doctor. For the full interview click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step12)].

On Saturday we had the opportunity to discuss with former astronaut Jean-Jacques Favier, we talked about our project and how it could be implemented in space. For the full interview click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step14)].

### Collaborations and partnerships

#### Concordia iGEM team

As both of our projects are related to space, we decided to establish partnership goals such as creating a contribution document for future iGEM teams or doing an experiment for them. We also did the plan for our contribution document and separate for both of our teams the work. This contribution is a guide for future iGEM teams.

### Entrepreneurship

We had our third meeting with Le Catalyseur where they presented us with what is a business model.

## Week of July 27th to 2nd of August

### Project

#### Clonings

##### tHmg1-CrtE

Both blocks have been purified and ligated together to produce the final construction.

##### CrtYBekI

New strategy for one of the intermediaries, so the fragments were amplified by PCR, cloned into pUC19 and transformed into *E. coli* stellar cells.

##### Red regulation

Fragments were amplified using new oligos.

##### Blue regulation

We continued testing different PCR methods for the fragments. For one of the blocks an InFusion cloning into pUC19 was done, but it didn’t work.

![](https://static.igem.org/mediawiki/2020/f/fe/T--Toulouse_INSA-UPS--2020--week31.jpg)

#### Cocolture

Yeast culture monitoring with different concentrations of glucose, acetate and ethanol to know its growth rate.
Preparation of *Clostridium ljungdahlii medium*.

### Human Practices

Interview with Romain Charles, astronaut’s support who participated on Mars 500 (a project that simulated the conditions that will be encountered by a crew on Mars). To read more about this interview click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step15)].

We were interviewed by La dépêche du Midi, a regional daily newspaper published in Toulouse.

### Collaborations and partnerships

![](https://static.igem.org/mediawiki/2020/8/8a/T--Toulouse_INSA-UPS--2020--GhanaMeeting.jpg)

#### AshesiGhana iGEM Team

They have sent emails to NGOs that are working on nutrition in Africa. We gave constructive and analytic feedback on their design process as well as informations about coastal erosion in France and Europe. We also have created a new channel in the shared slack for implementation because we think that we can help them on that part.

## Week of August 3rd to 9th

### Project

#### Clonings

##### tHmg1-CrtE

Colonies were verified by enzymatic digestion, we are ready to insert into the yeast.

##### CrtYBekI

Last week’s colonies were digested and we did an InFusion cloning to add a new fragment to the construction.

##### Red regulation

Amplified fragments were purified, cloned into pUC19 and transformed into *E. coli* stellar cells. Unfortunately none of the clones presented the insert.

##### Blue regulation

InFusion cloning was performed, but we did not get any positive results.

![](https://static.igem.org/mediawiki/2020/7/77/T--Toulouse_INSA-UPS--2020--week32.jpg)

### Collaborations and partnerships

#### Concordia iGEM team

![](https://static.igem.org/mediawiki/2020/0/02/T--Toulouse_INSA-UPS--2020--ConcordiaDB.jpg)

The goal of this meeting was to present to some of our team members the software Astrobio created by Concordia iGEM team. To give them a first feedback.

#### Pittsburgh iGEM team

With the iGEM Pittsburgh team, we exchanged on both of our projects as we are using two different regulation systems. We pointed out some advantages and drawbacks on both systems.

## Week of August 10th to 16th

### Project

#### Clonings

##### tHmg1-CrtE

We tried yeast transformation of the tHmg1-CrtE construction, but it didn’t work.

##### CrtYBekI

We got the results that last week cloning did not work.

##### Limonene

We retried the cloning with a new strategy, but unfortunately it didn’t work

##### Blue regulation

We retried the first part of the blue regulation, but we found out that some of the fragments are too small to be amplified by PCR.

![](https://static.igem.org/mediawiki/2020/9/98/T--Toulouse_INSA-UPS--2020--week33.jpg)

### Collaborations and partnerships

#### AshesiGhana iGEM team

We had feedback from the design team about advices that we gave them two weeks ago. We gave them some names of companies that are using bioluminescence bacteria (Glowee) and enzymes that are degrading plastic (Carbios). The survey about the acceptability of our Vitamin A production device has been sent to people in Ghana thanks to the iGEM Ghana team.

#### Pittsburgh iGEM team

We were invited to participate on their podcast « Bluetooth bacteria », that you can listen [[here](https://open.spotify.com/episode/2eupp1xmCxbtHeowtJIc1S?si=RHLVHMwCT5qZi-JTUncP0w)].

### Video

![](https://static.igem.org/mediawiki/2020/8/82/T--Toulouse_INSA-UPS--2020_citedelespace.png)

We visited La cité de l’espace, to decide which scenes we wanted to shoot there for our videos.

## Week from the 17th to 23rd of August

### Project

#### Clonings

##### CrtYBekI

The blocks were cloned into *E. coli* stellar cells by InFusion, we will know the results next week.

##### Limonene

Fragments were purified once again to try a new strategy.

 ![](https://static.igem.org/mediawiki/2020/b/bc/T--Toulouse_INSA-UPS--2020--week34.jpg)

#### Cococulture

##### Bacteria

We finally received the bacteria at the end of the week, but it had spent too much time at room temperature during the delivery process and it didn’t grow correctly.

##### Yeast

We tried different acetate concentrations and measured the growth rate.

### Collaborations and partnerships

#### Concordia iGEM team

![](https://static.igem.org/mediawiki/2020/6/64/T--Toulouse_INSA-UPS--2020--laptop.jpg)

The meeting goal was to give feedback from the other part of our team about their database. This feedback was really useful for them because the other team members did not have previous knowledge on how Astrobio database works.

We also attended their conference about microbes and cell agriculture for sustainable food production which was really interesting.

## Week of August 24th to 30th

### Project

#### Clonings

##### tHMG1-CrtE

*Saccharomyces cerevisiae* gal+ and gal- strains were transformed with the β-carotene boosters (Hmg1 and CrtE) at the beginning of the week. By the end of the week we had some clones! And none in the negative control plates. Clones were verified by genomic PCR, and we had positif results. We are done with this construction!

 ![](https://static.igem.org/mediawiki/2020/c/c3/T--Toulouse_INSA-UPS--2020--week35.jpg)

### Video

![](https://static.igem.org/mediawiki/2020/e/ef/T--Toulouse_INSA-UPS--2020--filming.jpg)

From Tuesday to Friday we shot with Rémy for our video deliverables. We finished shooting all the clips for the promotion video (and the narration) and we started some scenes of the presentation video.

## Week of August 31th to 6th of September

### COVID-19

From there on, we had two big problems. First, only five members of the team were left working in the lab, since the other three had to go back to class. Worse, the pandemic stroke back in the Toulouse student community and the five survivors had to miss some weeks when they were experiencing Covid-19 symptoms or while waiting for the test results.

### Project

#### Clonings

There were less and less hands in the lab and we were running out of time. We had a difficult decision to make and decided to stop working on the blue and red regulation, to focus more time and effort on the other constructions.

##### CrtYBekI

Last week clones could be OK! They were sent to be sequenced.

##### Rose and brazzein

We performed a bulk PCR test on the numerous clones and all turned out negative.

##### Limonene

We checked the fragment insertion for the lemon gene, but it gave negative results.

 ![](https://static.igem.org/mediawiki/2020/a/ad/T--Toulouse_INSA-UPS--2020--week36.jpg)

#### Cococulture

We started setting up the system for the coculture with the help of Christine Lafforgue, professor at INSA Toulouse.

##### Bacteria

We had a meeting with Jason Whitham to try to solve our problems on why *Clostridium ljungdahlii* wasn’t growing. We concluded that all our protocols need to be optimized. To read the full interview click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step17)].

##### Yeast

We followed three cultures of yeast so we can understand how the two substrates are consumed. We then ran an HPLC batch analysis of acetate and ethanol.

We also tested the growth of yeast in a media closer to the media that will be used for the coculture, unfortunately we forgot to add NTA (that helps to dissolve the metals) so nothing grew, but we will restart with a new medium.

### Human Practices

![](https://static.igem.org/mediawiki/2020/3/30/T--Toulouse_INSA-UPS--2020--VideoGameCosmos.jpg)

We discussed with Jason Whitham, expert in anaerobic fermentation, about how to make *Clostridium ljungdahlii* grow, he gave some very useful tips, to learn more about it click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step17)].
We sent the first part of our video game followed up with some questions to a pedagogical inspector who diffused it to many french high school teachers.

### Collaborations and partnerships

#### Concordia iGEM team

We talked about the design of the contribution document and mentioned using Canva.

#### Calgary iGEM team

iGEM Calgary contacted us to talk about our projects together because they also work on yeast that produces provitamin A. We had an interesting meeting talking about a potential collaboration.

## Week of September 7th to 13th

### Project

#### Clonings

##### tHmg1+CrtE

We prepared the preculture of the transformed yeast and the non-transformed in YNB+Acetate for the quantification of GGPP next week.

##### CrtYBekI

We now can combine the two parts of the vitamin A into one.

##### Rose and brazzein

We got the results of the sequencing, unfortunately two bases have been deleted in the ORF.

![](https://static.igem.org/mediawiki/2020/f/fa/T--Toulouse_INSA-UPS--2020--week37-38.jpg)

#### Coculture

##### Bacteria

We prepared the medium for the bacteria.

##### Yeast

We measured the growth rate and the specific consumption rates of ethanol and acetate.
We followed three cultures of yeast on the coculture medium in order to know the growth rate.

### Human Practices

We had a meeting with Stéphane Guillouet for the culture condition and monitoring with limitation of oxygen. We decided that we will perform a batch and then a chemostat. Click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#pdf_step1)] to know more about it.

### Collaborations and partnerships

#### Concordia iGEM team

The goal of this meeting was to agreed on the maximum number of pages for the contribution document.

### Entrepreneurship

We had our fourth meeting with Le Catalyseur where we discussed our business model.

## Week of September the 14th to 20th

### Project

#### Clonings

##### tHmg1+CrtE

The pre-cultures for the quantification of GGPP did not grow enough. So we had to restart the experiment.

##### CrtYBekI

Both parts of the construction were assembled. Next step: yeast (which already has tHMG1-CrtE) transformation.

![](https://static.igem.org/mediawiki/2020/a/ab/T--Toulouse_INSA-UPS--2020--week38.jpg)

#### Coculture

##### *Clostridium ljungdahlii*

![](https://static.igem.org/mediawiki/2020/c/c1/T--Toulouse_INSA-UPS--2020--Clostri.jpg)

We finally received the fresh strain of *Clostridium ljungdahlii*. We prepared the reactor for the experiments next week.

## Week of September 21st to 27th

### Project

#### Clonings

We had to make a difficult decision once again, and decided to fully focus on the production of β-carotene, so we stopped working on the two different tastes.

##### tHmg1+CrtE

Good news, our GGPP assess is successful and our construction is increasing its pool by a whopping five-times.

##### CrtYBekI

Our transformants are not orange as expected. Sadly, the sequencing easily explained it: our clones had a non-detected before mutation creating a shift in the ORF, preventing any expression of our enzyme.

![](https://static.igem.org/mediawiki/2020/f/f0/T--Toulouse_INSA-UPS--2020--week39.jpg)

#### Coculture

#### Bacteria

We inoculated the reactor with *Clostridium ljungdahlii* and monitored the growth by measuring the OD and HPLC analysis.

### Human Practices

![](https://static.igem.org/mediawiki/2020/6/6f/T--Toulouse_INSA-UPS--2020--CiteExhibition.jpg)

We did our conference with La Citée de l’espace and three experts (Brigitte Godard, the European astronaut’s doctor, Yves Gourinat, professor at ISAE-SUPAERO, and Gilles Truan, expert in synthetic biology) on Wednesday about microbiology and space, to learn more about it click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Education#online-conference)].

During the weekend, we went to La Citée de l’espace to present our exhibition and to talk with the general public about our project and the iGEM competition, click [[here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Education#itinerant-exhibition)] to read more about our itinerant exhibition.

### Entrepreneurship

We had our fourth meeting with Le Catalyseur where we continued the discussion about our business model.

### Video

Rémy came this week to shoot the last scenes for our presentation video.

## Week from the September 28th to 4th of October

### Project

#### Clonings

##### Concordia's experiment

We performed an experiment for iGEM Concordia as part of our partnership, where we looked at the fluorescence evolution after adding a stress factor. We could observe some fluorescence in all conditions, so we conclude that there is no right change in the gal10 promoter activity under these different stress conditions.

### Collaborations and partnerships

#### AshesiGhana iGEM team

We talked together about the gold criteria « Partnership » and we agreed to do it due to the numerous meetings we had and the work we have done. We decided to wait until 50 answers for the survey for the iGEM Ghana team to start analyzing the results. We also created a document to both work on each other wiki partnership page.

#### Concordia iGEM team

We chose a final template for our contribution document to start implementing it with the work we have done.

#### German Meetup

![](https://static.igem.org/mediawiki/2020/c/cf/T--Toulouse_INSA-UPS--2020--Centri.jpg)

We received the SIGMA centrifuge we won by getting the first prize in the poster presentation during the German Meetup organized by iGEM Marburg.

## Week from October 5th to 11th

### Human Practices

![](https://static.igem.org/mediawiki/2020/f/fe/T--Toulouse_INSA-UPS--2020--HighSchool.jpg)

We visited Leon Blum High School in Villefranche-de-Lauragais where we presented synthetic biology and our video game to the students. We received lots of useful feedback.

## Week of October 12th to 18th

![](https://static.igem.org/mediawiki/2020/d/d1/T--Toulouse_INSA-UPS--2020--Catalyseur.jpg)

### Entrepreneurship

We had our last meeting with Le Catalyseur, they helped us organize our last weeks of iGEM by defining all the tasks we still had to do, and assigning a responsible person for each of them.

### Wiki

We did our first “fake wiki freeze”.

![](https://static.igem.org/mediawiki/2020/c/cc/T--Toulouse_INSA-UPS--2020--Fake.jpg)

![](https://static.igem.org/mediawiki/2020/1/14/T--Toulouse_INSA-UPS--2020--Fake2.jpg)

## Week of October 19th to 25th

### Video

We finished recording the narration parts of our presentation video.

### Wiki

We did our second “fake wiki freeze”.

![](https://static.igem.org/mediawiki/2020/3/37/T--Toulouse_INSA-UPS--2020--Fake3.jpg)

![](https://static.igem.org/mediawiki/2020/b/bd/T--Toulouse_INSA-UPS--2020--Fake4.jpg)

## Week of October 25th to 31st

![](https://static.igem.org/mediawiki/2020/5/5e/T--Toulouse_INSA-UPS--2020_THE_END_AMOUR_SUR_VOUS.jpg)

### Wiki

We spent these last days finishing our wiki, by the wiki freeze all the team learnt how to script pages using HTML.

[Go to Implementation ![](https://static.igem.org/mediawiki/2020/5/5c/T--Toulouse_INSA-UPS--2020_fleche_next.png)](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Implementation)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Toulouse INSA-UPS/Engineering

![](https://static.igem.org/mediawiki/2020/2/21/T--Toulouse_INSA-UPS--2020_newbanner.jpg)

[![](https://static.igem.org/mediawiki/2020/9/98/T--Toulouse_INSA-UPS--2020_IGEMINI_blanc.svg) Toulouse INSA-UPS](https://2020.igem.org/Team%3AToulouse_INSA-UPS)

* [Home](https://2020.igem.org/Team%3AToulouse_INSA-UPS)
* Team

  [Team members](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Team)
  [Attributions](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Collaborations)
  [Partnership](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Partnership)
* Project

  [Description](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Description)
  [Design](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Design)
  [Modeling](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Model)
  [Experiments](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Experiments)
  [Results](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Results)
  [Contribution](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Contribution)
  [Engineering Success](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Engineering)
  [Notebook](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Notebook)
* Implementation

  [Implementation](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Implementation)
  [Sustainable Development Impact](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Sustainable)
  [Supporting Entrepreneurship](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Entrepreneurship)
* [Parts](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Parts)
* Human Practices

  [Overview](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices)
  [Integrated Human Practices](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#Integrated_Human_Practices)
  [Science Communication](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Education)
  [Contribution](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Contribution)
* [Safety](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Safety)
* 🏆

  [Achievements](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Achievements)
  [Awards](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Awards)
  [Poster](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Poster)
  [Promotion video](https://video.igem.org/videos/watch/02a9ba65-6ec1-4be7-b3e7-07cc5a3ff566)
  [Presentation video](https://video.igem.org/videos/watch/0195342d-4c1d-4908-ae87-328fbcbbbc22)

### Engineering success

* [Research and design a strategy](#research-and-design-a-strategy)
* [Design of our cloning strategy](#design-of-our-cloning-strategy)
* [Results of the experiments](#results-of-the-experiments)
* [Analysis and Validation of the constructed strains](#analysis-and-validation-of-the-constructed-strains)

![PF](https://static.igem.org/mediawiki/2020/e/e4/T--Toulouse_INSA-UPS--2020_petite_fusee.png)

# Engineering success for the improvement of the mevalonate pathway

Our aim was to create a nutritious and tasty yeast for food supplements. This aim involved the production of β-carotene, limonene and geraniol. These three compounds derive from the mevalonate pathway. The model developed to demonstrate the feasibility of the project highlighted that production yield is a controlling parameter of the system, which led us to design, build and validate a chassis strain optimized for the production of β-carotene, limonene and geraniol. This is why the improvement of this pathway was required to reach sufficient yields.
In this page, we present how we succeeded in the improvement of the mevalonate pathway. These successes resulted in the construction and the validation of the following parts:

[BBa\_K3570000](http://parts.igem.org/Part%3ABBa_K3570000): contains all biobricks need to improve the mevalonate pathway.

[BBa\_K3570006](http://parts.igem.org/Part%3ABBa_K3570006): contains the upstream sequence of DPP1 used as integration locus.

[BBa\_K3570007](http://parts.igem.org/Part%3ABBa_K3570007): contains the downstream sequence of DPP1 used as integration locus.

[BBa\_K3570008](http://parts.igem.org/Part%3ABBa_K3570008): contains the HIS3 selectable marker.

## **Research and design a strategy**

Our strategy to improve the mevalonate pathway is to integrate a truncated version of tHMG1 and the gene coding for CrtE from *Xanthophyllomyces dendrorhous* in the yeast genome.
tHMG1 is the rate-limiting enzyme in the mevalonate pathway [1]. A truncated version of tHMG1 is known to enhance the conversion of 3-hydroxy-3-methylglutaryl-CoA into mevalonate [2]. On the other hand, the production of β-carotene is usually low and should be prioritized over limonene and geraniol. β-carotene synthesis is highly dependent on the level of GGPP. The enzyme CrtE from *X. dendrorhous* is a GGPP synthase more efficient than the native GGPP synthase of *S. cerevisiae*.
The strain used was named BY4741 ∆Gal4 and has four auxotrophies (Leucine, Methionine, Uracil and Histidine) which have been used to select the integrations of our project components. The ∆Gal4 property was necessary for the regulation of the production of flavors (limonene/geraniol/brazein) which we will not be talked about here.

![](https://static.igem.org/mediawiki/2020/7/74/T--Toulouse_INSA-UPS--2020_full_pathway.png?fbclid=IwAR3RPFyNguMZOO9K7_6MAVGJv8kMBIQIaUPUJhbmSc2TlckgncVKx3iDJ1o)

## **Design of our cloning strategy**

The first part of our strategy consisted in cloning the genes of interest into a pUC19 vector for amplification in *E. coli*. We chose the bidirectional TDH3-TEF1 promoter and the terminators tCYC1 and tPGK. For the yeast genome integration, the integrative locus used was DPP1 and the selective locus used is HIS3. Note that neither the DPP1 targeting sequence nor the HIS3 sequence were present on the registry. This added some extra-challenge here but we needed 4 different loci and selection markers for our project and these could be of interest for future iGEMers.

![](https://static.igem.org/mediawiki/parts/5/51/T--Toulouse_INSA-UPS--2020_HMG1-crtE.png)

Fig. 2: tHMG1-CrtE-pUC19.

The cloning strategy was to clone the blocks into two vectors before bringing them together in a unique plasmid (figure 3). The blocks B14, B15 and B16 are to be cloned in pUC19 using InFusion kit from TaKaRa Bio to form pUC19-B14B15B16. pUC19 is linearized by *SbfI* and *BamHI* to insert blocks B14, B15 and B16. The other blocks B17, B18 and B19 are to be cloned in another pUC19 using InFusion kit to form pUC19-B17B18B19. pUC19 will then be linearized by *BamHI* and *EcoRI* to insert blocks B17, B18 and B19.
pUC19-B17B18B19 is used as a template vector to insert the sequence of B14B15B16 from pUC19-B14B15B16 using SbfI and BamHI restriction enzymes and T4 DNA ligase. The resulting plasmid pUC19-B14B15B16B17B18B19 should contain B14B15B16B17B18B19 which is our  [BBa\_K3570000](http://parts.igem.org/Part%3ABBa_K3570000) part. See our clonings experiments [here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Experiments#clonings-methods) .

B14 corresponds to the  [BBa\_K3570006](http://parts.igem.org/Part%3ABBa_K3570006)  part.
B18 corresponds to the  [BBa\_K3570008](http://parts.igem.org/Part%3ABBa_K3570008) part.
B19 corresponds to the  [BBa\_K3570007](http://parts.igem.org/Part%3ABBa_K3570007)  part.

![](https://static.igem.org/mediawiki/2020/4/4f/T--Toulouse_INSA-UPS--2020_fig16.png)

Fig. 3: Cloning strategy of tHMG1 and CrtE in pUC19.

The second part of our cloning strategy consists in the extraction of pUC19-B14B15B16B17B18B19 from *E. coli*, its digestion by *Sbf*I and *EcoR*I and transformation of*Sbf*fI-*EcoR*I DNA fragment into yeast cells in order to integrate the insert containing blocks B14 to B19 ([experiments)](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Experiments#transformation) in the in yeast genome. Transformed yeasts are grown on YNB LEU+, URA+, MET+ HIS- with 2% (g/100ml) glucose. GGPP is extracted from yeast cells and analyzed by LC-MS (See our experiments [here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Experiments#ggpp-extraction-and-quantification-by-lcms) ).

We will have to grow the transformed yeast on YNB leu+, ura+, met+ his- with 2% (g/100 ml) of glucose. GGPP will have to be extracted from yeast and analyzed by LC-MS.

## **Results of the experiments**

* Construction of pUC19-B14B15B16:

The gBlocks B14, B15 and B16 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (Figure 4).

![](https://static.igem.org/mediawiki/2020/6/6e/T--Toulouse_INSA-UPS--2020_fig18.png)

Figure 4: PCR verification of the digested pUC19 and the three gBlocks B14, B15 and B16. Expected size of DNA fragments are at 2.6 kb, 0.4 kb, 1.8 kb and 1.0 kb respectively

pUC19 was digested by *SbfI - BamHI* and prepared to receive the PCR products B14, B15 and B16 using InFusion kit. After transformation of Stellar cells, selection of clones on ampicillin plates, and plasmid extraction from 8 clones, we performed a digestion restriction profile analysis using *Sac*I (Figure 5).

![](https://static.igem.org/mediawiki/2020/8/8e/T--Toulouse_INSA-UPS--2020_fig18Acloning.png)

Figure 5: Verification using the InFusion kit: the expected sizes were 4.8 kb and 1.2 kb

We had six clones that had the expected profile. Since the sequence was also validated, we had successfully obtained the first plasmid of our tHmg1-CrtE construction.

* Assembly of the pUC19-B17B18B19:

The gBlocks B17, B18 and B19 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (Figure 6).

![](https://static.igem.org/mediawiki/2020/7/77/T--Toulouse_INSA-UPS--2020_fig19.jpeg)

Figure 6: PCR verification of the digested pUC19 and gBlocks B17, B18, B19

We digested the pUC19 vector by *BamHI* and *EcoRI* and purified the digested vector on gel. We proceeded to the InFusion kit reaction, transformation of Stellar cells, selection on ampicillin, and minipreps from 6 clones. The plasmids were assessed by restriction profiling with the enzymes *BamHI* and *EcoRI*.

![](https://static.igem.org/mediawiki/2020/9/98/T--Toulouse_INSA-UPS--2020_fig20.png)

Figure 7: Verification using the InFusion Kit: the expected sizes were 4.8 kb and 2.6 kb

Only one clone had the expected profile (figure 7). We sent it to sequencing and it was fortunately valid. We had successfully obtained the second plasmid of our tHMG1-CrtE insert.

* Assembly of tHmg1-CrtE insert:

The next step was to combine both plasmids by subcloning the fragment B14B15B16 into plasmid pUC19-B17B18B19.
To do this, we first prepared plasmids with the QIAGEN Plasmid Plus Midi Kit. Then, we digested both plasmids with SbfI and BamHI and purified the resulting DNA materials with the Monarch Genomic DNA Purification Kit by NEB. Fragments were ligated together with T4 DNA ligase by NEB followed by a transformation into Stellar cells (ampicillin selection). Over eight assessed colonies, two presented the expected restriction profile (number 1 and 3) when digested with *Sbf*I and *EcoR*I, albeit the digestion was not complete (Figure 8).

![](https://static.igem.org/mediawiki/2020/6/67/T--Toulouse_INSA-UPS--2020_fig21.png)

Figure 8: Ligation verification: the expected sizes are 6.6 kb and 2.6 kb.

* Yeast transformation

Since the construction was successful, we proceeded to the yeast transformation. The plasmid was digested with enzymes *Sbf*I and *EcoR*I and purified to transform the strain BY4741 *Saccharomyces cerevisiae*. The resulting cells were grown on YNB LEU+, URA+, MET+ HIS- with 2% glucose for 3 days. At the third try, we were able to observe around 20 colonies in our yeast transformation, about the same amount of colonies on the positive control and none on the negative control plate which consisted of wild BY4741 on YNB LEU+, URA+, MET+ HIS-.

Verification of integration of BBa\_K3570000 using the DPP1 homology sequence (BBa\_K3570006 and BBa\_K3570007) was performed by a genomic PCR using the TaKaRa PCR amplification Kit and the following primers: primer 1 (forward) hybridizes on our selectable marker HIS3 while primer 2 (reverse) hybridizes upstream of the DPP1 gene.

Primer 1: ATCAGGATTTGCGCCTTT

Primer 2: GCCGCCGAGGGTATTTTACTTCCG

We randomly chose eight clones from our transformation and one from the positive control plate (Figure 9). All clones have the expected size (1.2 kb), and the PCR negative control, where we inserted pRS313 does not show any band , which means that the [BBa\_K3570000](http://parts.igem.org/Part%3ABBa_K3570000) part is well integrated in the yeast genome. The integration in the yeast genome is a success that means that the parts  [BBa\_K3570008](http://parts.igem.org/Part%3ABBa_K3570008) (HIS3 selective marker),  [BBa\_K3570006](http://parts.igem.org/Part%3ABBa_K3570006)  and  [BBa\_K3570007](http://parts.igem.org/Part%3ABBa_K3570007) (for DPP1 homologous sequence) all work. Our modified strain is BY4741 DPP1::tHMG1-crtE.

![](https://static.igem.org/mediawiki/2020/0/03/T--Toulouse_INSA-UPS--2020_fig22.jpeg)

Figure 9: Verification of the integration of  [BBa\_K3570000](http://parts.igem.org/Part%3ABBa_K3570000) part into the yeast genome by PCR.  [BBa\_K3570000](http://parts.igem.org/Part%3ABBa_K3570000) size is 1.2 kb.

## **Analysis and Validation of the constructed strains**

The integration of the [BBa\_K3570000](http://parts.igem.org/Part%3ABBa_K3570000) part in the yeast genome should enhance the flow of the mevalonate pathway. This modification should result in an increase of the concentration of GGPP, which is the precursor of beta-carotene. GGPP have been extracted from BY4741 DPP1::tHMG1-crtE and analyzed by LC-MS (see our experiments [here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Experiments#ggpp-extraction-and-quantification-by-lcms) ).
Our strain BY4741 DPP1::tHMG1-crtE showed a five-fold increase in GGPP concentration in comparison with the wild-type yeast (figure 10). This important result demonstrates that the parts BBa\_K3570000, BBa\_K3570006, BBa\_K3570007 and BBa\_K3570008 work.

![](https://static.igem.org/mediawiki/parts/3/37/T--Toulouse_INSA-UPS--2020_GGPP.png)

Figure 10: Results of the GGPP analysis from culture on YNB with 2% Glucose by LC-MS

Data from the literature [2] suggest a stable turn-over of mevalonate pathway intermediates in native and engineered *S. cerevisiae* strains, i.e. a stable ratio between the GGPP production flux and its concentration. This would indicate that our strain optimization strategy successfully enhances by five-fold the capability of GGPP production, and thus the potential of β-carotene production. Moreover, the increased flux through the mevalonate pathway is expected to increase not only β-carotene production, but also the production of limonene and geraniol (a provitamin A, a flavor of lemon and of rose) synthesized from other mevalonate intermediates.

## **References**

[1]

Misawa, N., 2011. Pathway engineering for functional isoprenoids. Curr. Opin. Biotechnol. 22, 627–633. http://dx.doi.org/10.1016/j.copbio.2011.01.002

[2]

Hery Rabeharindranto, Sara Castaño-Cerezo, Thomas Lautier, Luis F. Garcia-Alles, Christian Treitz, Andreas Tholey, Gilles Truan, 2019. Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae. Metabolic Engineering Communications 8 (2019) e00086. https://doi.org/10.1016/j.mec.2019.e00086

[3]

Polakowski, T., Stahl, U., & Lang, C. (1998). Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast. Applied Microbiology and Biotechnology, 49(1), 66–71. https://doi.org/10.1007/s002530051138

[4]

Verwaal, R., Wang, J., Meijnen, J.-P., Visser, H., Sandmann, G., van den Berg, J. A., & van Ooyen, A. J. J. (2007). High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous. Applied and Environmental Microbiology, 73(13), 4342–4350. https://doi.org/10.1128/aem.02759-06

[5]

Peng, B., Williams, T. C., Henry, M., Nielsen, L. K., & Vickers, C. E. (2015). Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities. Microbial Cell Factories, 14(1). https://doi.org/10.1186/s12934-015-0278-5

[6]

[SGD:S000003424](https://www.yeastgenome.org/locus/S000003424)

[7]

[SGD:S000006284](https://www.yeastgenome.org/locus/S000006284)

[8]

Curran, K. A., Karim, A. S., Gupta, A., & Alper, H. S. (2013). Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metabolic Engineering, 19, 88–97. https://doi.org/10.1016/j.ymben.2013.07.001

[9]

*S. cerevisiae* genome, chromosome XVI, Ty4 LTR region. GenBank: CP046096.1

[10]

[RS313 plasmid](https://www.addgene.org/vector-database/3972/)

[Go to Notebook ![](https://static.igem.org/mediawiki/2020/5/5c/T--Toulouse_INSA-UPS--2020_fleche_next.png)](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Notebook)

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# Team:Toulouse INSA-UPS/Experiments

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* 🏆

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### Experiments

* [Materials](#materials)

+ [Plasmids and other DNA fragments](#plasmids-and-other-dna-fragments)
+ [Biological materials and tools](#biological-materials-and-tools)

* [Yeast modification protocols](#yeast-modifications-protocols)

+ [Cloning's methods](#clonings-methods)
+ [Transformation](#transformation)
+ [GGPP extraction and quantification by LC-MS](#ggpp-extraction-and-quantification-by-lcms)
+ [Geraniol HPLC analysis](#geraniol-hplc-analysis)

* [Fermentation protocols](#fermentation-protocols)

+ [Medium for the coculture](#medium-for-the-coculture)
+ [Growth of *C. ljungdahlii* on CO2 and H2](#growth-experiments-of-clostridium-ljungdahlii)
+ [Growth of *S. cerevisiae* on acetate and ethanol](#growth-experiments-of-saccharomyces-cerevisiae)
+ [Acetate and ethanol analysis](#acetate-and-ethanol-analysis)

![PF](https://static.igem.org/mediawiki/2020/e/e4/T--Toulouse_INSA-UPS--2020_petite_fusee.png)

# Materials

## Plasmids and other DNA fragments

### Plasmids

**pUC19**
This vector was used as a receiver for most of our intermediate clonings. It contains an ampicillin resistance gene, MCS and LacZ gene. This is a high copy number vector. This was a kind gift from Dr. Gilles Truan from TBI.

**pFL36**
This plasmid was used to retrieve the LEU2 auxotrophic marker. It contains the ampicillin resistance gene.This was a kind gift from Dr. Anthony Henras from IBCG.

**pMRI34**
This plasmid was used to retrieve the DPP up and down homologous sequence, as well as the crtE and tHMG1 genes. It also contains the ADH1 terminator and CYC1 terminator. This was a kind gift from Dr. Gilles Truan from TBI.

**pRS313**
This plasmid was used as a positive control during the yeast transformation because it has a HIS3 auxotrophic marker. It also contains ampicillin resistance gene.This was a kind gift from Dr. Anthony Henras from IBCG.

**pENZ-047**
This plasmid was used as a source for TDH3 and TEF1 promoters. It contains an ampicillin resistance gene. This was a kind gift from Dr. Gilles Truan from TBI.

**pHR0016**
This plasmid was used as a source of HO up and down homologous sequence, CrtYBekI gene (fusion protein of CrtYB + ek linker + CrtI) and the CYC1 terminator. It contains kanamycin resistance gene. This was a kind gift from Dr. Gilles Truan from TBI.

**pFL38**
This plasmid was used as a source of URA3 auxotrophic marker. It contains ampicillin resistance gene. This was a kind gift from Dr. Anthony Henras from IBCG.

**pL38**
This plasmid was used as a source to retrieve the GFP protein, C120 “forward” and “reverse” promoter. It contains an ampicillin resistance gene.This was a kind gift from Sylvain Pouzet PhD student at University Paris Diderot.

**pL105**
This plasmid was used as a source of NLS-VP16-EL222. It contains ampicillin resistance gene.This was a kind gift from Sylvain Pouzet PhD student at University Paris Diderot.

### IDT-ordered fragments

**Brazzein protein**
ATGGACAAATGCAAAAAAGTATACGAAAATTATCCAGTTTCTAAATGTCAACTAGCAAACCAGTGTAATTATGACTGTAAGTTGGATAAGCACGCTAGGTCAGGTGAATGCTTTTACGATGAGAAGAGAAACTTACAATGTATTTGCGATTATTGTGAATAC

**Geraniol synthase protein**
ATGTCTTCATCATCATCATCATCTTCATCCATGTCTCTGCCTTTGGCAACTCCATTGATCAAAGACAATGAATCTCTCATCAAGTTCTTGCGCCAACCCCTGGTGCTTCCTCATGAGGTTGATGACAGCACAAAAAGGAGGGAATTGTTGGAAAGAACAAGAAAAGAACTAGAATTAAATGCAGAAAAACCATTGGAGGCCTTGAAGATGATAGATATAATTCAAAGATTGGGATTATCATATCATTTTGAAGATGATATTAATTCAATTCTCACAGGATTTTCAAATATTAGCAGCCAAACTCATGAAGGTTTGTTGACTGCTTCACTTTGTTTTCGATTGCTTCGACACAATGGGCATAAGATCAATCCTGATATATTCCAAAAATTCATGGACAACAATGGAAAGTTTAAAGATTCATTAAAGGATGACACATTAGGCATGTTAAGCTTATATGAAGCTTCATATTTGGGAGCCAATGGAGAAGAAATATTGATGGAAGCCCAAGAGTTCACCAAAACTCACCTGAAAAACTCATTGCCAGCCATGGCACCATCTCTTTCTAAGAAGGTTTCTCAAGCTTTAGAGCAACCAAGACATAGAAGAATGTTGAGGTTAGAAGCTAGAAGATTTATTGAAGAATATGGTGCTGAAAATGACCATAATCCAGACCTTCTTGAGCTTGCAAAATTGGATTATAACAAAGTCCAATCTCTACACCAAATGGAATTGTCTGAGATAACAAGGTGGTGGAAACAATTAGGGCTTGTGGATAAACTCACCTTTGTTGGAAATCGACCCCTTGAATGCTTTCTTTGGACAGTGGGATTATTACCAGAGCCTAAGTATTCAGGTTGCAGAATTGAGCTTGCAAAAACCATAGCCATTTTGCTTGTCATTGATGATATCTTTGATACTCATGGTACCCTAGATGAGCTTCTTCTATTCACTAATGCCATTAAAAGATGGGATCTTGAGGCCATGGAAGATTTACCAGAATATATGAGAATTTGTTACATGGCATTGTACAATACTACTAATGAAATTTGCTAT

**YPRCdelta15 up homologous sequence**
GCCAGGCGCCTTTATATCATATAATTAAGACACAAAAGGATAAAACAAAGGTGTTAACTATTCTGCATACTCACTATCGTAAACTGTCCTGCAAATCGTGTAAATATGTATTTCATTTTTTTTGCAGTGAAAAAAGGCATGTAAAATACCGCATCAAGTAACTCTACTCCGCCTGTGGTTTCAAGACTAACGGCTTGAGACAAAATGGGAAGAAATGATTGCAGAAAAGCCATATGTGTAATAGCAAAAAGCTGGATAGTGCTTACCAGATGTTTACCTTAATTTCTTGGTGAATTAGAGAAGTACAGAAGTTTTACTATTAATCCCACCATAGAAATTTGTATAGGAAAGTAGTTTATTGGAGTTATTGGATATACTGTGTAAACTATTTCTTGAAATTGTAATCTTAAGATGCTTTGTTAATTCTATTAAAAATAGAAAATGATTTTCATATgTATTTcTTTATTTATATcTTGGCATTACTCTTCATCATTTTTTTCCCTCTAAGAAGCTTCCTTTCTTTTTATAAGGATAACAAAACCAAAAGGAATATTGGGTCAGATGAATGGACGCGAATGCAAGACAGAAGTCCAAATCACGTCAAGACAAAGAAAGAAAGAAAGAAAAACTAACACATTAATGTAGTTTTAAAATTTCAAATCCGAACAACAGAGCATAGGGTTTCGCAAA

**YPRCdelta15 down homologous sequence**
AATGGAAGGTCGGGATGAGCATATACAAGCACTAAGAAGAACAATACAGAACTCTACACGGTATTATTGTGCTACAAGTTGGAATAAAACCGAGTGTTTTGACGATACTAACGTTGTTAAGAAAGTAACTTGTTATCAAACTCATTACCAACTTGTGATTAATTGGTGAATAATATGATAATTGTCGAAATTCCATTGTTGGTAAAGCCTATAATATTATGTATACAGATTATACTAGAAATTCTTTGGAAAATATAAGAATCCCCAAAATTGAATCGGTATTTCTACATACTAATATTACCATTACTTCTCCTTTCGTTTTATATGTTTCATTCCTATTACATTATCGATCTTTGCATTTCAGCTTCCATTATATTTGATGTCTGTTTTATGTCCCCACGTTACACCGCATGTGACAGTATACTTCTAACATGAGTGCTACCGAATAGATGACATTTTAGACTTTCATTCCAACAACTTGGTTGACAGAATGTTACGTACCCTATATCTAATCTATATGAGGCCTGAATCTAACTGAAAGGTGGAATTTCAGTAATTTATCAAGCTTTAATAAGTTTGGGTAGTTTAACTGTGCAAAAAGGTATTTACCTTACATACTGAATCTTGTCTGTTTGGTAGCGGCTGCTTTAT

**GAL1/10 bidirectional promoter**
GTTTTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTTTATTACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAGTTTTTGCATTTATATATCTGTTAATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATCATCCTATGGTTGTTAATTTGATTCGTTCATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGATATGTATATGGTGGTAATGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTC

**PGK terminator**
ATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTATTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTTCGGGTTCAGCGTATTTTAAGTTTA

**GBD-PHYA fusion protein**
ATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAGTGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTACTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAAGGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGATTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGATAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCTAACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAGGTCAAAGACAGTTGACTGTATCGCCGGAATTTGTAATACGACTCACTATAGGGCGAGCCGCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGATGTCAGGCTCTAGGCCGACTCAATCCTCTGAGGGCTCAAGGCGATCACGCCACAGCGCTAGAATTATTGCGCAGACCACTGTAGATGCGAAACTCCATGCTGATTTCGAAGAATCAGGCTCTTCCTTTGACTACTCAACCTCTGTACGTGTCACTGGCCCAGTTGTCGAAAATCAACCACCAAGATCTGACAAAGTTACCACGACTTATTTACATCATATACAGAAAGGAAAGTTAATTCAACCCTTCGGTTGTTTACTTGCCTTGGATGAAAAAACCTTCAAAGTTATTGCATACTCTGAGAATGCATCTGAACTGTTGACAATGGCCAGTCATGCAGTTCCTAGTGTTGGCGAACACCCTGTTCTAGGCATTGGCACAGATATAAGGTCACTTTTCACTGCTCCTAGTGCGTCTGCATTGCAAAAAGCCCTTGGATTTGGAGATGTCTCTCTTTTGAATCCCATTCTTGTCCATTGCAGGACTTCTGCAAAACCCTTTTATGCGATTATCCACAGAGTTACAGGTAGCATCATCATCGACTTTGAACCCGTCAAGCCTTATGAAGTCCCCATGACAGCCGCTGGTGCCTTACAATCATACAAGCTCGCTGCCAAAGCAATCACTAGGCTGCAATCTTTACCCAGCGGGAGTATGGAACGTCTTTGTGATACAATGGTTCAAGAGGTTTTTGAATTAACGGGGTATGACCGCGTTATGGCTTATAAGTTTCATGAAGATGACCACGGTGAGGTTGTCTCCGAGGTTACAAAACCTGGGCTGGAGCCTTATTTAGGTTTGCATTATCCTGCCACCGACATCCCTCAAGCAGCCAGATTTCTGTTTATGAAAAACAAGGTCCGGATGATAGTTGATTGCAATGCAAAACATGCTAGGGTACTTCAAGATGAAAAGCTTTCCTTTGACCTTACATTGTGTGGCTCCACCTTGAGAGCACCACACAGCTGCCATTTGCAGTACATGGCCAACATGGATTCAATTGCATCTCTGGTTATGGCCGTTGTAGTTAACGAGGAAGATGGTGAAGGTGATGCTCCTGATGCTACTACACAGCCTCAAAAGAGAAAGAGACTATGGGGTTTAGTAGTTTGTCACAATACGACTCCGAGATTTGTTCCATTTCCTCTCAGGTATGCCTGTGAGTTTCTAGCTCAAGTCTTTGCCATACACGTCAATAAGGAGGTTGAACTCGATAACCAGATGGTGGAGAAGAACATTTTGCGCACGCAAACACTCTTGTGCGATATGTTGATGCGTGATGCTCCATTAGGTATTGTGTCGCAAAGCCCCAATATAATGGACCTTGTGAAATGTGATGGAGCAGCTCTATTGTATAAAGACAAAATTTGGAAACTGGGAACAACTCCAAGTGAGTTCCACTTGCAGGAGATTGCTTCATGGTTGTGTGAATACCACATGGATTCAACGGGTTTGAGCACTGATAGTTTGCATGACGCCGGTTTTCCAAGAGCTCTATCTTTAGGGGATTCGGTATGTGGTATGGCAGCCGTGAGAATATCATCGAAAGACATGATTTTCTGGTTCCGTTCTCATACCGCTGGTGAAGTGAGATGGGGAGGTGCGAAACATGATCCAGACGATAGGGACGACGCAAGGAGAATGCACCCAAGGTCATCGTTCAAGGCTTTCCTTGAAGTCGTCAAGACAAGGAGTTTACCTTGGAAGGACTATGAGATGGATGCCATACACTCCTTGCAACTTATTTTGAGAAATGCTTTCAAGGATAGTGAAACTACTGATGTAAATACAAAGGTCATTTACTCGAAGCTAAATGATTTGAAAATTGATGGTATACAAGAACTAGAAGCTGTGACCAGTGAAATGGTTCGTTTAATTGAGACTGCTACGGTGCCAATATTGGCGGTTGATTCTGATGGACTGGTTAATGGTTGGAACACGAAAATTGCTGAGCTGACTGGTTTATCGGTTGATGAAGCAATCGGTAAACATTTCCTCACATTGGTTGAAGATTCTTCAGTGGAAATCGTTAAAAGAATGCTAGAAAACGCATTAGAAGGTACTGAAGAGCAAAATGTCCAGTTTGAAATAAAGACACATCTGTCCAGAGCTGATGCTGGTCCAATATCCTTAGTTGTAAATGCATGCGCAAGTAGAGATCTCCATGAAAACGTAGTTGGGGTGTGCTTTGTAGCCCATGATCTTACTGGCCAAAAAACTGTGATGGACAAATTTACGCGCATTGAAGGTGATTACAAGGCAATCATCCAAAATCCAAACCCGCTGATCCCGCCAATATTTGGTACCGATGAATTTGGATGGTGCACAGAATGGAATCCAGCAATGTCAAAATTAACCGGTTTGAAGCGAGAGGAAGTGATTGACAAAATGTTATTAGGTGAAGTATTTGGGACGCAGAAGTCATGTTGTCGTCTAAAGAATCAAGAAGCCTTTGTAAACTTAGGCATTGTGTTAAACAATGCTGTCACCTCCCAAGATCCAGAAAAAGTATCGTTTGCTTTCTTCACAAGAGGTGGCAAGTATGTGGAGTGTTTGTTGTGTGTTAGTAAAAAATTAGACAGGGAAGGTGTAGTTACAGGTGTCTTCTGTTTCCTACAATTAGCCTCTCATGAGCTTCAGCAAGCGTTGCATGTTCAACGTTTAGCCGAACGAACCGCAGTCAAGAGACTAAAGGCTCTAGCATACATAAAAAGACAGATCAGAAATCCGCTATCTGGGATTATGTTTACAAGAAAAATGATAGAAGGTACTGAATTAGGACCAGAGCAAAGAAGAATTTTGCAAACTTCCGCGTTATGTCAGAAGCAACTAAGCAAAATCTTAGATGACTCGGATCTTGAAAGCATCATTGAAGGATGCTTGGATTTGGAAATGAAAGAATTCACCTTAAATGAAGTGTTGACTGCTTCCACATCTCAAGTAATGATGAAGAGTAACGGAAAATCCGTTCGGATAACAAATGAGACCGGAGAAGAAGTAATGTCTGACACTTTGTATGGAGACAGTATTAGATTACAACAAGTCTTGGCAGACTTCATGCTGATGGCTGTAAACTTTACACCATCCGGTGGTCAGCTAACTGTTTCAGCTTCCTTGAGAAAAGACCAGTTGGGTCGTTCTGTGCATTTAGCTAATCTAGAGATTAGATTAACGCATACCGGCGCTGGCATACCTGAGTTTTTACTAAACCAAATGTTTGGAACTGAAGAAGATGTATCAGAAGAAGGTTTGTCTTTAATGGTTAGCCGAAAACTGGTGAAACTGATGAATGGAGATGTTCAGTACTTGAGACAAGCTGGGAAATCAAGTTTCATTATCACTGCGGAATTAGCTGCAGCAAACAAGTAA

**GAD-FHY1 fusion protein**
GCGGAATTAATTCCCGAGCCTCCAAAAAAGAAGAGAAAGGTCGAATTGGGTACCGCCGCCAATTTTAATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCACTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCAAACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACTTCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAAAACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAATCACTACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCGAGCGCCGCCATGGAGTACCCATACGACGTACCAGATTACGCTATGCCTGAAGTAGAAGTGGATAACAACAACGAGAAGCCAAGTGAGATTAATTCCTTCCATCACATGATCATAAGTAGTAGTAAAAATGTGTTGAAAATGGAAGAAGTTGAAGTTAGCAAGAAGAGGAAATTTCAAACGGATCAATCTGATGAGTTATCGTTACTTCCACTGTCAAAACACACTTGTTTTGCCAATGTTGCGTGTTCGGAGAATACAAATGGTAATTCGGAGATAGATACAGAATACTCAATGTCTTCTTATGTCAATTCAACCACTTCTATGGAGTGCAACAATGATATAGAAATGAAAGAAGAATCCTCTGGTTCATGCGGTGAAGACAAAATGATCTCTTTCGAAAGCCATTTGGATTACATCTATGGCACTCAGAACTTAGAGGACTTTTCAGAGAAAGTCATTGAAAACATTTTGTATCTCGACGAACAAGAAGAAGAAGAAGAAGACGCTAAAGGGTGTAGTTCCAATGCAGCTAAGTTTGTACTTTCCTCTGGAAGATGGACCGTTAACCAAGACGATAGCACTTTGCATGAGACAAAGAAGCCCACCATTGATCAGGAATTTGAACAATACTTCTCAACGCTAATGTTGTAA

**ADH1 promoter+truncated ADH1 promoter**
GGAGTTGATTGTATGCTTGGTATAGCTTGAAATATTGTGCAGAAAAAGAAACAAGGAAGAAAGGGAACGAGAACAATGACGAGGAAACAAAAGATTAATAATTGCAGGTCTATTTATACTTGATAGCAAGACAGCAAACTTTTTTTTATTTCAAATTCAAGTAACTGGAAGGAAGGCCGTATACCGTTGCTCATTAGAGAGTAGTGTGCGTGAATGAAGGAAGGAAAAAGTTTCGTGTGCTTCGAGATACCCCTCATCAGCTCTGGAACAACGACATCTGTTGGTGCTGTCTTTGTCGTTAATTTTTTCCTTTAGTGTCTTCCATCATTTTTTTGTCATTGCGGATATGGTGAGACAACAACGGGGGAGAGAGAAAAGAAAAAAAAAGAAAAGAAGTTGCATGCGCCTATTATTACTTCAATAGATGGCAAATGGAAAAAGGGTAGTGAAACTTCGATATGATGATGGCTATCAAGTCTAGGGCTACAGTATTAGTTCGTTATGTACCACCATCAATGAGGCAGTGTAATTGGTGTAGTCTTGTTTAGCCCATTATGTCTTGTCTGGTATCTGTTCTATTGTATATCTCCCCTCCGCCACCTACATGTTAGGGAGACCAACGAAGGTATTATAGGAATCCCGATGTATGGGTTTGGTTGCCAGAAAAGAGGAAGTCCATATTGTACACCCGGAAACAACAAAAGGTTCCTCCAGGTCGAGATCCGGGATCGAAGAAATGATGGTAAATGAAATAGGAAATCAAGGAGCATGAAGGCAAAAGACAAATATAAGGGTCGAACGAAAAATAAAGTGAAAAGTGTTGATATGATGTATTTGGCTTTGCGGCGCCGAAAAAACGAGTTTACGCAATTGCACAATCATGCTGACTCTGTGGCGGACCCGCGCTCTTGCCGGCCCGGCGATAACGCTGGGCGTGAGGCTGTGCCCGGCGGAGTTTTTTGCGCCTGCATTTTCCAAGGTTTACCCTGCGCTAAGGGGCGAGATTGGAGAAGCAATAAGAATGCCGGTTGGGGTTGCGATGATGACGACCACGACAACTGGTGTCATTATTTAAGTTGCCGAAAGAACCTGAGTGCATTTGCAACATGAGTATACTAGAAGAATGAGCCAAGACTTGCGAGACGCGAGTTTGCCGGTGGTGCGAACAATAGAGCGACCATGACCTTGAAGGTGAGACGCGCATAACCGCTAGAGTACTTTGAAGAGGAAACAGCAATAGGGTTGCTACCAGTATAAATAGACAGGTACATACAACACTGGAAATGGTTGTCTGTTTGAGTACGCTTTCAATTCATTTGGGTGTGCACTTTATTATGTTACAATATGGAAGGGAACTTTACACTTCTCCTATGCACATATATTAATTAAAGTCCAATGCTAGTAGAGAAGGGGGGTAACACCCCTCCGCGCTCTTTTCCGATTTTTTTCTAAACCGTGGAATATTTCGGATATCCTTTTGTTGTTTCCGGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTCCTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAATAGAACAGATACCAGACAAGACATAATGGGCTAAACAAGACTACACCAATTACACTGCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATAATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGTCTCACCATATCCGCAATGACAAAAAAATGATGGAAGACACTAAAGGAAAAAATTAACGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCGAAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAACGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGTCTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATTGTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACCAAGCATACAATCAACTCCA

**ADH1 terminator**
AGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGTCTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAATTTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGCC

**Limonene synthase**
 ATGCGTCGTTCAGCAAACTACCAACCTTCAATTTGGGACCACGATTTTTTGCAGTCATTGAATAGCAACTATACGGATGAAGCATACAAAAGACGAGCAGAAGAACTGAGGGGAAAAGTGAAGATAGCGATTAAGGATGTAATCGAGCCTCTGGATCAGTTGGACCTGATTGATAACTTGCAAAGACTTGGATTGGCTCATCGTTTTGAGACTGAGATTAGGAACATATTGAATAATATCTACAACAATAATAAAGATTATAATTGGAGAAAAGAAAATCTGTATGCAACCTCCCTTGAGTTTAGACTACTTAGACAACATGGCTATCCTGTTTCTCAAGAGGTTTTCAATGGTTTTAAAGACGACCAGGGAGGCTTCATTTGTGATGATTTCAAGGGAATACTGAGCTTGCATGAAGCTTCGTATTACAGCTTAGAAGGAGAAAGCATCATGGAGGAGGCCTGGCAATTTACCAGCAAACATCTTAAAGAAGTGATGATCAGCAAGAACATGGAAGAGGATGTATTTGTAGCAGAACAAGCGAAGCGTGCACTGGAACTGCCTCTGCATTGGAAAGTGCCAATGTTAGAGGCAAGGTGGTTCATACACATTTATGAGAGAAGAGAGGACAAGAACCACCTTTTACTTGAACTGGCTAAGATGGAGTTTAACACTTTGCAGGCAATTTACCAGGAAGAACTAAAAGAAATTTCAGGGTGGTGGAAGGATACAGGTCTTGGAGAGAAATTGAGCTTTGCGAGGAACAGGTTGGTAGCGTCCTTCTTATGGAGCATGGGGATCGCGTTTGAGCCTCAATTCGCCTATTGTCGGAGAGTGCTCACAATCTCGATAGCCCTAATTACAGTGATTGATGACATTTATGATGTCTATGGAACATTGGATGAACTTGAGATATTCACTGATGCTGTTGAGAGGTGGGACATCAATTATGCTTTGAAGCACCTTCCGGGCTATATGAAAATGTGTTTTCTTGCGCTTTACAACTTTGTTAATGAATTTGCTTATTACGTTCTCAAACAACAGGATTTTGATTTGCTTCTGAGCATAAAAAATGCATGGCTTGGCTTAATACAAGCCTACTTGGTGGAGGCGAAATGGTACCATAGCAAGTACACACCGAAACTGGAAGAATACTTGGAAAATGGATTGGTATCAATAACGGGCCCTTTAATTATAACGATTTCATATCTTTCTGGTACAAATCCAATCATTAAGAAGGAACTGGAATTTCTGGAAAGTAATCCAGATATAGTTCACTGGTCATCCAAGATTTTCCGTCTGCAAGATGATTTGGGAACTTCATCGGACGAGATACAGAGAGGGGATGTTCCGAAATCAATCCAGTGTTACATGCATGAAACTGGTGCCTCAGAGGAAGTTGCTCGTCAACACATCAAGGATATGATGAGACAGATGTGGAAGAAGGTGAATGCATACACAGCCGATAAAGACTCTCCCTTGACTGGAACAACTACTGAGTTCCTCTTGAATCTTGTGAGAATGTCCCATTTTATGTATCTACATGGAGATGGGCATGGTGTTCAAAACCAAGAGACTATCGATGTCGGTTTTACATTGCTTTTTCAGCCCATTCCCTTGGAGGACAAACACATGGCTTTCACAGCATCTCCTGGCACCAAAGGCACCGGCGCTTGGTCACACCCGCAGTTCGAAAAATAA

**GAL1/10 bidirectional promoter**
GTTTTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTTTATTACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAGTTTTTGCATTTATATATCTGTTAATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATCATCCTATGGTTGTTAATTTGATTCGTTCATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACGAGGACGCACGGAGGAGAGTCTTCCTTCGGAGGGCTGTCACCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGATATGTATATGGTGGTAATGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTC

**PGK terminator**
ATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTTCGGGTTCAGCGTATTTTAAGTTTA

**YPRCtau3 up homologous sequence**
AAAGGAGGTGCACGCATTATGGAGAaCACTACGATACGATAGCTGCGTTGTTGTTGAAGGGGTTTCTTAAGGTTGTTTTCGTTGAAGGTAAATATTGGTCGTTTTTGTGCAGCATATTGTCCTCAAGATGCAAACTCTGCAGGTCCATTTGCAGTAAAGTGAGTTGCCTCTCGAAGAATCATTAATTTCGTATAACCGTCACTATTAAAGTCAGAAAATAAATTCTGTCGTAGACAATGTTACCATAATGTTCTTGTCCATTTTGCATACACTTTAAATATTCATTTGATTTCTCAGGGTTCATGATCATAATAAATTGCGCATTCGCAAGGCGGTAGTATTATAATGGGGTCCATCATTCTGTAGCAAGAAGTTACAGTACGCTGTTCAAGCGTTAAACAAGATAAGTAATCTCGAATGAAACATTCATATTTCGCATGAGCCAACATACAGTTGCTGAGTAATCTTCATTGCGCTTATTTATCGGCATTGAGATTGTAAAGGAAGTAAAACGCATTTTTGCAGATCTGTTCTCTTATGTATTTTTAATCGTCCTTGTATGGAAGTATCAAAGGGGACGTTCTTCACCTCCTTGGAA

**YPRCtau3 down homologous sequence**
GATGGGACGTCAGCACTGTACTTGTTTTTGCGACTAGATTGTAAATCATTCTTTATTTAATCTCTTTCTTTAACTACTGCTTAAAGTATAATTTGGTCCGTAGTTTAATAACTATACTAAGCGTAACAATGCATACTGACATTATAAGCCTGAACATTACGAGTTTAAGTTGTATGTAGGCGTTCTGTAAGAGGTTACTGCGTAAATTATCAACGAATGCATTGGTGTATTTGCGAAAGCTACTTCTTTTAACAAGTATTTACATAAGAATAATGGTGATCTGCTCAACTGATTTGGTGATAACTCTAACTTTTTTAGCAACAATTTAAAAGATAATTCGAACATATATAACAGTAGGAAGAATTTGTGTACGTCAAATTAAGATAATTTAGCATTACCAAAGTTATTAACCTAAACATAAAATATATATGAGACACATGTGGAAATCGTATGAAACAACTGTTATGAAACTGACAAGAATGAATATATAGAGTAAGCTCCGCTTGTAAAGAGGAATCACTTAAGTGTATAAATGTCTCGACGATTACTTTAGATCCAAGATTGATGATTGATATTACTCTGTAATACTTAAGCTCTTTTAATAGCTCACTGTTGTATTACGGGCTCGAGTAATACCG

### Yeast CENPK1137 genome

This yeast strain was used to obtain the MET17 auxotrophic marker and TDH1 promoter.

## Biological materials and tools

### Microbial strains

#### *Saccharomyces cerevisiae*

**BY4741**: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0

**BY4741 ∆GAL4**: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 GAL4∆0

**CENPK3711**: MATa/α ura3-52/ura3-52 trp1-289/trp1-289 leu2-3,112/leu2-3,112 his3 Δ1/his3 Δ1 MAL2-8C/MAL2-8C SUC2/SUC2

#### *E.coli*

* ***E.coli Stellar***: F–, endA1, supE44, thi-1, recA1, relA1, gyrA96, phoA, Φ80d lacZΔ M15, Δ(lacZYA-argF) U169, Δ(mrr-hsdRMS-mcrBC), ΔmcrA, λ–

#### *Clostridium ljungdahlii*

* [ATCC 55383 (PETC)](https://bacdive.dsmz.de/strain/2802)

### Media

**YPD*** Dissolve 10 g of BactoYeast extract in 500 mL water.
* Dissolve 20 g of BactoPeptone in the above solution.
* Dissolve 20 g Dextrose in the above solution.
* q.s. to 1000 mL with water.* Autoclave.

**YNB - Y 1250 (Sigma, Germany)*** Dissolve 6.8 g in 1000 mL of water.* Autoclave.

**LB*** Dissolve 5 g of yeast extract in 500 mL water.
* Dissolve 10 g peptone from casein in the above solution.
* Dissolve 10 g sodium chloride in the above solution.
* q.s. to 1000 mL with water.
* Autoclave.

**LB-agar*** Dissolve 5 g of yeast extract in 500 mL water.
* Dissolve 10 g peptone from casein in the above solution.
* Dissolve 10 g sodium chloride in the above solution.
* Dissolve 12 g agar-agar in the above solution.
* q.s. to 1000 mL with water.
* Autoclave.

**LB*** Dissolve 5 g of yeast extract in 500 mL water.
* Dissolve 10 g peptone from casein in the above solution.
* Dissolve 10 g sodium chloride in the above solution.
* q.s. to 1000 mL with water.
* Autoclave.

**Electrophoresis gel 1%:**
1 g of Agarose ([A9539](https://www.sigmaaldrich.com/ProductLookup.htmL?ProdNo=A9539&Brand=SIGMA)) per 100 mL of TAE (0.5X).

### Electrolyzer

The Electrolyzer 65 is Double-Cell PEM electrolyzer stackfrom [FuelCell Store](https://www.fuelcellstore.com/electrolyzer-65-e106), for production of hydrogen from distilled water. The center of the cell houses the hydrogen side of the two individual cells. The two outer sides of the cell have the function of supplying water and removing the oxygen produced. The two individual cells are connected electrically in series. Both cells have an electrode area of 16cm2.

Specifications:* Hydrogen Production Rate: 65 cm3/min
* Oxygen Production Rate: 32.5 cm3/min
* Hydrogen Storage Volume: 80 cm3/min
* Oxygen Storage Volume: 40 cm3/min
* Power Consumption: 16 Watts at 4.0 VDC
* Permissible Operating Voltage: 0 - 4 VDC
* Permissible Operating Current: 0 - 4.4 A
* Permissible Operating Pressure: 0 - 5 mbar
* Electrode Area: 2 cells at 16cm2 each
* Operating Medium: Distilled Water
* Dimensions (H x W x D): 9.9" x 9.9" x 4.7" (250 x 250 x 120 mm)
* Weight: 2.1 pounds (950 g)

# Yeast modifications protocols

## Cloning's methods

### Digestion and DNA purification

**1. Enzymatic plasmid digestion**

+ CutSmart Buffer 2 µl
+ DNA (pUC19) 1 µl
+ Enzyme 0.5 µl
+ Water 16.5 µl
+ 2h incubation at 37 °C

**2. DNA purification**
**Materials**

* DNA fragment previously digested
* Table-top microcentrifuge.
* Sodium Acetate 3 M.
* Ethanol 90%.
* 1.5 mL microcentrifuge tube.
* Nuclease free-water.
* Freezer at -80 °C.

**Methods**

1. Add twice the DNA volume of phenol, and vortex for 1 min.
2. Centrifuge at 13000rpm for 5 min.
3. Take 180 µL from the phase on top.
4. Add 0.1x of Sodium Acetate 3 M and 3x of Ethanol.
5. Mix immediately and thoroughly by inverting the tube 20 times.
6. Incubate at -80 °C for 4 h 30 min to precipitate DNA.
7. Centrifuge 15 min at 14,000rpm.
8. Remove the pellet.
9. Centrifuge 1 min at 14,000rpm.
10. Remove the pellet.
11. Incubate at room temperature for 5 min.
12. Resuspend pellet with 5 µL of nuclease-free water.

**3. Gel verification**
Agarose gel migration at 100V during 25 min using [Gel Loading Dye, Purple (6X)](https://international.neb.com/products/b7024-gel-loading-dye-purple-6x#Product%20Information) by NEB, and [1kb plus ladder](https://international.neb.com/products/n3200-1-kb-plus-dna-ladder) as molecular weight marker by NEB.

### Gel extraction

Agarose gel migration at 50 V during 60 min using [Gel Loading Dye, Purple (6X)](https://international.neb.com/products/b7024-gel-loading-dye-purple-6x#Product%20Information) by NEB, and [1kb plus ladder](https://international.neb.com/products/n3200-1-kb-plus-dna-ladder) as molecular weight marker by NEB.

Different kits were used to purify DNA from gel.

**1. Monarch® DNA Gel Extraction Kit**
We used the [Monarch® DNA Gel Extraction Kit](https://international.neb.com/products/t1020-monarch-dna-gel-extraction-kit#Product%20Information) by NEB to purify DNA.

**Material**

* Agarose gel band with the desired DNA
* [Monarch® DNA Gel Extraction Kit](https://international.neb.com/products/t1020-monarch-dna-gel-extraction-kit#Product%20Information) buffers
* [Monarch® DNA Gel Extraction Kit](https://international.neb.com/products/t1020-monarch-dna-gel-extraction-kit#Product%20Information) column and collecting tube
* 1.5 mL microcentrifuge tube
* Table-top microcentrifuge
* 50 °C water bath
* scale

**Methods**

1. Excise the DNA fragment from the agarose gel, taking care to trim excess agarose. Transfer to a 1.5 mL microfuge tube, and weigh the gel slice.
2. Add 4 volumes of Gel Dissolving Buffer to the gel slice.
3. Incubate the sample between 50 °C, vortexing periodically until the gel slice is completely dissolved (10 minutes).
4. Insert column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
5. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
6. Repeat step 5.
7. Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through.
8. Add 25μl of milliQ water to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA.

**2. NucleoSpin-Gel-and-PCR-Clean-up**

We also used the [NucleoSpin-Gel-and-PCR-Clean-up kit](https://www.mn-net.com/media/pdf/02/1a/74/Instruction-NucleoSpin-Gel-and-PCR-Clean-up.pdf) by NucleoSpin.

**Materials**

* Agarose gel band with the desired DNA
* [NucleoSpin-Gel-and-PCR-Clean-up kit](https://www.mn-net.com/media/pdf/02/1a/74/Instruction-NucleoSpin-Gel-and-PCR-Clean-up.pdf) buffers
* [NucleoSpin-Gel-and-PCR-Clean-up kit](https://www.mn-net.com/media/pdf/02/1a/74/Instruction-NucleoSpin-Gel-and-PCR-Clean-up.pdf) column and collecting tube
* 1.5 mL microcentrifuge tube
* Table-top microcentrifuge
* 50 °C water bath
* scale

**Methods**

1. Excise DNA fragment and solubilize gel slice in twice the weighted volume with NT1.
2. Incubate 10 min at 50 °C.
3. Centrifuge 30s at 11,000 g.
4. Add 700 µL of NT3, and centrifuge 30s at 11000 g.
5. Repeat step 4.
6. Centrifuge 1 min at 11000 g.
7. Add 20 µL of milliQ water, wait for 2 min at room temperature.

### DNA extraction

**1. QIAprep® Spin Miniprep Kit**
We followed the [QIAprep® Spin Miniprep Kit](https://www.qiagen.com/us/resources/download.aspx?id=56b0162c-23b0-473c-9229-12e8b5c8d590&lang=en) to extract DNA.

**Materials**

* 5 mL bacterial overnight culture
* QIAprep® Spin Miniprep Kit buffers
* 1.5 mL microcentrifuge tubes
* Table-top microcentrifuge

**Procedure**

1. Pellet 5 mL bacterial overnight culture by centrifugation at 6800 x g for 3 min at room temperature.
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a 1.5 mL microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 6 times.
5. Centrifuge for 10 min at 17,900 x g in a table-top microcentrifuge.

**2. QIAprep® Plasmid Midiprep kit**

We used the [QIAGEN Plasmid midi kit](https://www.qiagen.com/us/resources/download.aspx?id=c164c4ce-3d6a-4d18-91c4-f5763b6d4283&lang=en) to extract DNA from our intermediary clones before being inserted to the yeast.

**Materials**

* Overnight bacterial culture
* Buffers from [QIAGEN Plasmis midi kit](https://www.qiagen.com/us/resources/download.aspx?id=c164c4ce-3d6a-4d18-91c4-f5763b6d4283&lang=en)
* 70% ethanol
* nuclease-free water
* Ice
* Centrifuge

**Procedure**

1. Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 min at 4 °C
2. Resuspend pellet in 4 mL of buffer P1.
3. Add 4 mL of buffer P2 mix thoroughly by vigorously inverting 6 times and incubate at room temperature (25 °C) for 5 min.
4. Add 4 mL of prechilled buffer P3 mix thoroughly by vigorously inverting 6 times.
5. Incubate on ice for 15 min.
6. Centrifuge at 4,000 x g for 60 min at 4 °C. Re-centrifuge the supernatant at 4,000 x g for 30 min at 4 °C.
7. Equilibrate a QIAGEN-tip with 4 mL Buffer QBT and let it flow through the column.
8. Apply the supernatant from step 6 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
9. Wash the QIAGEN-tip with 10 mL buffer QC, let it flow through the column and repeat this step.
10. Elute DNA with 5 mL Buffer QF into a 15 mL falcon tube.
11. Add 3.5 mL of roomtemperature isopropanol to the eluted DNA and mix to precipitate DNA. Centrifuge at 4,000 x g for 60 min at 4 °C. Carefully decant the supernatant.
12. Wash the DNA pellet with 2 mL room-temperature 70% ethanol and centrifuge at 4,000 x g for 20 min. Carefully decant supernatant.
13. Air-dry pellet for 10 min and redissolve DNA in 100 µL of nuclease-free water.

### PCR

**1. PCR amplification**
We used [CloneAmp HiFi PCR Premix.](https://www.takarabio.com/products/pcr/pcr-master-mixes/high-fidelity/cloneamp-hifi-pcr-premix)

**Materials**

* PCR thermocycler
* PCR tubes
* Primers (both forward and reverse)
* Template DNA
* CloneAmp HiFi PCR Premix

**Procedure**
Keep each of the reaction components on ice during the preparation. All components should be mixed and briefly centrifuged.

![](https://static.igem.org/mediawiki/2020/e/ed/T--Toulouse_INSA-UPS--2020_exp1.png)

Mix the master mix by tapping then centrifuge briefly the mix.

![](https://static.igem.org/mediawiki/2020/e/e0/T--Toulouse_INSA-UPS--2020_exp2.png)

**2. PCR purification**
We used [QIAquick PCR Purification Kit](https://www.qiagen.com/fr/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/qiaquick-pcr-purification-kit/?clear=true#orderinginformation) to purify our DNA from PCR.

**Materials**

* Binding buffer(PB)
* Washing buffer (PE)
* Elution buffer (EB)
* pH indicator I
* 3 M sodium acetate
* 2 mL collection tube
* QIAquick column
* 1.5 mL microcentrifuge tube
* 96-100 % ethanol
* Microcentrifuge capable of achieving 17,900 x g

**Procedure**

1. **Before starting**. Add ethanol to Buffer PE and 1:250 volume pH indicator I to Buffer PB.
2. Binding DNA.
3. Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is violet or orange add 10 µL 3 M sodium acetate.
4. Place the QIAquick column in a 2 mL collection tube.
5. Apply the sample to the QIAquick column and centrifuge at 17,900 x g for 60 sec.
6. **Washing DNA.**
7. Add 750 µL of Buffer PE and centrifuge at 17,900 x g for 60 sec.
8. Centrifuge once more at 17,900 x g for 60 sec.
9. Place the QIAquick column in a 1.5 mL microcentrifuge tube.
10. **Eluting DNA.**
11. Add 30 µL Buffer EB to the center of the QIAquick membrane.
12. Wait at room temperature for 1 min.
13. Centrifuge at 17,900 x g for 1 min.

**3. Taq polymerase**
We used [TaKaRa Taq DNA polymerase](https://www.googleadservices.com/pagead/aclk?sa=L&ai=DChcSEwiJrMPfyc_sAhWGstUKHfkxC-YYABAAGgJ3cw&ae=2&ohost=www.google.com&cid=CAESQeD2-bjXNdzGxJFTW3Iocj0cm8sCEH7xrR1ZOJaS7q6Nd-fBpisVH_0ABfO_GX7YpMQ1rE7SK0lDs34JOyXapBuQ&sig=AOD64_0LDtdfnBf_G-6G8Jt-nUebP2mxHQ&q&adurl&ved=2ahUKEwjitbvfyc_sAhWHERQKHRucAJcQ0Qx6BAgHEAE&dct=1https://www.googleadservices.com/pagead/aclk?sa=L&ai=DChcSEwiJrMPfyc_sAhWGstUKHfkxC-YYABAAGgJ3cw&ae=2&ohost=www.google.com&cid=CAESQeD2-bjXNdzGxJFTW3Iocj0cm8sCEH7xrR1ZOJaS7q6Nd-fBpisVH_0ABfO_GX7YpMQ1rE7SK0lDs34JOyXapBuQ&sig=AOD64_0LDtdfnBf_G-6G8Jt-nUebP2mxHQ&q&adurl&ved=2ahUKEwjitbvfyc_sAhWHERQKHRucAJcQ0Qx6BAgHEAE&dct=1) to verify the presence of DNA fragments (mainly for construction or integration validation).

**Materials**

* PCR thermocycler
* PCR tubes
* Primers (both forward and reverse)
* Template DNA
* dNTP Mixture
* 10X PCR Buffer

**Procedure**

![](https://static.igem.org/mediawiki/2020/c/c4/T--Toulouse_INSA-UPS--2020_exp3.png)

1. We recommend keeping the components on ice while preparing the mix.

Notes: Gently mix the reaction.

2. Transfer PCR tubes into a thermal cycler.

![](https://static.igem.org/mediawiki/2020/7/78/T--Toulouse_INSA-UPS--2020_exp4.png)

**4. Genomic PCR for verification**
We used the [TAKARA PCR mix](https://www.takarabio.com/assets/documents/User%20Manual/R011_e.v1906Da.pdf) to verify our yeast transformation.

**Materials**

* 0.02N NaOH
* [TAKARA PCR mix](https://www.takarabio.com/assets/documents/User%20Manual/R011_e.v1906Da.pdf) PCR Buffer (Mg2+Plus)
* [TAKARA PCR mix](https://www.takarabio.com/assets/documents/User%20Manual/R011_e.v1906Da.pdf) dNTPs
* [TAKARA PCR mix](https://www.takarabio.com/assets/documents/User%20Manual/R011_e.v1906Da.pdf) TAQ
* Primers
* Nuclease-free water
* Thermocycler
* 0.2 mL PCR tube

**Method**

1. Add 10 µL of 0.02 M NaOH in a 0.2 mL PCR tube.
2. Add a yeast fresh transformed colony in the 0.2 mL PCR tube.
3. Gently mix.
4. Incubate 5 min at 95 °C, then incubate on ice until used.
5. Prepare the PCR mix (in a total volume of 50 µL):

![](https://static.igem.org/mediawiki/2020/8/8b/T--Toulouse_INSA-UPS--2020_exp5.png)

6. Add 2 µL of the yeast to the mix
7. Place the tubes in a thermal cycler, and execute the following program:

![](https://static.igem.org/mediawiki/2020/3/31/T--Toulouse_INSA-UPS--2020_exp6.png)

### Cloning

**1. In-Fusion**
**Materials**

1. Insert: PCR Fragment purified (ClonAMP®)
2. Vector: Linearized plasmid purified (Cutsmart®)
3. [In-Fusion® cloning kit Takara](https://www.takarabio.com/products/cloning/in-fusion-cloning)
4. PCR Thermocycler

**Methods**
![](https://static.igem.org/mediawiki/2020/1/18/T--Toulouse_INSA-UPS--2020_exp7.png)

**2. Ligation (T4 DNA ligation)**
We used the [T4 DNA ligase](https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202) by NEB to ligate our vector and insert.

**Material**

* DNA vector
* DNA insert
* T4 DNA ligase buffer
* T4 DNA ligase
* nuclease free water
* 65 °C warm bath
* 1.5 mL microcentrifuge tube
* thermocycler

**Methods**

1. Set up the following reaction in a microcentrifuge tube on ice (molar ratio of 1:3)
   ![](https://static.igem.org/mediawiki/2020/c/c5/T--Toulouse_INSA-UPS--2020_exp8.png)
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16 °C overnight.
4. Heat inactivate at 65 °C for 10 minutes.
5. Chill on ice and transform 5 μl of the reaction into 50 μl competent cells.

A control is performed by adding nuclease-free water instead of the insert.

## Transformation methods

**1. *E. coli* stellar transformation**
We used [StellarTM Competent Cells Protocol](http://www.takara.co.kr/file/manual/pdf/pt5055-2.pdf) (PT5055-2) from ClonTech

**Materials**

* Plasmids (given to us)
* Stellar Competent Cells
* 14 mL round-bottom tube
* SOC medium
* Selective medium on plates
* 37 °C incubator
* 42 °C water bath

**Procedure**

1. Thaw Stellar Competent Cells in an ice bath and then move 50 μl of competent cells into a 14-mL round-bottom tube.
2. Add 5 ng of DNA for transformation.
3. Place tubes on ice for 30 min.
4. Heat shock the cells for exactly 45 sec at 42 °C in a water bath.
5. Place tubes on ice for 1–2 min.
6. Add 450 μl of SOC medium.
7. Incubate by shaking for 1 hr at 37 °C.
8. Plate an appropriate amount of culture on a selective medium (ampicillin for pRS313, pRS315, pFL36, pFL38, L-83, L-105 and pENZ-047, and Kanamycin for pMRI34 and pHR0016)
9. Incubate overnight at 37 °C.

**2. Yeast**

**2.1 Yeast competent cells**

**Materials**

* 75 mL of YPD medium.
* 50 mL falcon-tube.
* Centrifuge
* 26 mL of LiAc/TE.

**Methods**

1. Overnight preculture from a fresh colony of yeast in 25 mL of YPD.
2. Dilute overnight precultures to low OD600 (e.g. 0.05) in 50 mL fresh YPD medium.
3. Mesure concentration every 2h to 3h until it reaches an OD of around 0.8.
4. Transfer 50 mL to a 50 mL falcon-tube and centrifuge 5 min at 3000rpm (room-temperature).
5. Remove flow-thru and add 25 mL of LiAc/TE, mix thoroughly by inverting the tube 10 times.
6. Centrifuge 5 min at 3000rpm (at room-temperature).
7. Remove flow-thru.
8. Add 400 µL of LiAc/TE, mix thoroughly by inverting the tube 10 times.

Yeast competent cells should be used on the same day that they have been prepared.

**2.2 Yeast transformation**

**Materials**

* Transforming DNA
* Competent yeast cells
* 10 mg/mL carrier DNA (SS-DNA)
* 50% PEG in 100mM LiAc/TE
* NaCl
* YNB plates (with all the amino acids except for the amino acid corresponding to the selectable marker)
* 30 °C warm bath
* 42 °C warm bath
* 30 °C incubator
* table-top microcentrifuge
* 1.5 mL microcentrifuge tube

**Method**

1. Prepare mix in 1.5 mL microcentrifuge tube:

![](https://static.igem.org/mediawiki/2020/4/49/T--Toulouse_INSA-UPS--2020_exp9.png)

Positive control was performed by adding 1 µL of pR313 instead of the transforming DNA, and negative control had no DNA.

1. Vortex solution.
2. Incubate 45 min at 30 °C.
3. Add 13 µL of DMS0 and vortex solution.
4. Incubate 15 min at 42 °C.
5. Add 450 µL of NaCl and vortex solution.
6. Centrifuge at 10,000 rpm for 1 min.
7. Remove flowthru and resuspend pellet with 80 µL of NaCl.
8. Plate solution on YNB plates (with all the amino acids except for the amino acid corresponding to the selectable marker)
9. Incubate at 30 °C for two to three days.

## GGPP extraction and quantification by LC/MS

**Sampling and Sample preparation**
GGPP was sampled by fast filtration. The cells transformed with our insert containing tHMG1 and CrtE and the wild type were grown on YNB + 2% Glucose. 10 mL of broth were filtered through 0.45 µm Sartolon polyamide (Sartorius, Goettingen, Germany) and washed with 5 mL of YNB with leucine, methionine and uracile. The filters were rapidly plunged into liquid nitrogen and then stored at −80 °C until extraction. Intracellular metabolites were extracted by incubating filters in closed glass tubes containing 5 mL of an isopropanol/H20 NH4HCO3 100 mM (50/50) mixture at 70 °C for 10 min. Cellular extracts were cooled on ice and sonicated during 1 min. Cell debris was removed by centrifugation (5000×g, 4 °C, 5 min). Supernatants were evaporated overnight (SC110A SpeedVac Plus, ThermoFisher, Waltham, MA, USA), resuspended in 200 μL of methanol: NH4OH 10 mM (7:3) at pH 9.5 and stored at − 80 °C until analysis.

**LC-MS analyses of GGPP**
Analyses were carried out on a LC–MS platform composed of a Thermo Scientific™ Vanquish™ Focused UHPLC Plus system with DAD, coupled to a Thermo Scientific™ Q Exactive™ Plus hybrid quadrupole-Orbitrap™ mass spectrometer (ThermoFisher).

Analysis of GGPP was performed on a Thermo Scientific™ Hypersil C18 GOLD™, 3 µm, and 2.1 × 100 mm column. The column was kept at 25 °C and the flow rate was set to 0.3 mL/min during the first 2 min and 0.4 mL/min for the rest of the chromatographic run. The solvent system consisted of (A) 20 mM NH4FA, pH 9.5, in water and (B) 20 mM NH4FA, pH 9.5, in 9:1 (v/v) acetonitrile–water, with the following gradient: 0–12 min from 100% A to 100% B, 5.5 min kept with 100% B and within 0.5 min the return to the initial condition and 4 min equilibration of the column. The injection volume was 10 µL.

Mass detection was carried out in a negative electrospray ionization (ESI) mode. The settings of the mass spectrometer were as follows: spray voltage 3.2 kV, capillary and desolvation temperature were 350 and 400 °C respectively, maximum injection time 200 ms. Nitrogen was used as sheath gas (50 a.u.) and auxiliary gas (15 a.u.). The automatic gain control (AGC) was set at 106 and resolution at 70,000 from m/z 100 to 700. MS analyses were performed bytargeted selected ion monitoring (tSIM) mode with 0.5 m/z isolation window for SIM and ±10 ppm for inclusion tolerances. tSIM MS scans the targeted masses in different time segments selected based on the retention times of the analytes. Data acquisition was performed using Thermo Scientific Xcalibur software. Calibration standards (prepared at concentrations from 0.08 nM to 10 µM) were used to construct calibration curves enabling to determine the concentration of each compound in the samples to be measured.

**Reference**
Sara Castaño‑Cerezo, Hanna Kulyk‑Barbier, Pierre Millard, Jean‑Charles Portais, Stéphanie Heux, Gilles Truan, Floriant Bellvert, 2019. Functional analysis of isoprenoid precursors biosynthesis by quantitative metabolomics and isotopologue profiling. Metabolomics (2019) 15:115

<https://doi.org/10.1007/s11306-019-1580-8>

## Geraniol HPLC analysis

Geraniol was quantified by High-Pressure Liquid Chromatography (HPLC), using a 1220 Infinity II LC system (Agilent, Germany). Separation was carried out with a Synergi 4 µm Fusion-RP C18 Å 250x4.6 mm column (Phenomenex) column at 30 °C, with a flow rate of 1 mL/min, using a linear gradient starting from 30 % acetonitrile / 70 % H2O up to 100 % acetonitrile at 30 minutes. Geraniol was detected by UV at 200 nm. Two peaks corresponding to cis- and trans-geraniol were detected at 16.5 and 17 minutes. The injection volume was 20 µL. Standard mixtures (at concentration from 0.05 to 0.5 mM) were used to construct calibration curves (Fig. 1).

![](https://static.igem.org/mediawiki/2020/b/bd/T--Toulouse_INSA-UPS--2020_exp10.png)

Figure 1. Geraniol calibration curve.Two peaks corresponding to cis- and trans-geraniol were detected at 16.5 and 17 minutes.

# Fermentation protocols for the coculture system

## Medium for the coculture between *Clostridium ljungdahlii* and *Saccharomyces cerevisiae*.

**Medium and conditions**
The range of temperature for the yeast is [25;37] °C and the range of temperature for the bacteria is [30;40] °C. This is why we fixed the coculture temperature at 33 °C. The optimal pH of *S. cerevisiae* is 5.5 and *C. ljungdahlii* can grow at this pH.

We elaborated a medium for the coculture based on the media used for *Clostridium ljungdahlii* and *Saccharomyces cerevisiae* [1],[2]. To design it we receive help from Jason M. Whitham. All the compounds used in *S. cerevisiae* media are approximately in the same quantity in the media used for *C. ljungdahlii*. The medium is therefore composed of YNB (what the yeast needs) and we added everything else the bacteria needs. Here is how we elaborated the first coculture medium:

Dissolve ingredients of the pre-medium, warm the solution if necessary to dissolve YNB. Add 10 mL of the trace element solution which should be at pH=4. Sparge medium with 80% N2 and 20% CO2 gas mixture for several minutes to make it anoxic and adjust pH to 5.5. Autoclave at 121 °C for 15 min. Add 10 mL of vitamins (sterilized by filtration) from sterile stock solutions prepared under 100% N2 gas atmosphere. Adjust pH of complete medium to 7 by adding the appropriate volume of NaOH (at 1 mM). Before yeast inoculation, be sure to have a pO2 at 5%.

![](https://static.igem.org/mediawiki/2020/e/ea/T--Toulouse_INSA-UPS--2020_table.png)

**Growth experiments with *S. cerevisiae***
We grew CENPK1137 cells at 33 °C, pH 5.5 and 150rpm as followed:

1. pre-medium without Na2S + trace elements solution + 0.38% ethanol + 0.5% acetate
2. pre-medium + trace elements solution + 0.38% ethanol + 0.5% acetate
3. pre-medium without Na2S + trace elements solution + vitamins solution + 0.38% ethanol + 0.5% acetate

**Growth experiments with *C. ljungdahlii***
We made all our *C. ljungdahlii* growths on the coculture medium as we describe it in the protocole “Growth experiments of *Clostridium ljungdahlii*”.

**References**
[1] 879. CLOSTRIDIUM LJUNGDAHLII MEDIUM
https://www.dsmz.de/microorganisms/medium/pdf/DSMZ\_Medium879.pdf

[2] BD Difco™ Yeast Nitrogen Base without Amino Acids
https://www.fishersci.com/shop/products/bd-difco-dehydrated-culture-media-yeast-nitrogen-base-without-amino-acids-6/p-4901538

## Growth experiments of *Clostridium ljungdahlii*

**Strain and conditions**
*Clostridium ljungdahlii* (Tanner et al. 1993) was obtained from DSMZ (<https://www.dsmz.de/>, Germany). Growth experiments were carried out in serum bottles filled with water, culture medium or other solutions. All bottles were sparged with nitrogen and/or carbon dioxide before autoclave to ensure anaerobic conditions.

![](https://static.igem.org/mediawiki/2020/2/24/T--Toulouse_INSA-UPS--2020_exp11.png)

Figure 2. Serum bottles inoculated with *Clostridium ljungdahlii*

**Storage**
2.5 mL of 50% glycerol / 50% water solution were added in 10 mL serum bottles under a nitrogen atmosphere to ensure anaerobic conditions. Sterilization was performed by autoclave. We added 2.5 mL of an active culture in exponential growth phase on fructose (5 g/l), and serum bottles were stored at -80 °C.

**Medium preparation**
Dissolve ingredients of the pre-medium, warm the solution if necessary to dissolve YNB. Add 10 mL of the trace element solution which should be at pH=4. Sparge medium with 80% N2 and 20% CO2 gas mixture for several minutes to make it anoxic and adjust pH to 5.5. Autoclave at 121 °C for 15 min. Add 10 mL of vitamins (sterilized by filtration) from sterile stock solutions prepared under 100% N2 gas atmosphere. Adjust pH of complete medium to 7 by adding the appropriate volume of NaOH (at 1 mM).

![](https://static.igem.org/mediawiki/2020/e/ea/T--Toulouse_INSA-UPS--2020_table.png)

**Experimental setup for growth of *C. ljungdahlii* on CO2 and H2**
Growth experiments of *C. ljungdahlii* on CO2 and H2 were performed using a tailor-made reactor of 300 mL with two heads. The stoppers have been made impermeable to gases with silicone. CO2 was provided from a gas bottle at a constant flow rate under approximately 2 bar pressure (Air Liquide, France) and H2 from a water electrolyzer (Electrolyzer 65, FuelCellStore, USA). We added a constant flow of nitrogen to reduce the risk of H2 accumulation and increase our safety. The set-up of the system is shown on figure 1.

![]( https://static.igem.org/mediawiki/2020/7/74/T--Toulouse_INSA-UPS--2020_exp12.png)

Figure 3: Scheme of the set-up for *Clostridium ljungdahlii* growth on CO2 and H2

![]( https://static.igem.org/mediawiki/2020/9/98/T--Toulouse_INSA-UPS--2020_exp13.png)
![]( https://static.igem.org/mediawiki/2020/1/1e/T--Toulouse_INSA-UPS--2020_exp14.png)

Figure 4: Set-up for *Clostridium ljungdahlii* growth on CO2 and H2

We filled the reactor with 200 mL of culture medium (without fructose and vitamins) sparged with CO2 and N2. Vitamins were added after autoclave. The reactor was inoculated with 15 mL of an active *C. ljungdahlii* culture in mid-exponential growth phase (after 2 days of incubation). Growth was initially warried out at the optimal temperature of 37 °C (we will also try at 33 °C, i.e. the coculture temperature).

Every two hours, 2 mL of broth were sampled. 1 mL was used for OD measurement at 600nm, using a Genesys spectrophotometer (Thermo Fisher, USA). The other milliliter was centrifuged 5 minutes at 13 000 rpm, and the supernatant was filtered and stored at -20 °C until analysis.

**References**

* Michael E. Martin, Hanno Richter, Surya Saha, Largus T. Angenent, 2016. Traits of Selected Clostridium Strains for Syngas Fermentation to Ethanol. Biotechnology and Bioengineering, Volume113, Issue3, March 2016, Pages 531-539. DOI 10.1002/bit.25827

- Jason M. Whitham, Joel J. Pawlak, Amy M. Grunden, 2016. *Clostridium ljungdahlii*: A Review of the Development of an Industrial Biocatalyst. Current Biotechnology Volume 5 , Issue 1 , 2016. DOI: 10.2174/2211550105666151208211335

## Growth experiments of *Saccharomyces cerevisiae*

**Strain**
The strain used for the growth experiments was *Saccharomyces cerevisiae* CENPK3711.

**Medium and culture conditions**
*S. cerevisiae* was grown on YNB medium (Sigma) with different concentrations of ethanol and/or acetate as sole carbon source(s) (Figure 1). Temperature was set at 33 °C, which is appropriate to grow both yeast (growing at temperature between 25 and 37 °C) and bacteria is (30-40 °C).

*S. cerevisiae* cells were inoculated on fresh petri dishes of YNB with 2 % of glucose, which was used to inoculate pre-cultures in glass tubes containing 5 mL of YNB with acetate and/or ethanol (150 rpm). After 24h, these pre-cultures were diluted 10 times serially in 4 tubes. The second pre-cultures were carried out in the same conditions for 24 h. Growth experiments were carried out in 500 mL shake flasks containing 50 mL of medium at 150 rpm. Three biological replicates were carried out for each experiment.

![]( https://static.igem.org/mediawiki/2020/7/7a/T--Toulouse_INSA-UPS--2020_exp15.png)

Figure 5. Concentration of carbon sources (acetate and/or ethanol, expressed as weight percent) used to grow *Saccharomyces cerevisiae* on YNB in each experiment

**Sampling**
Every two hours, 2 mL of broth were sampled. 1 mL was used for OD measurement at 600nm, using a Genesys spectrophotometer (Thermo Fisher, USA). 1 mL was centrifuged 5 minutes at 13 000 rpm, and the supernatant was filtered and stored at -20 °C until HPLC analysis.

## Acetate and ethanol analysis **HPLC analysis** Ethanol and acetate were quantified by High-Pressure Liquid Chromatography (HPLC), using a UltiMate 3000 SD HPLC Dionex™ system (Thermo Scientific™). Separation was carried out with an Aminex HPX-87H 300 x 7.8 mm column (BioRad, France). Elution was carried out at 50 °C and a flow rate of 0.6 mL/min with an isocratic solution of 5 mM of H2SO4 as mobile phase. Detection was performed by refractometry with aSP6040 (Spectra-Physics) detector. The injection volume was 20 µL. Standard mixtures (at concentration from 0.5 to 20 g/L) were used to construct calibration curves (Figures 1 and 2). We used this method for the analysis of supernatant of yeast cultures. ![]( https://static.igem.org/mediawiki/2020/4/41/T--Toulouse_INSA-UPS--2020_exp16.png) Figure 6. Acetate standard curve ![]( https://static.igem.org/mediawiki/2020/4/4f/T--Toulouse_INSA-UPS--2020_exp17.png) Figure 7. Ethanol standard curve. **NMR analysis** The extracellular medium of *Clostridium ljungdahlii* was analyzed by NMR to identify and quantify all the (protonated) compounds produced during growth on H2 and CO2. 180 µL of supernatant collected in mid-exponential growth phase were mixed with 20 µL of TSP-d4 (used as internal standard and spectrum reference) dissolved in D2O at a concentration of 10 mM, and introduced in a 3 mm tube. All NMR experiments were carried out on a Bruker Avance III spectrometer (Bruker, Germany) operating at 800 MHz proton frequency and equipped with a TCI cryogenic probe head. Temperature of the samples was 293 K. Spectra were processed with Bruker TopSpin 4.0.7. For the 1H 1D spectra, the standard Bruker pulse sequence noesypr1d for a 1D NOESY with water pre-saturation was used. Key parameters were as follows: spectral width 13 ppm/10417 Hz; complex points, TD 32768; interscan delay, d1 10 s; NOE mixing time, d8 100 ms; number of scans, ns = 32; dummy scans, ds = 4. Total experiment time was 25 minutes. For the 1H-1H 2D spectra, the standard Bruker pulse sequence dipsi2phpr for a 2D TOCSY with water pre-saturation was used. Key parameters were as follows: spectral width 13 ppm/10417 Hz in dimension F2 and 10 ppm/8012 Hz in dimension F1; complex points, TD 16384 in F2 and 128 in F1; interscan delay, d1 1 s; TOCSY mixing time, d9 60 ms. Total experiment time was 25 minutes. Only ethanol and acetate were detected in significant amounts (> 100 µM).

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# Team:Toulouse INSA-UPS/Results

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### Results

* [Yeast Modification Results](#yeast-modification-results)

+ [Flavors production](#cloning-of-geraniol-and-limonene)
+ [Optogenetic systems](#cloning-of-optogenetic-systems)
+ [β-carotene precursors production](#cloning-of-beta-carotene-precursors)
+ [β-carotene production](#cloning-for-beta-carotene)

* [Fermentation for the coculture system](#fermentation-experiments-for-the-coculture-system)

+ [Definition of a coculture medium](#definition-of-a-coculture-medium)
+ [Growth experiments with *C. ljungdahlii* on CO2 and H2](#growth-experiments-with-clostridium-CO2-H2)
+ [Growth of *S. cerevisiae* on acetate and ethanol](#growth-of-saccharomyces-acetate-ethanol)
+ [Conclusion on coculture](#coculture-conclusion)

![PF](https://static.igem.org/mediawiki/2020/e/e4/T--Toulouse_INSA-UPS--2020_petite_fusee.png)

# Results

## Information

In order to supplement the astronauts' food with tasty nutritious yeast, we had to do several clonings to insert key genes into the yeast genome. However, our expectations were high and we did not have the time to complete all of our clonings. We made a cloning progress chart here below at the time when we left the lab. We managed to enhance the pool of precursors of β-carotene as you can see below and in our [Engineering success page](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Engineering). We are also proud to prove with our encouraging coculture results that our project has a promising future.

![](https://static.igem.org/mediawiki/2020/c/c0/T--Toulouse_INSA-UPS--2020--CLONINGS.jpg)

Cloning process chart

# Yeast modification results

Introducing new metabolic pathway in engineered *Saccharomyces cerevisiae* to constitutively synthetize vitamin and flavors in a controlled manner.

The goal of this section is to provide engineered yeast in order to constitutivly produce β-carotene and geraniol and limonene in an optogenetic controlled system. Thus, all cloning for gene of interest were performed in *Escherichia coli* as described in the Experiments section. Our strategy was to assemble several blocks (some of our clonings with 4 to 5 blocks) in a single cloning step by using the In-Fusion kit (ligation-independent cloning) from TaKaRa Bio. However, due to the limitation of the technology, we soon found out that assembling 4 or 5 blocks in a single reaction was not conclusive. Thus, we subcloned the genes of interest with no more than two blocks at the same time, which increasing dramatically the cloning steps.

**Materials and methods:**

Most of our blocks were synthesized as gBlocks by IDT (Integrated DNA Technologies). They were all given a number to facilitate their identification and our organization. The sequences that were not synthesized by IDT are coming from plasmids obtained from different laboratories.

All the protocols used in this cloning section can be found here ([Experiments page](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Experiments)).

## Molecular engineering for flavors production

### Cloning for geraniol and brazzein production

**Summary and cloning strategy ([BBa\_K3570001](http://parts.igem.org/Part%3ABBa_K3570001)):**

In our project, we aimed to give to the astronauts the opportunity to choose the flavor of the yeast. Thus, we explored the possibility to use geraniol and brazein to make sweet rose flavored yeast. For geraniol and brazzein, we aimed to construct two different vectors (figure 1) and to fuse them together into a single plasmid:

![](https://static.igem.org/mediawiki/2020/3/3d/T--Toulouse_INSA-UPS--2020_fig1cloning.png)

Figure 1: Cloning strategy for geraniol and brazzein production

The first intermediary plasmid is composed of blocks B1, B2 and B3 in the pUC19 chassis to create pUC19-B1B2B3, so it is composed of the upstream integration site ([BBa\_K3570014](http://parts.igem.org/Part%3ABBa_K3570014)), the brazzein gene and its FLAG tag, the geraniol synthase ([BBa\_K3570012](http://parts.igem.org/Part%3ABBa_K3570012)) and their common bidirectional promoter GAL1/10. The other intermediary plasmid is composed of blocks B4 and B5 in the pUC19 to create pUC19-B4B5 (so including the leucine selection marker for the yeast *S. cerevisiae* [BBa\_K1430001](http://parts.igem.org/Part%3ABBa_K1430001) and the downstream integration site [BBa\_K3570015](http://parts.igem.org/Part%3ABBa_K3570015)).

**Results and discussion:**

* **Assembly of pUC19-B1B2B3 (block 1,2 and 3):**

The gBlocks B1, B2 and B3 have been amplified by PCR then purified. At the same time, digestion of our vector pUC19 by *Sbf*I and *Xba*I was performed and purified on gel with the same kit. Before we proceed to cloning, we verified all our purified PCR fragments (Figure 2).

![](https://static.igem.org/mediawiki/2020/0/0b/T--Toulouse_INSA-UPS--2020_fig2Acloning.png)

Figure 2: Agarose electrophoresis in order to verify gBlocks B1, B2, B3 and pUC19 digested with SbfI and XbaI. On the right the expected results and on the left the obtained gel. All gBlocks and the pUC19 present the expected sizes.

The purified DNA fragments had the expected length and were subsequently used for cloning. In-Fusion, transformation of Stellar cells, selection on ampicillin, miniprep of a few clones were done. Then, the plasmid construction was checked out with the restriction enzymes *Sbf*I and *Sac*I in order to release the expected fragment of 3748 bp and 2672 bp. After several attempts, we accepted that this cloning strategy was very unlikely to yield proper clones. As previously proposed, a new design strategy consisting of two steps instead of one step was adopted (Figure 3).

![](https://static.igem.org/mediawiki/2020/8/80/T--Toulouse_INSA-UPS--2020_fig3.png)

Figure 3: Revisited cloning strategy for pUC19-B1B2B3. First, the B1 block is cloned into pUC19 vector and then B2 and B3 blocks are cloned in the resulted vector from step 1.

First, the pUC19-B1 was built by the In-Fusion kit and three of the obtained colonies were verified by digestion with *Sal*I and *Sbf*I (figure 4).

![](https://static.igem.org/mediawiki/2020/8/86/T--Toulouse_INSA-UPS--2020_Fig4cloning.png)

Figure 4: Agarose electrophoresis of DNA fragments in order to validate the cloning experiments. DNA plasmids from 3 colonies were double digested with *Sbf*I and *Sal*I.

Colonies 2 and 3 have the expected digested profile on the gel: pUC19-B1 was successfully obtained. It was digested by *Sal*I and *Xba*I to insert the B2 and B3 fragments. After the In-Fusion reaction and transformation, the colonies were verified by PCR and we expected a fragment at 872 bp (Figure 5).

![](https://static.igem.org/mediawiki/2020/1/1f/T--Toulouse_INSA-UPS--2020_Fig5cloning.png)

Figure 5: Agarose electrophoresis of PCR amplification on ampicilin selected clones after cloning gBlocks B1, B2 and B3 into pUC19 by the In-Fusion kit. On the right the expected results and on the left the obtained gel. The expected size is 872 bp.

As it can be seen, only clone #4 presented a strand at 872 bp. The plasmid was sent for sequencing. Unfortunately, it presented a deletion in the ORF of the geraniol synthase (B2) involving a shift in the reading frame. We tried again this cloning step without success. At this point, we decided to stop linearizing the vector by digestion and switch to preparing the linear vector by PCR. This would prevent getting a majority of colonies presenting the empty vector. This PCR was performed using different concentrations of the template plasmid (figure 6).

![](https://static.igem.org/mediawiki/2020/0/02/T--Toulouse_INSA-UPS--2020_fig6cloning.png)

Figure 6: Agarose electrophoresis of PCR amplification of pUC19-B1 with different template quantities (from 10 ng to 100 ng of DNA). On the right the expected results and on the left the obtained gel.

This showed that the linearized pUC19-B1 could be amplified with template concentrations as low as 10 ng. In-Fusion was then performed to integrate B2 and B3. The selected colonies were checked by PCR and we expected a 1205 bp fragment (figure 7).

![](https://static.igem.org/mediawiki/2020/2/2e/T--Toulouse_INSA-UPS--2020_fig7cloning.png)

Figure 7: Agarose electrophoresis of PCR amplification on ampicilin selected clones after cloning gBlocks B2, B3 into pUC19-B1 with the In-Fusion kit. On the right, the expected results and on the left, the obtained gel. The expected band should be at 1.2 kb.

According to the results, none of the clones had the expected size that would correspond to a correct insertion to pUC19-B1 of B2 and B3. Unfortunately at this time, a re-emergence of coronavirus restricted our work ([Impact of COVID-19](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Description#COVID-19)). We had to choose which cloning we would carry on. As this part was not our first priority, its progress simply stopped here.

* **Assembly of the pUC19-B4B5 (Block 4 and 5)**

In parallel with pUC19-B1 construction, we tried to build pUC19-B4B5 which contains the leucine auxotrophic marker and the downstream integration site.

The expected sizes of the amplified DNA fragments were obtained for both fragments (data not shown). These were integrated by the In-Fusion kit into the pUC19 plasmid. Four clones were verified by digestion with *EcoR*I which should reveal fragments at 1366 bp and 3807 bp if correct (figure 8).

![](https://static.igem.org/mediawiki/2020/3/34/T--Toulouse_INSA-UPS--2020_fig8A.png)

Figure 8: Agarose electrophoresis in order to verify ampicilin selected clones after cloning pUC19-B4B5 with the In-Fusion kit and digested with EcoRI. On the right, the expected results and on the left, the obtained gel. The expected sizes are 1.3 kb and 3.8 kb.

The three first colonies presented correct profiles and their plasmids were sent for sequencing. Clone M28 presented the correct sequence.

The next step should have been to include fragment B1B2B3 in this pUC19-B4B5 but it had to be abandoned because of the re-emergence of the coronavirus.

### Cloning for limonene production

**Summary and cloning strategy:**

We needed to insert the gene coding for limonene synthase in the yeast. As for geraniol and brazzein production cloning, our first idea was to clone all our four blocks in one step (strategy not shown). Since this failed, we had to redefine our strategy in 2 steps, each would sequentially integrate two blocks in pUC19. This was not more conclusive and we ended up inserting the block one by one in pUC19.

The final strategy for the cloning of limonene synthase is presented Figure 9.

![](https://static.igem.org/mediawiki/2020/5/5e/T--Toulouse_INSA-UPS--2020_fig9cloning.png)

Figure 9: Final cloning strategy for limonene production. To build our construction for limonene production, the four DNA fragments needed were inserted one by one in the pUC19 plasmid.

**Results and discussion:**

The block B10, B11 and B13 were synthesized as gBlocks by IDT, amplified by PCR with CloneAmp HiFi PCR, and purified by NucleoSpin Gel or NEB Monarch Gel extraction kits. The auxotrophic marker (block B12) was amplified by PCR with CloneAmp HiFi PCR using *S. cerevisiae* genomic DNA as template (data not shown).
So we started by inserting block B10 in the pUC19 plasmid. Four colonies presented plasmids with the expected digestion profiles (Figure 10).

![](https://static.igem.org/mediawiki/2020/c/cf/T--Toulouse_INSA-UPS--2020_fig10.png)

Figure 10: Agarose electrophoresis in order to verify ampicilin selected clones after cloning pUC19-B10 with the In-Fusion kit and digesting with SbfI and SacI. On the right the expected results and on the left the obtained gel. The expected sizes are 2.6 kb and 1.2 kb, the non-digested plasmid should be 3.8 kb.

The first four tracks of this gel correspond to four different clones tested by digestion. The last line contains undigested plasmid. The sequences proved to be valid. The next step was to integrate fragment B11. The obtained colonies were assessed by PCR but unfortunately, none presented the expected amplicon size (Figure 11).

![](https://static.igem.org/mediawiki/2020/a/ae/T--Toulouse_INSA-UPS--2020_fig11cloning.png)

Figure 11: Agarose electrophoresis in order to verify ampicilin selected clones after cloning pUC19-B10B11 with the In-Fusion kit. On the right, the expected results and on the left, the obtained gel. The expected size is 2kb. None of the clones presented the expected size.

At this step, we had to stop the experiments: we still had three fragments to insert in our vector before being able to integrate it in the yeast, and we lacked time and workers to undertake it with the COVID-19 lurking around.

## Molecular engineering for Optogenetic Systems

Our aim is to give the choice to astronauts between either raw yeast, lemon or sweet rose taste. This is why we thought to use optogenetic systems to regulate the production of limonene, geraniol and brazzein using light. The red optogenetic system required two fused proteins encoded by *GBD-PhyA* and *GAD-FHY1* genes. For the blue ontogenetic system, it required only the *NLS-EL22-vp16* gene.

### Cloning of the red optogenetic system

**Summary and cloning strategy ([BBa\_K3570004](http://parts.igem.org/Part%3ABBa_K3570004)):**

The insert containing genes for the PhyA-FHY1 optogenetic system was designed to be cloned into the limonene production plasmid, or the brazzein/geraniol production one (if you have read the previous cloning descriptions, you can guess how this will end up!). The following blocks B6, B7, B8 and B9 (figure 12) were designed to be cloned by the In-Fusion kit into a pUC19.

![](https://static.igem.org/mediawiki/2020/9/9d/T--Toulouse_INSA-UPS--2020_fig12.png)

Figure 12: Cloning strategy for the red optogenetic system

**Results and discussion:**

As for the previous clonings, the first strategy consisted in inserting 4 blocks all together. Since all the colonies we obtained contained empty pUC19 vectors, we tried to avoid the problem by preparing the vector from PCR amplification instead of digestion using restriction enzymes.
At this time, however, because of the coronavirus re-emergence restricting our work ( [COVID-19 impact](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Description#COVID-19) ), we were not able to finish our construction and decided to stop this cloning (not very surprising!).

### Clonings for the blue optogenetic system

**Summary and cloning strategy ([BBa\_K3570005](http://parts.igem.org/Part%3ABBa_K3570005)):**

The sequence was obtained from the plasmid EZ-l105 and EZ-l83 (Zhao *et al*. 2018) given by Sylvain Pouzet. The block B28 was the pPGK1–VP16–EL222–tCYC1 sequence from EZ-l105 (pPGK1: [BBa\_K122000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K122000), VP16-EL222: [BBa\_K3570021](http://parts.igem.org/Part%3ABBa_K3570021) and tCYC: [BBa\_K2637017](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2637017)). The blocks B27 and B30 were retrieved from the plasmid EZ-l83, they were the sequences pC120 and pC120-GFP-tADH1 respectively (GFP: [BBa\_E0040](http://parts.igem.org/Part%3ABBa_E0040) and tADH1: [BBa\_J63002](http://parts.igem.org/wiki/index.php?title=Part:BBa_J63002)). The strategy was to integrate B28 in the pUC19 chassis then to integrate B30 and B27.

![](https://static.igem.org/mediawiki/2020/1/1f/T--Toulouse_INSA-UPS--2020_fig13.png)

Figure 13: Cloning strategy for the blue optogenetic system

**Results and discussion:**

Since the B27 PCR amplification was not conclusive because of its small length (232 bp), it was suppressed from the design and we decided to build only pUC19-B28B30 by integrating sequentially B28 and B30.

Both blocks (B28 and B30) have been amplified by PCR. First, the pUC19-B28 construction was built by the In-Fusion kit and the plasmids from the obtained colonies were verified by PCR (figure 14).

![](https://static.igem.org/mediawiki/2020/6/66/T--Toulouse_INSA-UPS--2020_figcloning.png)

Figure 14: Agarose electrophoresis of PCR amplification of pUC19-B28. On the right the expected results and on the left the obtained gel. The expected band size is at 1.9 kb.

As can be seen, all the plasmids presented the correct profile. They were sent for sequencing and it appeared that plasmid #3 had no mutation. It was then used as a chassis to integrate B30 with the the In-Fusion kit. The colonies obtained were checked by PCR, expecting a fragment at 1419 bp (figure 15).

![](https://static.igem.org/mediawiki/2020/8/88/T--Toulouse_INSA-UPS--2020_fig14.png)

Figure 15: Agarose electrophoresis of PCR amplification of pUC19-B28B30. On the right, the expected results and on the left, the obtained gel. The expected size should be at 1.4 kb.

The results showed that tracks 1, 3, 4, 5, 11 and 12 presented the correct profile. Thus, pUC19-B28B30 was obtained.

As you can guess, the optogenetic parts were put in standby at this step because we were late in our cloning program and too few to perform all the experimentations because of the COVID-19. Besides, the blue optogenetic system was designed to be integrated in the limonene or the geraniol constructions that we failed to produce.

## Molecular engineering for β-carotene precursors

### Cloning of tHMG1 and CrtE

**Summary and cloning strategy ([BBa\_K3570000](http://parts.igem.org/Part%3ABBa_K3570000)):**

All the compounds we want to produce with the yeast derived from the mevalonate pathway, this is why the improvement of this pathway was required to reach sufficient yields.

The cloning strategy was to clone the blocks into two vectors before bringing them together in a unique plasmid. The blocks B14, B15 and B16 would be cloned in a pUC19 using the In-Fusion kit to form pUC19-B14B15B16 containing the DPP1 upstream sequence, tHMG1 and TDH3-TEF1. The other blocks B17, B18 and B19 containing CrtE, the histidine auxtrotrophic marker and the DPP downstream sequence would be cloned in parallel in another pUC19 using the same cloning method to form pUC19-B17B18B19. pUC19-B17B18B19 would be used as a template vector to insert the sequence of B14B15B16 from pUC19-B14B15B16 (Figure 16)

![](https://static.igem.org/mediawiki/2020/4/4f/T--Toulouse_INSA-UPS--2020_fig16.png)

Figure 16: Cloning strategy for β-carotene precursors production

**Results and discussion:**

Assembly of pUC19-B14B15B16:

The gBlocks corresponding to B14, B15 and B16 have been amplified by PCR, purified and verified on agarose gel (Figure 17).

![](https://static.igem.org/mediawiki/2020/6/6e/T--Toulouse_INSA-UPS--2020_fig18.png)

Figure 17: Agarose electrophoresis verification of PCR amplified gBlocks B14, B15, B16 and digested pUC19 with SbfI and BamHI. On the right, the expected results and on the left, the obtained gel. The expected sizes were 2.6 kb, 0.4 kb, 1.8 kb and 1.0 kb respectively. All have the expected size.

pUC19 was double digested by *Sbf*I and *BamH*I and prepared to receive the PCR products B14, B15 and B16 by the In-Fusion kit. After transformation of Stellar cells, selection on ampicillin, and minipreps of eight clones, we checked the restriction profiles of the constructions. The resulted plasmids containing two *Sac*I restriction sites were then verified by digestion with the enzyme *Sac*I (Figure 18).

![](https://static.igem.org/mediawiki/2020/8/8e/T--Toulouse_INSA-UPS--2020_fig18Acloning.png)

Figure 18: Agarose electrophoresis of PCR amplification of pUC19-B14B15B16 cloned with the In-Fusion kit and digested using SacI. On the right, the expected results and on the left, the obtained gel.

We succeeded to isolate six clones displaying a plasmid with the expected profile. Sequencing of the plasmids confirmed that we obtained the correct pUC19-B14B15B16 construction also known as the first step for tHmg1-CrtE construction.

**Assembly of the pUC19-B17B18B19**

The gBlocks B17, B18 and B19 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (Figure 19).

![](https://static.igem.org/mediawiki/2020/7/77/T--Toulouse_INSA-UPS--2020_fig19.jpeg)

Figure 19: Agarose electrophoresis of PCR amplification of the pUC19 digested using BamHI and EcoRI, and gBlocks B17, B18 and B19. On the right, the expected results and on the left, the obtained gel. The expected sizes were 2.6 kb, 1.4 kb, 1.4 kb and 0.4 kb respectively. All have the expected size.

pUC19 vector was double digested using *BamH*I and *EcoR*I and prepared to receive the PCR products B17, B18 and B19 with the In-Fusion kit. After transformation of Stellar cells, selection on ampicillin, and minipreps of 6 clones, we checked the restriction profiles of the constructions. The resulted plasmids were verified by digestion with *BamH*I and *EcoR*I (Figure 20).

![](https://static.igem.org/mediawiki/2020/9/98/T--Toulouse_INSA-UPS--2020_fig20.png)

Figure 20: Agarose electrophoresis of PCR amplification of the pUC19-B17B18B19 cloned by In-Fusion kit and digested using BamHI and EcoRI. On the right, the expected results and on the left, the obtained gel. The expected sizes were 4.8 kb and 2.6 kb.

Only one clone had the expected profile (figure 20, lane 4). This clone has been sequenced by Eurofins and it was fortunately valid. We had successfully obtained the second plasmid pUC19-B17B18B19 of our tHMG1-CrtE construction.

**Assembly of tHMG1-CrtE:**

The next step was to combine both plasmids by subcloning the fragment B14B15B16 into plasmid pUC19-B17B18B19.

To do this, we first extracted the DNA with the QIAGEN Plasmid Plus Midi Kit. Then, we digested both plasmids with *Sbf*I and *BamH*I and purified with the Monarch Genomic DNA Purification Kit by NEB. The fragments were ligated together with T4 DNA ligase by NEB followed by a transformation into Stellar cells (ampicillin selection). Over the eight assessed colonies, two colonies presented the expected restriction profile when digested with *Sbf*I and *EcoR*I (Figure 21).

![](https://static.igem.org/mediawiki/2020/6/67/T--Toulouse_INSA-UPS--2020_fig21.png)

Figure 21: Agarose electrophoresis of ampicilin selected clones of pUC19-B14B15B16B17B18B19 ligated with T4 DNA ligase digested with SbfI and EcoRI. On the right, the expected results and on the left, the obtained gel. The expected sizes were 6.8 kb and 2.6 kb. Two of them present the expected size (with some residual undigested clone left at 9.4 kb).

#### Yeast transformation:

Since the construction was successful, we proceeded to the yeast transformation by integration in the DPP1 locus. The plasmid was digested with enzymes SbfI and EcoRI and the fragment containing our construction was purified to transform the *Saccharomyces cerevisiae* strain BY4741 . The resulted cells were grown on YNB LEU+, URA+, MET+ HIS- with 2% glucose for 3 days. At the third try, we were able to observe around 20 colonies in our yeast transformation, about the same amount of colonies on the positive control and none on the negative control plate.

Verification of integration of BBa\_K3570000 using the DPP1 homology sequence (BBa\_K3570006 and BBa\_K3570007) was performed by a genomic PCR using the TaKaRa PCR amplification Kit and the following primers: primer 1 and primer 2.

Primer 1: ATCAGGATTTGCGCCTTT

Primer 2: GCCGCCGAGGGTATTTTACTTCCG

We randomly chose eight clones from our transformation and one from the positive control plate (Figure 22).

![](https://static.igem.org/mediawiki/2020/0/03/T--Toulouse_INSA-UPS--2020_fig22.jpeg)

Figure 22: Verification of DPP1 integration. On the right, the expected results and on the left, the obtained gel. The expected size is 1.2 kb.

All clones have the expected size (1.2 kb), and the PCR negative control where we inserted pRS313 did not show any band, which means that the [BBa\_K3570000](http://parts.igem.org/Part%3ABBa_K3570000) part is well integrated in the yeast genome. The integration in the yeast genome is a success that means that the parts  [BBa\_K3570008](http://parts.igem.org/Part%3ABBa_K3570008) (HIS3 selective marker),  [BBa\_K3570006](http://parts.igem.org/Part%3ABBa_K3570006)  and  [BBa\_K3570007](http://parts.igem.org/Part%3ABBa_K3570007) (for DPP1 homologous sequence) all work. Our modified strain is BY4741 DPP1::tHMG1-crtE.

#### **Analysis and Validation of the constructed strains**

The integration of the [BBa\_K3570000](http://parts.igem.org/Part%3ABBa_K3570000) part in the yeast genome should enhance the flow of the mevalonate pathway. This modification should result in an increase of the concentration of GGPP, which is the precursor of beta-carotene. GGPP have been extracted from BY4741 DPP::tHMG1-crtE and analysed by LC-MS (see our experiments [here](https://2020.igem.org/Team%3AToulouse_INSA-UPS/Experiments#ggpp-extraction-and-quantification-by-lcms) ).
Our strain BY4741 DPP::tHMG1-crtE showed a five-fold increase in GGPP concentration in comparison with the wild type yeast (figure 23). This important result demonstrates that the part BBa\_K3570000 works.

![](https://static.igem.org/mediawiki/parts/3/37/T--Toulouse_INSA-UPS--2020_GGPP.png)

Figure 23: Results of the GGPP analysis from culture on YNB with 2% Glucose by LC-MS

Data from the literature [2] suggest a stable turn-over of mevalonate pathway intermediates in native and engineered *S. cerevisiae* strains, i.e. a stable ratio between the GGPP production flux and its concentration. This would indicate that our strain optimization strategy successfully enhances by five-fold the capability of GGPP production, and thus the potential of β-carotene production. Moreover, the increased flux through the mevalonate pathway is expected to increase not only β-carotene production, but also the production of limonene and geraniol (a provitamin A, a flavor of lemon and of rose) synthesized from other mevalonate intermediates.

## Molecular engineering for β-carotene production

In order to make yeast produce β-carotene, the precursor of vitamin A, we also needed to insert a fusion protein: CrtYB(ek)I.

### Cloning of the fusion protein CrtYB(ek)I

**Summary and cloning strategy ([BBa\_K3570002](http://parts.igem.org/Part%3ABBa_K3570002)):**

Our strategy was to divide our part into two plasmids: one composed of blocks B20 (HO up), B221 (TDH1) and B22 (CrtYB(ek)I) and tCYC) in pUC19 vector and forms pUC19-B20B21B22; and another composed of B23 (URA3 auxotrophic marker) and B24 (HO down) also in pUC19 vector leading to pUC19-B23B24. Then we would use pUC19-B23B24 as a template vector to insert B20B21B22.

![](https://static.igem.org/mediawiki/2020/3/3a/T--Toulouse_INSA-UPS--2020_fig23.png)

Figure 24: Cloning strategy for pUC19-B21B22 and pUC19-B23B24. First, the B21 and B22 blocks are cloned into pUC19 vector, B23 and B24 blocks are cloned in pUC19, then the two constructions are ligated together.

**Results and discussion:**

**Assembly of pUC19-B20B21B22:**

The blocks B20, B21 and B22 have been amplified by PCR, purified and verified on agarose gel (data not shown). pUC19 was digested by *Sbf*I - *Xma*I and purified on gel. We proceeded to the In-Fusion reaction, transformation of Stellar cells, selection on ampicillin, and miniprep of clones. Unfortunately, this cloning repeatedly failed. So we decided to first clone B21 and B22 in the pUC19. The resulting colonies were checked by PCR (Figure 24).

![](https://static.igem.org/mediawiki/2020/6/6f/T--Toulouse_INSA-UPS--2020_fig24.png)

Figure 24: Agarose electrophoresis of PCR amplification verification of the B21 insertion in pUC19 using the In-Fusion kit. On the right, the expected results and on the left, the obtained gel. The two Crt - columns are negative controls.

To verify our cloning, we tested two different PCRs on our four clones: one is expected to provide a 4300 bp fragment and another one, a 300 bp fragment. We observed that 2 clones seemed to match our expectations. Both were sequenced and appeared to us as valid (or so we felt…). After following the same protocol to insert the last block B20, we obtained many colonies and tested the restriction profiles of six of them (figure 25).

![](https://static.igem.org/mediawiki/2020/c/c9/T--Toulouse_INSA-UPS--2020_fig25.png)

Figure 25: Agarose electrophoresis of the ampicilin selected clones after the insertion of B20 into pUC19-B21 digested by SbfI and XbaI. On the right, the expected results and on the left, the obtained gel.

The six columns contain different DNA plasmids of 6 different colonies that have been digested by *Sbf*I and *Xba*I. Among the 6 clones, only two presented the expected size (1300 bp). We sent them to be sequenced by Eurofins, and the sequence again appeared as valid to us…

* **Assembly of pUC19-B23B24:**

The blocks B23, and B24 have been amplified by PCR with CloneAmp HiFi PCR and then purified by NucleoSpin Gel and PCR Clean-up (data not shown). The pUC19 vector was digested by *EcoR*I and*Xma*I and purified on gel. We proceeded as usual to the In-Fusion reaction, transformation of Stellar cells, selection on ampicillin, and minipreps of a few, …well…, of the unique clone. Its plasmid was checked by several restriction profiling (figure 26).

![](https://static.igem.org/mediawiki/2020/d/d2/T--Toulouse_INSA-UPS--2020_fig26.png)

Figure 26: Agarose electrophoresis of the pUC19-B23B24 digested with SbfI and XmaI, XmaI and EcoRI, and EcoRI and EcoRV respectively on each lane. On the right, the expected results and on the left, the obtained gel.

Luckily enough (for a change!), this single clone got the right digestion profile so we sent it to be sequenced and it was fine.

* **Assembly of pUC19-CrtYB-CrtI:**

The next step was to integrate B20B21B22 into pUC19-B23B24 as a vector with a classical cloning method using restrictions enzymes and ligase. Plasmids purified using QIAGEN Plasmid Plus Mini Kit were double digested by *Sbf*I and *Xma*I. The vector was purified and insert B20B21B22 was ligated into linearized pUC19-B23B24 using the T4 DNA ligase from NEB. Selected transformant *E. coli* cells were recovered from solid selective media and the presence of the gene of interest was assessed using a colony PCR followed by a transformation into Stellar cells (ampicillin selection). The next day, we obtained hundreds of colonies so we chose six of them for a DNA extraction on the following day. To verify our colonies, we performed a PCR and a digestion. From the six clones tested by PCR, four were positive (data not shown). We tested two of these clones by a series of digestion to validate our cloning (Figure 27).

![](https://static.igem.org/mediawiki/2020/5/5f/T--Toulouse_INSA-UPS--2020_fig27.png)

Figure 27: Agarose electrophoresis of the pUC19-B23B24B20B21B22 digested with SbfI and EcoRI (D1), SbfI and XmaI (D2), and EcoRI and EcoRV (D3).

Our clones presented the expected digestion profile, which allowed us to proceed to the transformation step in the yeast that had previously integrated tHMG1-CrtE.

* Yeast transformation:

As we managed to boost the precursors of Vitamin A, the final step to make yeast produce β-carotene was to insert the CrtYB-CrtI construction.

We tried a first round of transformation in the previously obtained tHMG1-CrtE yeast strain. We expected orange-colored yeast but our clones were desperately white. At this step, we had another careful look at our sequencing results. Knowing that something was wrong, we identified a mutation at the very beginning of block B22. Unfortunately, this mutation triggered a shift in the reading frame, preventing the expression of CrtYB-CrtI.

Because of the COVID-19 regulations and the university courses that were starting, we did not have time to continue our experiments. Further experiments will be necessary to correct the mutation by site-directed mutagenesis in order to produce β-carotene.

### OVERALL CONCLUSION:

Time and COVID-19 have been our main foes this summer which prevented us from finishing some of our cloning. Moreover, we were over-optimistic about what could be accomplished by a team of eight students regarding the cloning work. But we can still conclude our experiences in a positive way. We were able to insert a part of our design intended for the production of β-carotene in yeast. This allowed us to show that it is possible to improve the production of GGPP in order to produce β-carotene. Unfortunately, due to a mutation, we could not allow the yeast to express β-carotene but we could find a way to correct this error in the future.

# Fermentation experiments for the coculture system

All the clonings we have tried to do aim to build a nutritious yeast that will be used as food supplement for the astronauts. We have designed a coculture between *Clostridium ljungdahlii* and *Saccharomyces cerevisiae* to produce this nutritious yeast from CO2 and H2.
 In this part of our results, we are presenting the experiments and the results to prove that a such coculture is feasible. These experiments can be broken down into three main areas. Firstly, the definition of a culture medium common to both microorganisms. We tested this medium on the yeast and on the bacteria individually in order to find out whether it is suitable for both. According to our model, coculture is feasible. However, we need to simulate the different possible solutions proposed by the model in the yeast and the bacteria individually. These experiments have allowed us to refine the model and collect parameters to size the coculture reactor. The latest experiments are the coculture experiments.

![](https://static.igem.org/mediawiki/2020/0/09/T--Toulouse_INSA-UPS--2020--DescriptionCoculture.jpg)

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