iGEM 2021 — Team Toulouse_INSA-UPS

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# Team:Toulouse INSA-UPS/Notebook

![](https://static.igem.org/mediawiki/2021/8/80/T--Toulouse_INSA-UPS--2021_banniere_derniere.svg)

[![](https://static.igem.org/mediawiki/2021/3/38/T--Toulouse_INSA-UPS--2021_ELIXIO_blanc.png) Toulouse INSA-UPS](https://2021.igem.org/Team%3AToulouse_INSA-UPS)

* [Home](https://2021.igem.org/Team%3AToulouse_INSA-UPS)
* Project

  [Description](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Description)
  [Design](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Design)
* Methods

  [Materials](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Materials)
  [Protocols](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Methods)
  [Safety](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Safety)
* [Parts](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Parts)
* Results

  [Modeling](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Model)
  [Results](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Results)
  [Engineering Success](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Engineering)
  [Proof of Concept](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Proof_Of_Concept)
  [Notebook](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Notebook)
* Entrepreneurship

  [Overview](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Entrepreneurship_Overview)
  [Implementation](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Implementation)
  [Supporting entrepreneurship](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Entrepreneurship)
  [Sustainability](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Sustainable)
* Human practices

  [Integrated
  Human Practices](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices)
  [Education
  and Communication](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Communication)
* Team

  [Team
  Members](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Team_members)
  [Attributions](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Attributions)
  [Collaborations](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Collaborations)
  [Partnership](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Partnership)
* Achievements

  [Contribution](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Contribution)
  [Achievements](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Achievements)
  [Promotion video](https://video.igem.org/w/dVKw4vWtmtzMZGVwHpKofg)
  [Presentation video](https://video.igem.org/w/gvAj6w8BKog22bfZVXvFiT)
* [🏆](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Awards)

## Notebook

---

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

December
January
February
March
April
May
June
July
August
September
October

December

Week 51
Week 52
Week 53

January

Week 1
Week 2
Week 3
Week 4

February

Week 5
Week 6
Week 7
Week 8

March

Week 9
Week 10
Week 11
Week 12
Week 13

April

Week 14
Week 15
Week 16
Week 17

May

Week 18
Week 19
Week 20
Week 21

June

Week 22
Week 23
Week 24
Week 25
Week 26

July

Week 27
Week 28
Week 29
Week 30

August

Week 31
Week 32
Week 33
Week 34

September

Week 35
Week 36
Week 37
Week 38
Week 39

October

Week 40
Week 41
Week 42

## Week of December 14th to 20th

### Project

This week marks the creation of the 2021 team. After the responses to our
applications we had the first meeting with our PI, in which a first distribution of
roles was proposed:

Maxence: Finance/Modelo/Wiki
Margaux: Entrepreneurship
Romane: Wiki/Modelo
Thomas: Experimentation
Romain: Human Practices
 Camille: Organisation/Communication

We started to get in touch with each other and presented the ideas of the topics we
had proposed in our applications.

## Week of December 21th to 27th

### Project

This week corresponded to the first week of the end-of-year vacations. We continued to
develop our application projects while trying to find other potential topics.

## Week of December 28th to 03rd

### Project

During the last week of the year we continued our research work on topics.

## Week of January 04th to 10th

### Project

First week of the year and first real meeting with all the supervisors. The final roles have
been assigned:

Maxence: Finance/Modelo
Margaux: Entrepreneurship
Romane: Wiki/Modelo
Thomas: Experimentation /Wiki
Romain: Human Practices
Camille: Organisation/Communication

## Week of January 11th to 17th

### Project

We continued the brainstorming effort and began holding more meetings per week.

![](https://static.igem.org/mediawiki/2021/6/63/T--Toulouse_INSA-UPS--2021_week51_test.jpeg)

## Week of January 18th to 24th

### Project

The brainstorming effort continued this week. This is the first week where we started making
slideshows to further present our topic ideas.

First meeting of the student team in real life, we strengthened our ties around board games!

![](https://static.igem.org/mediawiki/2021/0/0b/T--Toulouse_INSA-UPS--2021_week3_test.jpeg)

## Week of January 25th to 31th

### Project

This was the first week where a subject on the violet was discussed during a brainstorming
meeting. We also started to have meetings with scientists to discuss the topic.

### Team Building

We met the 2020 team where we had a lot of fun playing together!

![](https://static.igem.org/mediawiki/2021/f/f4/T--Toulouse_INSA-UPS--2021_week4.png)

## Week of February 01st to 07th

### Project

To optimize the brainstorming, and to refocus our research, we started to have mini-meetings
during the week with the supervisors by dividing up the topics.

## Week of February 08th to 14th

### Project

This is the first time that the creation of a sustainable and modular fragrance has been
discussed in a meeting.

### Team Building

Second meeting of the team in real, we played board games.

## Week of February 15th to 21th

### Project

At this point of the brainstorming we had only 2 potential topics left, one on azo-dyes
treatment and one on violet.
We came close to choose the violet topic but left the
azo-dye topic for one last week.

## Week of February 22th to 28th

### Project

We continued to dig into our 2 potential topics. Towards the end of the week we met with a
chemist for the azo-dyes topic and we concluded that it would be complicated to do.

## Week of March 01st to 07th

### Project

This week marks the final selection of our topic on violet.
We met for the first time with L'Occitane (a cosmetics company). This meeting helped us to
evolve our subject and to focus more on the agreements in perfumery rather than on a
perfume.
From this week on we were sure to participate in the competition this year.

### Team Building

We organized a meal to get together.

![](https://static.igem.org/mediawiki/2021/3/3b/T--Toulouse_INSA-UPS--2021_week9.jpeg)

## Week of March 08th to 14th

### Project

This week a new supervisor joined the team. Philippe Urban working on yeast whose team
provided us with our yeast strain. The INSA students spoke to us for the first time about a
friend of theirs who was interested in our subject and who could join us.

## Week of March 15th to 21th

### Project

This week was very important for the team as we welcomed a new member, Manon. She was
assigned to the communication department to help Camille.
The project has also evolved tremendously. During the weekly meeting we presented to the
supervisors the idea of using *S. elongatus* to co-culture with our yeast.

![](https://static.igem.org/mediawiki/2021/3/3d/T--Toulouse_INSA-UPS--2021_week11.jpeg)

## Week of March 22th to 28th

### Project

It’s during this week that we began to construct our design and our bricks, first with
schemes. Thomas helped us with Benchling to work on the design. We met for the first time
Denis Jallet, our future advisor, who confirmed our idea of working with cyanobacteria.

## Week of March 29th to 04th

### Project

Denis accepted to join our team as an advisor to help us with the cyanobacteria in our
project. We made our first GANTT to see if our project could be realisable in our
experimentation time.

## Week of April 05th to 11th

### Project

It’s during this week that for the first time we put our design on Benchling. We also focus
our search to talk with cyanobacteria experts and see what materials we would need.

## Week of April 12th to 18th

### Project

This week we met many specialists to help us with our design and advise us about the
materials for the cyanobacteria.
We started to search for the name of our project and our
logo!

![](https://static.igem.org/mediawiki/2021/1/15/T--Toulouse_INSA-UPS--2021_week15.jpeg)

## Week of April 19th to 25th

### Project

For the first time, we were able to meet our supervisors in real life to talk about the
design of our project. The composition of the team was modified with the departure of
Romain. This week we met our 3 advisors Amandine, Thibault and Younes!
We contacted for
the first time iGEM IISER Pune to establish a collaboration.
We worked on our 2-minutes
presentation video.

## Week of April 26th to 02nd

### Project

We presented the storyboard of our 2-minutes video to Eglantine, who was willing to help us
with the animation part.
Since we were ahead of schedule with the design and organization
of the project, the supervisors decided to suspend the weekly meetings and put the project
on hold for a while so that we could focus on our final exams.
This week, the project was
named Elixio!

## Week of May 03rd to 09th

### Project

We did not have a meeting this week.
Although the project was paused to allow us to
concentrate on our exams, some tasks were not stopped. We continued to work on the video
presentation and we had to order material for the beginning of the experiments.

## Week of May 10th to 16th

### Project

We did not have a meeting this week.
The effort on the project process continued.
Some
orders for the beginning of the experiments have started to arrive. Thomas and Camille went
to Ambre's laboratory to repel strains containing plasmids of interest.

![](https://static.igem.org/mediawiki/2021/f/f7/T--Toulouse_INSA-UPS--2021_week19.jpeg)

## Week of May 17th to 23th

### Project

The 2 members of the team coming from the university finished their year during this
week.
We started going to the lab to drop off equipment and to sort out what was left of
the 2020 team.

![](https://static.igem.org/mediawiki/2021/7/73/T--Toulouse_INSA-UPS--2021_week20.jpeg)

## Week of May 24th to 30th

### Project

After the break for exams we resumed our weekly meetings. INSA members have finished their
exams too.

### Collaboration

For the first time we had a meeting with iGEM Pune to meet them and see how we could work
together

![](https://static.igem.org/mediawiki/2021/9/97/T--Toulouse_INSA-UPS--2021_week21.png)

## Week of May 31th to 06th

### Project

This marks the beginning of our internship!

![](https://static.igem.org/mediawiki/2021/a/ad/T--Toulouse_INSA-UPS--2021_week22_fisrt.jpeg)

**pFLEUR:** Amplification of the first block HygR and Z3eV by PCR to open the
vector of integration pCfB3038 XII-1.

**pVIOLETTE:** Amplification of NsR, ofCCD1, Lcye and the vector of integration
pCfB3040 XII-4 by PCR . First In Fusion performed and failed. A second test was carried out
at the end of the week.

**pFRAMBOISE-fused:** Amplification of the first block NeoR and pTet and PCR to
open the vector of integration pCfB3034 X-3. First In Fusion performed.

**pCONCOMBRE:** amplification of blocks LOX and HPL by PCR. LacI amplification
by PCR colony on a cyanobacteria strain. The integration vector PCR failed due to a problem
in the design of the primers, new ones have been ordered.

![](https://static.igem.org/mediawiki/2021/e/ed/T--Toulouse_INSA-UPS--2021_week22av.png)

**Cyanobacteria cultures:** The first kinetics of cyanobacteria growth have been
done this week.

### Human Practices

Margaux and Camille made a podcast on the radio Campus FM to talk about our project and to
highlight biotechnologies.
Manon, Margaux and Camille participated to the Exposcience, an
exhibition allowing young students to present scientific projects. This allowed us to share
our project with the general public.

![](https://static.igem.org/mediawiki/2021/6/69/T--Toulouse_INSA-UPS--2021_week22radio.jpeg) ![](https://static.igem.org/mediawiki/2021/3/33/T--Toulouse_INSA-UPS--2021_week22expo.png)

### Safety training

Thomas, Manon, Maxence and Romane have been trained in the use of the autoclave.

### Sanitary precaution

In order to carry out our experiments in the best conditions, we decided to vaccinate the 6
of us as soon as possible. Maxence had already received his 2 doses of vaccine. This week
Manon and Margaux received their first and last dose of vaccine.

## Week of June 07th to 13th

### Project

**pFLEUR:** Second attempt of In Fusion by changing the quantities of the DNA
fragments, 2 clones obtained were sent for sequencing.

**pVIOLETTE:** Last In Fusion failed. Attempted a new strategy by performing
PCR overlaps to assemble the LcyE and ofCCD1 fragments, but that did not work.

**pFRAMBOISE-fused:** Amplification of the vector obtained with the first In
Fusion and attempt at a new In Fusion to finish the construction.

**pCONCOMBRE:** In Fusion analysis by PCR and enzymatic digestion showed that
the construct was assembled. Sequencing showed mutations in the construct.

![](https://static.igem.org/mediawiki/2021/c/cb/T--Toulouse_INSA-UPS--2021_week23av.png)

**Cyanobacteria cultures:** We continued to perform the culture tests on our
cyanobacteria.

### Safety training

We have received training in laboratory safety.

### Sanitary precaution

Romane had her first and last dose of vaccine and Thomas and Camille their first.

## Week of June 14th to 20th

### Project

**pFLEUR:** First attempt at In Fusion failed.

**pVIOLETTE:** Failure of the second attempt of In Fusion. Fragments for the In
Fusion were re-amplified and purified on gel. Another In Fusion was attempted.

**pFRAMBOISE-fused:** Success of the last In Fusion. Amplification of the other
fragments, RtTA and Crty, to continue the construction.

**pCONCOMBRE:** Amplification of the vector with the new primers. Attempt of an
In Fusion with the 3 fragments and the vector.

![](https://static.igem.org/mediawiki/2021/1/14/T--Toulouse_INSA-UPS--2021_week24av.png)

**Yeast Cre-Lox:** We tried to perform a cre-lox on our yeast strain to remove a
resistance.

**Cyanobacteria cultures:** We continued to perform the culture tests on our
cyanobacteria.

### Video

We started shooting footage for our 2-minutes presentation video.

![](https://static.igem.org/mediawiki/2021/0/00/T--Toulouse_INSA-UPS--2021_week24film.jpeg) ![](https://static.igem.org/mediawiki/2021/7/74/T--Toulouse_INSA-UPS--2021_week24film2.jpeg)

## Week of June 21th to 27th

### Project

**pFLEUR:** Sequencing showed that the construct was good for one strain. In
Fusion to perform the second part of the construct was attempted.

**pVIOLETTE:** New primers have been ordered to try a new strategy, an In
Fusion in 2 steps, to achieve the construction. The first In Fusion of the next strategy was
attempted.

**pFRAMBOISE-fused:** Analysis of clones obtained with the latest In
Fusion.

**pCONCOMBRE:** Further sequencing showed that the construct had no mutation
and was finished. The helper strain was constructed for triparental conjugation to introduce
the construct into our cyanobacteria strain.

![](https://static.igem.org/mediawiki/2021/f/f0/T--Toulouse_INSA-UPS--2021_week25av.png)

**Yeast Cre-Lox:** The first attempt at cre-lox failed. We also realized that
the glycerol stock we were working with was contaminated. So we re-stocked our strain to try
the cre-lox again.

**Cyanobacteria cultures:** The effort to control cyanobacteria cultures
continued.

### Video

We finished the shooting of our 2-minutes presentation video, only remaining to edit it.

![](https://static.igem.org/mediawiki/2021/6/69/T--Toulouse_INSA-UPS--2021_week25film.jpeg) ![](https://static.igem.org/mediawiki/2021/2/27/T--Toulouse_INSA-UPS--2021_week25film2.jpeg)

### Collaboration

A meeting between our dry lab and the one in Pune was held to see how we could help each
other.

We also participated in the first Phototroph meetup, by presenting our
subject.

![](https://static.igem.org/mediawiki/2021/7/77/T--Toulouse_INSA-UPS--2021_week25meetup2.png)

## Week of June 28th to 04th

### Project

**pFLEUR:** The only clone obtained with the last In Fusion had a mutation in
one of the fragments.

**pVIOLETTE:** The first In Fusion of the new strategy succeeded. The second
step, another In Fusion to integrate the last fragment into the construction, was
attempted.

**pFRAMBOISE-fused:** We ordered primers to sequence the construct.

**pCONCOMBRE:** Attempt to integrate pConcombre into the cyanobacteria.

![](https://static.igem.org/mediawiki/2021/1/10/T--Toulouse_INSA-UPS--2021_week26av.png)

**Yeast Cre-Lox:** We had problems with phleomycin. This antibiotic was normally
used to select the strains that have integrated the plasmid of interest allowing us to
perform the cre-lox. We had to reorder phleomycin.

**Cyanobacteria cultures:** After several weeks of cultivation we
started trying to measure the sucrose produced by our cyanobacteria.

### Human Practices

We tested the game in the team, with the students and the supervisors.

![](https://static.igem.org/mediawiki/2021/c/cd/T--Toulouse_INSA-UPS--2021_week26hp.jpeg)

## Week of July 05th to 11th

### Project

**pFLEUR:** A new sequencing was attempted on the strain obtained with the last
In Fusion.

**pVIOLETTE:** The last In Fusion failed.

**pFRAMBOISE-fused:** Clones were sent for sequencing.

**pCONCOMBRE:** The PCR colonies to see if there were integrations in the
cyanobacterial genome failed.

![](https://static.igem.org/mediawiki/2021/4/46/T--Toulouse_INSA-UPS--2021_week27av.png)

**Yeast Cre-Lox:** We received a new stock of phleomycin, a new cre-lox was
attempted.

**Cyanobacteria cultures:** We changed the pH of our culture medium to try to
increase the growth rate of the cyanobacteria.

### Video

We were able to see the first drafts of the editing of our 2 minutes video.

### Communication

We shared a press release in order to present our project with the public.

### Collaboration

We started the meetings to begin organizing the Symposium on Phototrophic Organisms with iGEM
Pune, HKU iGEM and iGEM Stony Brook.

![](https://static.igem.org/mediawiki/2021/0/09/T--Toulouse_INSA-UPS--2021_week27col.png)

### Human practices

We started programming the production of our game. We also considered the interventions we
could make to test it, in a high school or with visually impaired people.

## Week of July 12th to 18th

### Project

**pFLEUR:** Sequencing confirmed that the last In Fusion was successful, this
genetic construct was completed.

**pVIOLETTE:** Another In Fusion failed. Another strategy is attempted.

**pFRAMBOISE-fused:** The last sequencing was missing 20 bp. To be sure that
the construction was finished, new primers were ordered and a new sequencing was performed.

**pCONCOMBRE:** New strategies are being attempted to verify PCR integration,
including DNA extraction from cyanobacteria.

![](https://static.igem.org/mediawiki/2021/3/33/T--Toulouse_INSA-UPS--2021_week28av.png)

**Yeast Cre-Lox:** We still had problems with phleomycin, we tried to perform
some tests by adjusting the pH of the antibiotic.

**Cyanobacteria cultures:** We changed the pH of our culture medium to
try to increase the growth rate of the cyanobacteria.

![](https://static.igem.org/mediawiki/2021/d/db/T--Toulouse_INSA-UPS--2021_week28bio.jpeg)

### Communication

Journalists contacted us by phone, two articles about our project appeared in the press.

![](https://static.igem.org/mediawiki/2021/4/46/T--Toulouse_INSA-UPS--2021_week28com.png) ![](https://static.igem.org/mediawiki/2021/5/57/T--Toulouse_INSA-UPS--2021_week28com2.png)

### Collaboration

We had another meeting for the organisation of the Symposium in which we divided the tasks.

### Human practices

We found a scent rehabilitation program for people affected by covid or affected by
chemotherapy. We thought it would be interesting to contact someone in charge of this
program to learn more.

## Week of July 19th to 25th

### Project

**pVIOLETTE:** Fragment amplification for the new In Fusion strategy.

**pFRAMBOISE-fused:** The sequencing validated that the construction was finished.

**pCONCOMBRE:** Failure of the verification PCRs. Another method of DNA extraction
is attempted and new primers for PCRs are ordered.

**Yeast integration:** As the pFLEUR construction was finished we tried to
integrate it into the genome of *S. cerevisiae*.

![](https://static.igem.org/mediawiki/2021/2/2b/T--Toulouse_INSA-UPS--2021_week29av.png)

**Yeast Cre-Lox:** As we could not make our cre-lox with the phleomycin
resistant plasmid we ordered another plasmid to make it with the hygromycin resistance.

**Cyanobacteria cultures:** We detected sucrose in our culture medium
by HPLC.

### Communication

A journalist came to write a report on us at the laboratory.

![](https://static.igem.org/mediawiki/2021/3/37/T--Toulouse_INSA-UPS--2021_week29com.jpeg)

### Collaboration

Meeting between our dry lab and those of Pune to make an assessment of our partnership.
We
also had a meeting with all the French teams to discuss the organization of the French meet
up and we decided to do it online.

We participated to the second phototrophic meetup.
Denis and Amandine participated as
speakers.

![](https://static.igem.org/mediawiki/2021/e/e2/T--Toulouse_INSA-UPS--2021_week29col.png)

### Human practices

We have finished the cards in our deck.
Robertet, our sponsors, offered to provide us with scent vials for our game.

### Sponsor

We had a visit from Robertet, one of our sponsors. We gave them an update on our progress and
showed them where we were working.

![](https://static.igem.org/mediawiki/2021/7/76/T--Toulouse_INSA-UPS--2021_week29spo.jpeg)

## Week of July 26th to 01st

### Project

**pVIOLETTE:** We received new gblocks to perform a new In Fusion strategy.
The In Fusion with the new strategy failed.

**pFRAMBOISE-notfused:** Preparation of the fragments to build by In Fusion the
backup construction, pFRAMBOISE-notfused

**pCONCOMBRE:** A method to extract DNA from cyanobacteria was finally successful
but other primers had to be ordered for genotyping.

**Yeast integration:** The pFLEUR integration failed and we had a problem with the
selection marker of this integration (hygromycin).

![](https://static.igem.org/mediawiki/2021/0/0b/T--Toulouse_INSA-UPS--2021_week30av.png)

**Yeast Cre-Lox:** Failure of the last tentative.

**Cyanobacteria cultures:** We were able to improve the saturation OD of our
cultures with a new BG-11 culture medium.

As we were having problems working with our yeast strain we met Hélène Martin-Yken who gave
us a lot of advices and a new strain of *S. cerevisiae*.

### Collaboration

We had another meeting to talk about the organisation of the Symposium.

### Human practices

We finished writing the rules of our game.

## Week of August 02nd to 08th

### Project

**pVIOLETTE:** The same In Fusion as last week was retried, after verification by
sequencing we finally finished this construction!

**pFRAMBOISE-notfused:** The construction was sent to sequencing.

**pCONCOMBRE:** The PCRs finally worked, and the construct seemed to be integrated
into the genome of our cyanobacterium but new primers have been ordered to confirm it.

**Yeast integration:** We tried a new protocol to attempt to integrate our
constructs in our yeast, without success.

![](https://static.igem.org/mediawiki/2021/f/f8/T--Toulouse_INSA-UPS--2021_week31av.png)

**Yeast Cre-Lox:** After several failures of the Cre-Lox, we realized that the
selection with neomycin, to see whether or not the Cre-Lox worked, was not the right one and
that we should use G418 instead.

**Cyanobacteria cultures:** We managed again to increase the OD of our
cultures.

## Week of August 09th to 15th

### Project

**pFRAMBOISE-notfused:** The sequencing results showed that the construction was
globally successful, one area was not sequenced. New primers were ordered to cover the
unsequenced area.

**pCONCOMBRE:** The new primers did not remove the doubt about the integration, new
primers were ordered.

**Yeast integration:** We tried again to integrate pViolette and pFleur in our
yeast strain without success.

![](https://static.igem.org/mediawiki/2021/f/f8/T--Toulouse_INSA-UPS--2021_week31av.png)

**Yeast Cre-Lox:** We still had selection problems with antibiotics and we
performed more Cre-Lox trials.

**Cyanobacteria cultures:** We had a better growth rates in our cultures.

### Collaboration

We participated in the Symposium allowing us to present our project and to attend workshops.

![](https://static.igem.org/mediawiki/2021/9/90/T--Toulouse_INSA-UPS--2021_collab_symposium.png)

## Week of August 16th to 22th

### Project

**pFRAMBOISE-notfused:** The sequencing showed that the construction was correct
and finished.

**pCONCOMBRE:** With the new primers one arm of the integration still could not be
amplified. As all fragments were integrated and the strain was well resistant to the antibiotic
selection, we considered that the integration was successful.

**Yeast integration:** We tried again to integrate our constructions in the yeast
without success.

![](https://static.igem.org/mediawiki/2021/c/cd/T--Toulouse_INSA-UPS--2021_week32av.png)

## Week of August 23th to 29th

### Project

It was Manon's last week in the lab with us.

**Yeast integration:** We tried a new protocol to integrate one of our
constructions in our yeast.

![](https://static.igem.org/mediawiki/2021/c/cd/T--Toulouse_INSA-UPS--2021_week32av.png)

**Cyanobacteria cultures:** We measured the sucrose secretion with our new
lights, but it was still not optimal.

**Co-culture:** We have performed our first co-culture tests. We grew our
strain with yeast in the co-culture medium.

### Video

We showed the team presentation video to our supervisors for the first time.

We went
to Grasse for the weekend, the capital of perfume. We visited the company of Robertet, one
of our sponsors. We took advantage of the city heritage to start shooting shots for our
20-minutes presentation video.

![](https://static.igem.org/mediawiki/2021/c/cc/T--Toulouse_INSA-UPS--2021_week34gra.jpeg) ![](https://static.igem.org/mediawiki/2021/8/81/T--Toulouse_INSA-UPS--2021_week34gra2.jpeg)

### Team building

As we were close to Canne we took the opportunity to go and eat in a restaurant by the
sea.

![](https://static.igem.org/mediawiki/2021/8/81/T--Toulouse_INSA-UPS--2021_week34can.jpeg)

## Week of August 30th to 05th

### Project

**Yeast integration:** After weeks of trials with the new protocol, we finally
succeeded in integrating two of our constructions, pVIOLETTE and pFLEUR, into two yeast
strains.

![](https://static.igem.org/mediawiki/2021/d/d8/T--Toulouse_INSA-UPS--2021_week35.png)

**Cyanobacteria cultures:** We continued to try to improve the sucrose secretion
of our strains.

**Co-culture:** The yeasts grew well in the co-culture medium. We wondered how
we would count our 2 organisms when we put them together in the culture.

### Video

At the beginning of the week we came back to Toulouse after our weekend at Grasse.

![](https://static.igem.org/mediawiki/2021/9/93/T--Toulouse_INSA-UPS--2021_week35gra.jpeg)

## Week of September 06th to 12th

### Project

This week Thomas, Camille and Manon were back to school.
Maxence, Margaux and Romane
continued the experiments on all 3 of them.
The priority was to induce the production of
our molecules and to try to detect them. We had for the first time access to the
GC-MS.

**Yeast integration:** After successfully integrating pVIOLETTE we then
successfully integrated pFLEUR into the same yeast strain. We also succeeded to integrate in
another strain pFRAMBOISE-notfused

![](https://static.igem.org/mediawiki/2021/6/67/T--Toulouse_INSA-UPS--2021_week36av.png)

**Cyanobacteria cultures:** The effort continued to improve sucrose secretion.

### Collaboration

Thomas and Camille presented during the French meetup that we partly organized with all the
French teams.

![](https://static.igem.org/mediawiki/2021/e/e4/T--Toulouse_INSA-UPS--2021_week36col.png)

### Human Practices

We received the pieces of our game.

![](https://static.igem.org/mediawiki/2021/d/d4/T--Toulouse_INSA-UPS--2021_week36col2.jpeg)

## Week of September 13th to 19th

### Project

**Yeast integration:** The last transformation of the summer was done to build our
final yeast strain by integrating pFRAMBOISE-fused.

**Detection:** We were finally able to detect the β-ionone produced by a yeast
strain carrying the pFRAMBOISE-notfused construction. We were also able to detect carotene which
are degradation compounds of lycopene and precursors of our ionones.

![](https://static.igem.org/mediawiki/2021/4/45/T--Toulouse_INSA-UPS--2021_week37.png)

**Cyanobacteria cultures:** We were able to detect an increased secretion of
sucrose.

### Video

We filmed more shots of our video in the lab.

![](https://static.igem.org/mediawiki/2021/c/cb/T--Toulouse_INSA-UPS--2021_week37vid.jpeg)

### Human Practices

We were able to produce our game board! Thomas himself used the laser printer to produce
them.

![](https://static.igem.org/mediawiki/2021/e/e2/T--Toulouse_INSA-UPS--2021_week37hp.jpeg)

## Week of September 20th to 26th

### Project

That was the last week of experiments of the project.

**Yeast integration:** After our 4 months of experimentation and in the last
week of our internship we finally succeeded to produce our final yeast strain with the 3
integrations!

**Detection:** This week we were able to detect the 2 others ionones of our
project, the ɑ-ionone and the dihydro-β-ionone.

![](https://static.igem.org/mediawiki/2021/7/70/T--Toulouse_INSA-UPS--2021_week38av.png)

**Cyanobacteria cultures:** We have done our last cyanobacteria cultures.

### Video

We continued to film shots of our 20-minutes video presentation.

![](https://static.igem.org/mediawiki/2021/2/21/T--Toulouse_INSA-UPS--2021_week38vid.jpeg)

## Week of September 27th to 03rd

### Project

As the experiments were finished we could focus on the valorization of our results. We all
got together to do a fake wiki freeze in order to make progress on the wiki writing.

 ![](https://static.igem.org/mediawiki/2021/6/69/T--Toulouse_INSA-UPS--2021_week39wik.jpeg) ![](https://static.igem.org/mediawiki/2021/a/a0/T--Toulouse_INSA-UPS--2021_week39wik2.jpeg)

### Human practices

We had the opportunity to test our game in an association for visually impaired people. This
allowed us to get feedback on the game and to see how we could adapt it so that they could
play it.

 ![](https://static.igem.org/mediawiki/2021/6/65/T--Toulouse_INSA-UPS--2021_week39hp.jpeg)

### Communication

We went to present our project and for the first time our results to the CBI.

![](https://static.igem.org/mediawiki/2021/3/3d/T--Toulouse_INSA-UPS--2021_week39com.jpeg)

## Week of October 04th to 10th

### Project

We made a second fake Wiki freeze in order to continue to progress on our
website

### Video

We continued shooting for our 20 minutes video presentation.

![](https://static.igem.org/mediawiki/2021/2/25/T--Toulouse_INSA-UPS--2021_week40.jpeg)

### Human practices

We tested our game with some friends. They gave us very good feedback on our
game.

![](https://static.igem.org/mediawiki/2021/d/dd/T--Toulouse_INSA-UPS--2021_week40_hp.jpeg)

## Week of October 11th to 17th

### Project

We made a third fake Wiki freeze in order to definitely finish our website!

![](https://static.igem.org/mediawiki/2021/1/1d/T--Toulouse_INSA-UPS--2021_week41_wiki.jpeg)

### Human practices

We went to a high school class to talk to them about synthetic biology and our project and to
let them test our board game.

![](https://static.igem.org/mediawiki/2021/b/b8/T--Toulouse_INSA-UPS--2021_manon_presentation_highschool.jpg)

## Week of October 18th to 24th

### Project

After weeks of work, we finally succeeded in finishing our website !

Thank you for reading this notebook to the end. As a reward, here is one of our best
photos taken in the lab during summer!

![](https://static.igem.org/mediawiki/2021/5/54/T--Toulouse_INSA-UPS--2021_week42elephants.jpeg)

Content

Content

[Go
to Implementation ![](https://static.igem.org/mediawiki/2021/b/b5/T--Toulouse_INSA-UPS--2021_fleche_next.jpg)](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Implementation)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Toulouse INSA-UPS/Engineering

![](https://static.igem.org/mediawiki/2021/8/80/T--Toulouse_INSA-UPS--2021_banniere_derniere.svg)

[![](https://static.igem.org/mediawiki/2021/3/38/T--Toulouse_INSA-UPS--2021_ELIXIO_blanc.png) Toulouse INSA-UPS](https://2021.igem.org/Team%3AToulouse_INSA-UPS)

* [Home](https://2021.igem.org/Team%3AToulouse_INSA-UPS)
* Project

  [Description](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Description)
  [Design](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Design)
* Methods

  [Materials](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Materials)
  [Protocols](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Methods)
  [Safety](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Safety)
* [Parts](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Parts)
* Results

  [Modeling](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Model)
  [Results](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Results)
  [Engineering Success](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Engineering)
  [Proof of Concept](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Proof_Of_Concept)
  [Notebook](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Notebook)
* Entrepreneurship

  [Overview](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Entrepreneurship_Overview)
  [Implementation](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Implementation)
  [Supporting entrepreneurship](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Entrepreneurship)
  [Sustainability](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Sustainable)
* Human practices

  [Integrated
  Human Practices](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices)
  [Education
  and Communication](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Communication)
* Team

  [Team
  Members](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Team_members)
  [Attributions](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Attributions)
  [Collaborations](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Collaborations)
  [Partnership](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Partnership)
* Achievements

  [Contribution](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Contribution)
  [Achievements](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Achievements)
  [Promotion video](https://video.igem.org/w/dVKw4vWtmtzMZGVwHpKofg)
  [Presentation video](https://video.igem.org/w/gvAj6w8BKog22bfZVXvFiT)
* [🏆](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Awards)

**Engineering success**
[Overview](#a1)
[Cycle 1: Designing pVIOLETTE and first cloning attempts](#a2)
[Cycle 2: Two steps cloning strategy](#a3)
[Cycle 3: Ordering a new gene fragment](#a4)
[Cycle 4: Construction of the LycoYeast-VIOLETTE strain](#a5)
[Conclusion](#a6)

# Overview

---

**Synthetic biology** is often described as the design and construction of new biological
elements, devices, and systems, as well as the redesign of existing natural biological
systems for application purposes (Nature, 2021). As in any other engineering field,
synthetic biologists follow iterations of the **DBTL cycle (Design, Build, Test, Learn)**
to
successfully develop innovative biological systems (Figure 1).

![](https://static.igem.org/mediawiki/2021/2/25/T--Toulouse_INSA-UPS--2021_cyclesynbio.png)

***Figure 1: Iterative design-build-test-learn (DBTL)
cycle in Synthetic Biology (Waldby et al. 2018).***

In the framework of our project ELIXIO, we were able to **design** various genetic
constructs,
successfully **assemble** them, **insert them into their final chassis** and finally
**demonstrate the
production of three of the main molecules** of the violet scent.

While these results may seem very positive for a team of only six students and in a time
frame as short as four months, this engineering success was not always as straight-forward
as presented in our [Results](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Results) section.

To illustrate this, we have chosen to detail here the example of our **pVIOLETTE**
construction
for the production of **α-ionone**, which proved to be a significant challenge at
various
levels. In the end, **four iterations** of the DBTL cycle had to be performed before
reaching
the final proof of concept.

# Cycle 1: Designing pVIOLETTE and first cloning attempts

---

### Design

α-ionone is produced in two steps from lycopene, it is first converted to
ε-carotene (lycopene ε-cyclase activity) which is transformed into
α-ionone (carotenoid cleavage dioxygenase activity). A previous study showed that for
the first step, LcyE from *Latuca sativa* and for the second step, the CCD1 from
*Osmanthus fragrans* (OfCCD1) could be used to produce α-ionone (Chen et al.
2019).
When researching the literature on the production of α-ionone, it was found that the
second
step was rate limiting (Chen et al. 2019; Werner et al. 2019). The main hypothesis to
explain that is the poor accessibility of the OfCCD1 enzyme, which is cytoplasmic, to its
substrate which usually is trapped inside the membrane. To address this, OfCCD1 was fused to
the membrane-bound LcyE in a previous study in *E. coli* to facilitate substrate
channeling. This strategy allowed a >2.5-fold enhancement of α-ionone in *E.
coli*
(Chen et al. 2019). Inspired by this work, we chose to design the same fusion enzyme and
codon-optimize it for expression in *S. cerevisiae* to see if this strategy could also
work in our yeast (Figure 2).

![](https://static.igem.org/mediawiki/2021/6/61/T--Toulouse_INSA-UPS--2021_pviolette.png)

***Figure 2: Final design of pVIOLETTE.***

### Build

The different gene fragments were ordered from IDT and the cloning vector was provided to us
by [Sara Castaño Cerezo](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#a3). There were three different gene fragments
to assemble in the
vector:

* Nourseothricin resistance gene expression cassette (NsrR hereafter)
* PGAL1 promoter, LcyE gene and half of the LGS linker (LcyE hereafter)
* Other half of the LGS linker, OfCCD1 gene and TVPS13 terminator
  (OfCCD1 hereafter)

The different fragments as well as the cloning vector were amplified by PCR. It was first
tried to assemble the construction in one step through InFusion cloning. Even if the
positive cloning control worked, no clone was ever obtained on this cloning. After
trying several times and changing experimenters, it became clear that this strategy
would probably not work.

### Learn

Our hypothesis was that attempting to clone three inserts into a vector in one step was
perhaps a bit bold.

# Cycle 2: Two steps cloning strategy

---

### Design

Because of these results, it was decided to attempt to clone pVIOLETTE in two steps. In the
first step, the two inserts OfCCD1 and NsrR would be assembled in the vector (this
intermediate plasmid was named pVIOLET). In a second step, the last LcyE insert would be
inserted to finally have the final construct pVIOLETTE (Figure 3).

![](https://static.igem.org/mediawiki/2021/d/d3/T--Toulouse_INSA-UPS--2021_cycle2_eng_sucess.png)

***Figure 3: Two steps cloning strategy for
pVIOLETTE.***

### Build

New primers were ordered, and the different fragments were again amplified by PCR. The
intermediate construct pVIOLET was successfully assembled and verified by performing a
restriction profile and by sequencing.

For the cloning next step, however, the various tests never resulted in positive clones.
Because of this observation, it appeared that an inherent feature of the LcyE block sequence
made cloning impossible.

### Learn

We analyzed the sequence of the fragment and realized that the LGS linker had a very high GC
content compared to the rest of the sequence. According to the Takara Infusion kit handbook,
a GC-rich homology zone is less likely to work for InFusion cloning.

# Cycle 3: Ordering a new gene fragment to move the homology zone out of the linker

---

### Design

Based on this observation, it appeared that our whole cloning strategy had to be changed
again. A new 500 bp fragment (NewLinker) flanking the linker was designed together with new
LcyE (NewLcyE) and OfCCD1 (NewOfCCD1) fragments (Figure 4). The previous pVIOLET
intermediate plasmid was used as template for an inverse PCR with the aim to obtain the
linear vector with the NsrR cassette (new vector named pVIO).

![](https://static.igem.org/mediawiki/2021/0/0f/T--Toulouse_INSA-UPS--2021_cycle3_eng_succ.png)

***Figure 4: New cloning strategy with the final
homology regions out of the linker.***

### Build

Then an InFusion was made with the three insert NewLcyE, NewLinker and NewOfCCD1 with the
linear vector pVIO. With this strategy, the homology regions were not in the GC-rich linker
anymore. This times, many clones were obtained after incubation of the cloning plates.
After, screening the clones with colony PCR, performing restricting digestion profiles and
sequencing the positive clones, it appeared that this strategy was successful.

The next step was to integrate this vector in the genome of our parental yeast strain
LycoYeast. We tried three different yeast transformation protocols, tried changing
experimenters, but were never able to obtain yeast mutants.

### Learn

After discussion with yeast experts, it appeared that to obtain chromosomal integration of
our construct into the S. cerevisiae genome, a much larger amount of DNA had to be put in to
hope to obtain mutants. Even though in the yeast transformation protocols we used, the
amounts of DNA used for transformation rarely exceed 1 μg, they told us to use as much as
possible to increase our chances.

# Cycle 4: Construction of the LycoYeast-VIOLETTE strain

---

### Build

With this new information in mind, we tried transforming LycoYeast by adding 5μg of linear
pVIOLETTE in the transformation mix. This times, many clones were obtained and the correct
integration was checked by PCR. This new cycle of engineering therefore allowed us to
finally obtain our LycoYeast-VIOLETTE strain.

### Test

The next step was to finally test our strain. To do see, we cultivated it in a galactose
containing medium (YPGal) which is supposed to induce the expression of our fusion enzyme.
We noticed that in the induced cultures, a color change was observed compared to the
parental LycoYeast. This was expected since lycopene is red while carotenes are orange. We
were also delighted to find that a characteristic ionone sweet smell could be noticed!

To confirm these observations, a rigorous methodology of measurement was applied with the
development and application of analytical methods of carotenes in HPLC and ionones in GC-MS
(Figure 5).

![](https://static.igem.org/mediawiki/2021/a/ac/T--Toulouse_INSA-UPS--2021_eng_succ_alphaio.png)

***Figure 5: Strain characterization of
LycoYeast-VIOLETTE.** Upon galactose-induction, lycopene is converted to
ε-carotene (HPLC data on the left) which is then converted to α-ionone
(GC-MS data on the right).*

### Learn

These results allow us to conclude that the enzymatic fusion that we have designed [BBa\_K3930024](http://parts.igem.org/Part%3ABBa_K3930024) is
functional in *S. cerevisiae*, which could serve as an avenue to improve the production
of α-ionone by biotechnological means in this organism.

# Conclusion

---

We approached this project with the same optimism and big ideas that all iGEMers have. Our
instructors repeatedly warned us that most experiments are doomed to fail. Engineering a
biological system is indeed a complex, challenging, and often slow process. Here we have
described, using the example of α-ionone, how by performing successive iterations of
the DBTL cycle we were able to create a violet-scented yeast (Figure 6).

![](https://static.igem.org/mediawiki/2021/e/ea/T--Toulouse_INSA-UPS--2021_eng_success_conclusion.png)

***Figure 6: Summary of the different engineering
steps completed before reaching the final proof of concept for the production of
α-ionone.** D=Design, B=Build, T=Test, L=Learn.*

# References

---

Chen X, Shukal S, Zhang C. 2019. Integrating Enzyme and Metabolic
Engineering Tools for Enhanced α-Ionone Production. J Agric Food Chem. 67(49):13451–13459.
doi:10.1021/acs.jafc.9b00860.

Waldby C, Gray P, Griffiths P, Vickers C, Meek S, Small I. 2018. Synthetic Biology in
Australia: an outlook to 2030. Australian Council of Learned Academies Expert Working Group
Horizon Scanning Project.

Werner N, Ramirez-Sarmiento CA, Agosin E. 2019. Protein engineering of carotenoid cleavage
dioxygenases to optimize β-ionone biosynthesis in yeast cell factories. Food Chemistry.
299:125089. doi:10.1016/j.foodchem.2019.125089.

[Go to Proof of concept ![](https://static.igem.org/mediawiki/2021/b/b5/T--Toulouse_INSA-UPS--2021_fleche_next.jpg)](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Proof_Of_Concept)

![PF](https://static.igem.org/mediawiki/2021/e/ed/T--Toulouse_INSA-UPS--2021_flechequimonte.png)

*The whole **Toulouse INSA-UPS team** would like to thank our sponsors. Especially:

[![Robertet](https://static.igem.org/mediawiki/2021/e/e1/T--Toulouse_INSA-UPS--2021_logo_Robertet.png)](https://www.robertet.com/)
[![TBI](https://static.igem.org/mediawiki/2021/a/ac/T--Toulouse_INSA-UPS--2021_TBI.png)](http://www.toulouse-biotechnology-institute.fr/en/index.html)
[![TWB](https://static.igem.org/mediawiki/2021/f/fc/T--Toulouse_INSA-UPS--2021_TWB.jpg)](https://www.toulouse-white-biotechnology.com/en/)
[![Adisseo](https://static.igem.org/mediawiki/2021/4/46/T--Toulouse_INSA-UPS--2021_Addiseo.jpg)](https://www.adisseo.com/)
[![CBI](https://static.igem.org/mediawiki/2021/e/e3/T--Toulouse_INSA-UPS--2021_CBI.png)](https://cbi-toulouse.fr/eng/)
[![Fondation INSA](https://static.igem.org/mediawiki/2021/f/ff/T--Toulouse_INSA-UPS--2021_INSA.png)](http://fondation.insa-toulouse.fr/fr/index.html)
[![FSI](https://static.igem.org/mediawiki/2021/c/c3/T--Toulouse_INSA-UPS--2021_FSI.jpg)](https://www.fsi.univ-tlse3.fr/la-fsi)
[![CRITT Bio industries](https://static.igem.org/mediawiki/2021/f/ff/T--Toulouse_INSA-UPS--2021_CRITT.jpg)](https://www.bioindustries.net/)*

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Toulouse INSA-UPS/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Toulouse INSA-UPS/Results

![](https://static.igem.org/mediawiki/2021/8/80/T--Toulouse_INSA-UPS--2021_banniere_derniere.svg)

[![](https://static.igem.org/mediawiki/2021/3/38/T--Toulouse_INSA-UPS--2021_ELIXIO_blanc.png) Toulouse INSA-UPS](https://2021.igem.org/Team%3AToulouse_INSA-UPS)

* [Home](https://2021.igem.org/Team%3AToulouse_INSA-UPS)
* Project

  [Description](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Description)
  [Design](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Design)
* Methods

  [Materials](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Materials)
  [Protocols](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Methods)
  [Safety](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Safety)
* [Parts](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Parts)
* Results

  [Modeling](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Model)
  [Results](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Results)
  [Engineering Success](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Engineering)
  [Proof of Concept](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Proof_Of_Concept)
  [Notebook](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Notebook)
* Entrepreneurship

  [Overview](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Entrepreneurship_Overview)
  [Implementation](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Implementation)
  [Supporting entrepreneurship](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Entrepreneurship)
  [Sustainability](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Sustainable)
* Human practices

  [Integrated
  Human Practices](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices)
  [Education
  and Communication](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Communication)
* Team

  [Team
  Members](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Team_members)
  [Attributions](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Attributions)
  [Collaborations](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Collaborations)
  [Partnership](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Partnership)
* Achievements

  [Contribution](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Contribution)
  [Achievements](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Achievements)
  [Promotion video](https://video.igem.org/w/dVKw4vWtmtzMZGVwHpKofg)
  [Presentation video](https://video.igem.org/w/gvAj6w8BKog22bfZVXvFiT)
* [🏆](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Awards)

**Results**
[Cultivation and co-culture](#a1)

* [Yeast](#c1)
* [Cyanobacteria](#c2)
* [Co-culture](#c3)

[Cloning](#a2)

* [Yeast](#b1)
* [Cyanobacteria](#b2)

[Strain construction](#a3)

* [Yeast](#d1)
* [Cyanobacteria](#d2)

[Results production](#a4)

* [Color change](#e1)
* [Carotenoid](#e2)
* [Molecules of interest](#e3)
* [Enzymatic activity](#e4)

[Conclusion](#a5)

# Overview

---

In this section, we present all the experimental works performed during our summer. We start
here with the cultivation and co-culture set up which have been done in parallel to the
cloning efforts, integration in yeast and analytical analysis, presented thereafter.

# Cultivation and co-culture

---

This part describes our efforts to characterize and optimize the growth of our two
microorganisms and the preliminary work to set-up a co-culture. It is divided into a yeast
growth, cyanobacterium growth and co-culture sub-parts.

### Yeast growth on sucrose

The first step towards the synthetic microbial consortium we have designed was to study and
characterize the growth of our parent yeast strain LycoYeast on sucrose.

##### Growth of LycoYeast in the co-culture medium using exogenous sucrose

We first wanted to see if we could grow LycoYeast in a modified version of the cyanobacteria
medium (BG11) and by adding exogenous sucrose as a carbon source. Different co-culture media
formulations are described in the literature to grow cyanobacteria and yeast together (Hays
et al. 2017; Li et al. 2017). We used BG11 media supplemented with 1.2 g/L yeast nitrogen
base without amino acids (YNB w/o aa), and 100 mM NaCl (denoted BG-11[CoY]). We added 5 g/L
of exogenous sucrose, a concentration that should be reachable by our sucrose-secreting
cyanobacterium strains (Lin et al. 2020). The growth was followed by measuring the OD at 600
nm. The results are presented in Figure 1.

![](https://static.igem.org/mediawiki/2021/f/fc/T--Toulouse_INSA-UPS--2021_fig1_results.png)

***Figure 1: Growth of LycoYeast in BG-11[CoY] supplemented with 5 g/L exogenous
sucrose.***

After a long lag phase, it appears that our yeast is able to grow, although quite slowly, in
the co-culture medium with sucrose as its sole carbon source which is encouraging for the
continuation.

##### Determination of the maximal sucrose uptake rate of LycoYeast

To calibrate our **dynamic co-culture model**, we then chose to measure the maximum uptake
rate of sucrose in our LycoYeast strain. It has indeed been shown that sucrose consumption
by *S. cerevisiae* is a key point for the stabilization of the consortium with *S.
elongatus* (Hays et al. 2017). The value of this parameter has also been shown to be
highly strain-dependent (Rodrigues et al. 2021). This approach furthermore allowed us to
test whether our model could predict the growth rate of *S. cerevisiae on sucrose*.

To do so, *S. cerevisae* was grown in YP+Sucrose medium which contained 10 g/L of
sucrose. The biomass concentration was quantified over time by measuring the optical density
(OD) at 600 nm and converted to gDCW/L using a previously established correlation
factor of
0.53gDW/ODunit (Sonderegger and Sauer 2003). At every OD measurement, a sample of
culture
medium was also taken and analyzed after centrifugation by HPLC to quantify the sucrose
concentration. Time course concentrations of biomass and glucose were then fitted using
PhysioFit (Peiro et al. 2019) to estimate the maximum sucrose uptake rate of our yeast. The
results obtained for the best fit are presented in Figure 2.

![](https://static.igem.org/mediawiki/2021/f/fc/T--Toulouse_INSA-UPS--2021_fig2_results.png)

***Figure 2: Raw and fitted data of the growth of
LycoYeast using sucrose as a carbon source.** The biomass concentration
(left) is expressed in gDCW/L while the sucrose concentration (right) is in mM. The
time unit is in hour.*

We obtained the following values (confidence intervals at 95%):
**μmax=0.33 ± 0.02 h-1**
**qsucrose,max=-3.0 ± 0.3 mmol.gDCW-1.h-1**
The values we previously used for our model were those determined by Rodrigues et al. (2021)
for *S. cerevisiae* CEN.PK113-7D who had measured a qsucrose,max of **-8.2 ± 0.4
mmol.gDCW-1.h-1**. Our experimental sucrose uptake
value was used to
constrain our yeast genome scale model and predict the growth rate in this condition. The
predicted value (**0.44 h-1**) was in agreement with the experimental growth
rate
(**0.33 h-1**) measured in our condition. Updating this parameter has
significantly
improved the predictive capabilities of our model (predicted growth rate of 1.3
h-1 with the initial uptake rate taken from the literature vs 0.44 h-1
with the updated parameter). This demonstrates the value of this experiment to calibrate our
model and improves its predictive power.

### Cyanobacteria growth and sucrose secretion

##### Growth

This was the first time that an iGEM Toulouse team worked with cyanobacteria. We did not have
any equipment specifically dedicated to this and had to set up everything, which was done
step by step. Throughout the summer, we conducted liquid cultures of cyanobacteria with the
objective of attempting to reproduce the rapid growth phenotype of the Syn UTEX 2973 strain
as well as the high sucrose secretion of the strains provided to us (Yu et al. 2015; Lin et
al. 2020). This goal proved to be much more difficult than expected, we most often obtained
growth profiles that were linear rather than exponential, and with growth rates several
orders of magnitude lower than expected. Numerous tests were performed in an attempt to
optimize the culture parameters of these cyanobacteria. The results of these different
approaches are presented below.

**Culture configuration & CO2 transfer**

The cultures were routinely grown in small cell culture flasks which have the advantage of
providing optically thin cultures with good light penetration as well as avoiding
contamination. Since the growth was not very satisfying in these conditions (Figure 3),
different cultures in erlen flasks of different volumes and different configurations were
tested (with or without baffles, different types of lids). These modifications did not
improve the growth of our strains. The cultures at that time were carried out with a light
intensity of approximately 250 μmoles photon.m-2.s-1 and at
atmospheric CO2 (0.04%). In these conditions, Yu et al. (2015) obtained a
doubling time of
6.3 hours which is way below our estimated doubling time (which was around 29 hours). Our
idea was then to carry out cultures in bioreactors, in which we could control the pH during
the culture and enrich in CO2. Using 5% CO2, and a light intensity of
850 μmoles
photon.m-2.s-1, we were able to improve the growth rate relatively
with a maximum doubling time of 10 hours but it was still extremely low compared to what
would be expected under these conditions (doubling time of between 1.5 hours to 2 hours for
Yu et al. (2015) and Ungerer et al. (2018)). As it can be seen in Figure 3, the saturation
besides occur at an OD750nm of approximately 1 which is very low. After
discussing with experts met at the [Phototrophs Meetups](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Communication#a7), it appeared that this was not
normal and that something else was limiting the growth since *Syn UTEX 2973* usually
easily reach OD750nm 10 to 20.

![](https://static.igem.org/mediawiki/2021/f/f0/T--Toulouse_INSA-UPS--2021_fig3_results.png)

***Figure 3: Presentation of the different cultivation
configurations tested with examples of results** A) Cell culture flasks
cultures of 20 mL (Temperature 38°C, Light 250 μmoles
photon.m-2.s-1, CO2 (0,04%) B) Culture in erlen flasks
(Temperature 38°C, Light 250 μmoles photon.m-2.s-1, CO2
0,04%)
C) Bioreactor culture (Temperature 38°C, Light 850 μmoles
photon.m-2.s-1, CO2 5 %)*

**BG11 medium composition & pH of the cultures**

Our first hypothesis was that the composition of the BG11 medium we were using at that time
was suboptimal. We had bought this medium in the form of a ready-made powder sold by
Phytotech and did not really have any information about its composition. This lack of
standardization of the composition of the BG11 medium was a subject of discussion at the
[Phototrophs Meetups](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Communication#a7) and also concerned the regulation of the pH in the medium since
it appeared for example that our commercial medium was not buffered.

We therefore decided to try to prepare this medium ourselves. Since iGEM Marburg 2019 had
proposed an optimized version named BGM medium for this strain, we followed their protocol.
While with this medium the saturation phase occurred at a higher OD750nm
(approximately 5 rather than 1 in the bioreactor), the growth was still very slow.

![](https://static.igem.org/mediawiki/2021/f/f6/T--Toulouse_INSA-UPS--2021_fig4_results.png)

***Figure 4: Growth of WT Syn UTEX 2973 in BGM medium (Temperature 38°C, Light
250 μmoles photon.m-2.s-1, CO2 0,04%)***

**Light quantity and quality**

After discussing this with  [Dr.
Julie Zedler](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Attributions#a5), we came to question the quality of the
light spectrum provided to our cultures. The spectrum of the LED lights we were using
(Aquabeauty 70W) seemed indeed to be very blue (Figure 5). This type of light is not optimal
because unlike plants, cyanobacteria have in addition to chlorophyll (which absorbs in the
blue) phycocyanins that absorb in the red (Ungerer et al. 2018). So, we decided to get a new
LED panel (Greenception GC4) with a spectrum more adapted to our growths (we chose the
reference provided by the iGEM Marburg 2019 team with which they were able to obtain a fast
growth phenotype). The results obtained with these new lamps were again disappointing, not
significantly improving the growths.

![](https://static.igem.org/mediawiki/2021/b/b5/T--Toulouse_INSA-UPS--fig5_light_spectrum.png)

***Figure 5: Comparison of the emitted light spectrum of the first lights we
were using (A) and the second ones that were purchased (C) with the absorption
spectra of our cyanobacterium strain Syn UTEX 2973 (B). The black arrow
indicates the absorption peak related to phycobiliproteins, in red-orange for
(B).***

Overall, we were not able to find the growth rate-limiting element of our strains in the time
available, despite all the tests conducted. These difficulties highlight the need for
standardization in these fields and justify the interest of the Phototroph Guide to which we
contributed. These autotrophic chassis are very promising for the future of industrial
biotechnologies, but it can be difficult to work with them without specific equipment for
the culture of phototrophic microorganisms.

##### Sucrose secretion

Consistent with what was described above, we were unable to reproduce the high sucrose
productions obtained by Lin et al. (2020) with the strains we used. This seems logical in
view of the differences in growth rates already detailed. Sucrose could still be produced
and detected in Syn UTEX 2973 CscB and CscB SPS SPP strains. At the same time as the
different tests to optimize growth, these mutants were also grown under the different
conditions and sucrose production induced. The highest production was obtained with the CscB
strain for which a titer of 1,36 g/L in 7 days (BGM medium with the new LED panels at light
intensity 650 μmoles photon.m-2.s-1). For the CscB SPS SPP strain,
the
highest titer was 300 mg/L obtained after 8 days in the same conditions. In both cases, it
remains too low considering these strains had reported productivities
of 1.9 g.L−1day−1 for the CscB strain and 1.4
g.L−1day−1 for the CscB SPS SPP one (Lin et al. 2020).

![](https://static.igem.org/mediawiki/2021/2/28/T--Toulouse_INSA-UPS--2021_sucrose_secretion.png)

***Figure 6: Sucrose calibration curve (left) and example of HPLC analysis
result (right).** Both the WT and CscB strains were cultivated for 7 days
in BG11 with 150 mM NaCl and 1 mM IPTG. The titer of sucrose was determined to be
1.36 g/L which corresponds to the highest production that we obtained. The peak at
7.2 minutes most likely is sucrose-6-phosphate which is the precursors of sucrose.*

### Tips

The transporter CscB is a sucrose/H+ symporter which implies that sucrose export is
dependent upon culture pH. A pH of a least 7.8 is necessary to promote sucrose secretion
(Ducat et al. 2012).

### Co-culture

We demonstrated that the yeast was able to grow on sucrose in the co-culture medium. Our
cyanobacteria strains, although very slowly, produce sucrose. The next step was to try to
put the two organisms together and see if the sucrose produced by the cyanobacteria was
sufficient to keep the yeast alive and growing.

To do this, we inoculated the BG-11[CoY] medium with the CscB strain by adding 1 mM IPTG to
induce sucrose production. As with previous cultures, sucrose concentrations were extremely
low, in the order of 100 mg/L after 4 days. We were not very hopeful that our yeast could
grow with such low amounts of sugar. However, we still inoculated our LycoYeast strain,
which had previously been grown in the sucrose-enriched co-culture medium, at a very low
density (OD600nm=0.03). The same procedure was applied with a culture of WT
*Syn UTEX 2973* as a control. The co-culture temperature was set at 35°C. Serial
dilutions of these co-culture mixes were plated on YPD petri dishes. The plates were
incubated 2 days at 30°C without light and the yeast colonies were counted.

No significant yeast growth could be observed in our experimental setup. Still, we observed
that the yeast remained alive for at least 3 days, which shows that the two organisms can
cohabit in the same compartment. This attempt has allowed the development of the protocols
necessary for the establishment of the co-culture, which is a critical step forward. We are
hopeful that we could establish co-culture, provided we could reproduce the sucrose
productivity described by Lin et al. (2020), thus requiring us to identify the limiting
parameter in our cyanobacteria cultures.

## Conclusion

We showed both yeast and cyanobacterium strains to be compatible with growth in BG11[CoY]
medium. The yeast strain is able to grow from sucrose on this medium. However, the
sucrose production by the cyanobacterium and its growth remains to be optimized. From
our preliminary results, well-controlled bioreactor growth seems the best solution.

# Cloning

---

In this section, we will present you how we created the three plasmids to produce violet
scent molecules in the yeast (pFLEUR, pFRAMBOISE and pVIOLETTE), and the plasmid to add a
green leaf scent to the cyanobacterium (pCONCOMBRE).

In order to produce our molecules of interest, cloning was classically performed in two
steps:

* Amplification of the desired fragments (from IDT and Twist Bioscience blocks,
  available plasmids or genome sequences) by PCR.
* Assembly of the PCR amplified fragments and vectors by InFusion. All our routine
  protocols can be found in the [Protocol page](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Methods). The list of all the primers used for
  PCR is on our [Material page.](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Materials)

## ***Saccharomyces cerevisiae***

#### pFLEUR

pFLEUR (Fig.7) is a pCfB3038 XII-1 based vector (EasyClone-MarkerFree kit) designed to carry:

* the DBR1 and LIS genes, for the production of linalool and dihydro-β-ionone.
* the inducible promoters Z3eV and PCUP1, driving the expression of DBR1 and LIS
* the resistance marker HygR to select yeast integrants

![](https://static.igem.org/mediawiki/2021/9/9f/T--Toulouse_INSA-UPS--2021_pfleur.png)

***Figure 7: pFLEUR plasmid for production of linalool and
dihydro-β-ionone.** Two integrative sites flank the construction for
integration into the yeast chromosome XII locus 1.*

##### Cloning strategy

Fragments and vectors were amplified by PCR using the high fidelity Q5 polymerase and further
assembled by In-Fusion.
*According to our advisors and the Takara kit handbook advice, we separated the cloning
in two steps for an optimum of efficacy:*

* First, the Z3eV and HygR fragments were inserted into the pCfB3038 XII-1 vector,
  generating the intermediate construct pFLE.
* Second, DBR1 and LIS fragments were inserted in pFLE linearized by inverse PCR,
  resulting in the final pFLEUR construct.

##### Vectors construction

* **pFLE:**

  Expected size of the amplicons:

  |  |  |
  | --- | --- |
  | **Name of the amplicon** | **Expected size (pb)** |
  | Z3eV | 2065 |
  | HygR | 1860 |
  | pCfB3038 XII-1 | 3991 |

  #### [Click here for detailed results](#collapse1)

  + Z3eV and HygR fragments were PCR amplified from IDT gblock
    while pCfB3038
    XII-1 was linearized by PCR from Addgene template plasmid.
    Amplification
    products size was checked on EtBr stained agarose gel.
    Amplicons were
    further purified on gel when other amplification products
    were detected.

    **Figure 8abc shows amplification products
    matching with
    expected sizes.**
  + Z3eV and HygR fragments were inserted into pCfB3038 XII-1 by
    InFusion.
    Transformants were selected on ampicillin plates.
  + 14 transformants were screened by colony PCR with primer
    pairs flanking
    the inserted fragments (F Z3eV + R Z3eV and F HygR + P
    reverse screen
    pFleur).

    **2 positive transformants (clones 3 and 13) were
    detected.**
  + Plasmids were extracted from clones 3 and 13 and digested by
    restriction
    enzymes (SfoI and ScaI) to check the correctness of the
    assembly.

    **Figure 8d shows correct restriction maps for
    clones 3 and
    13.**
  + Plasmids from clones 3 and 13 were sent to Eurofins Genomics
    to check the
    inserted sequences. A missense mutation was detected in the
    DBR1 coding
    sequence for clone 3, while a minor point mutation was
    detected in the
    HygR terminator sequence.

  **pFLE from clone 13 was then chosen to
  further insert
  DBR1 and LIS fragments.**

  ![](https://static.igem.org/mediawiki/2021/3/3c/T--Toulouse_INSA-UPS--2021_fig8_results.png)

  ***Figure 8: pFLE assembly.** PCR amplicon sizes of HygR (A),
  Z3eV (B)
  and pCfB3038 XII-1 (C) and the restriction profile of pFle (D) were
  checked with
  agarose electrophoresis gel and revealed with EtBr. A theoretical gel is
  presented
  on the right of each gel and the NEB 1 kb DNA ladder is on the right or
  the left of
  each of them (note that a different ladder is presented on the
  theoretical
  gel).*

* **pFLEUR:**

  Expected size of the amplicons:

  |  |  |
  | --- | --- |
  | **Name of the amplicon** | **Expected size (pb)** |
  | pFle | 7916 |
  | LIS | 2512 |
  | DBR1 | 1396 |

  #### [Click here for detailed results](#collapse2)

  + LIS and DBR1 fragments were PCR amplified from IDT gblock while
    pFLE was
    linearized by inverse PCR on pFLE Miniprep. Amplification
    products size
    was checked on EtBr stained agarose gel. Amplicons were further
    purified
    on gel when other amplification products were detected.

    **Figure 9ab shows amplification products matching
    with
    expected sizes.**
  + LIS and DBR1 fragments were inserted into pFLE by InFusion.
    Transformants
    were selected on ampicillin plates.
  + 7 transformants were screened by colony PCR with primer pairs
    flanking
    the LIS block (F LIS+ R LIS).

    **1 positive transformant (clone 7) was
    detected.**
  + Plasmid was extracted from clone 7 and digested by restriction
    enzymes
    (ScaI and SalI) to check the correctness of the assembly.

    **Figure 9 shows correct restriction maps for clones
    7.**
  + Plasmid from clone 7 was sent to Eurofins Genomics to check the
    inserted
    sequence. No mutation was detected for clone 7.

  **We managed the assembly of pFLEUR with the
  plasmid
  coming from clone 7. The pFLEUR construction for production of linalool
  and
  dihydro-β-ionone is ready to be integrated in the yeast
  genome**

  ![](https://static.igem.org/mediawiki/2021/6/6d/T--Toulouse_INSA-UPS--2021_figure9_results2.png)

  ***Figure 9: pFLEUR assembly.** PCR amplicon sizes of LIS, DBR1
  (A) and
  pFLE linearised (B) and pFLEUR restriction profile (D) were checked with
  agarose
  electrophoresis gel and revealed with EtBr. A theoretical gel is presented
  on the
  right of each gel and the NEB 1 kb DNA ladder is on the right or the left of
  each of
  them (note that a different ladder is presented on the theoretical
  gel).*

#### pFRAMBOISE

pFRAMBOISE-fused (Fig.10) is a pCfB3034 X-3 based vector (EasyClone-MarkerFree kit) designed to
carry:

* the CrtY-phCCD1 genes fusion with a LGS linker, responsible for the production of both
  β-carotene and β-ionone.
* the inducible promoters Teto7, driving the expression of CrtY-phCCD1.
* the resistance marker NeoR to select for yeast integrants.

A backup plan, pFRAMBOISE-notfused, was also designed. In this case, CrtY is under the
control of the Teto7 promoter, and the phCCD1 with a fynAnchor is constitutively expressed
under a TEF1 promoter.

###### pFRAMBOISE-fused

![](https://static.igem.org/mediawiki/2021/0/0b/T--Toulouse_INSA-UPS--2021_pframbBbon.png)

###### pFRAMBOISE-notfused

![](https://static.igem.org/mediawiki/2021/b/ba/T--Toulouse_INSA-UPS--2021_pframbAbon.png)

***Figure 10: pFRAMBOISE-fused and pFRAMBOISE-notfused plasmids for production
of
β-carotene and β-ionone.**Two integrative sites flank the two
constructions for integration in the chromosome X locus 3.*

##### Cloning strategy

The cloning strategy is the same as pFLEUR:

* First, the Teto7 promoter together with the NeoR fragment were inserted in the
  pCfB3034 X-3 vector, generating the intermediate construct pFRAMB.
* Second, CrtY-phCCD1 and rtTA fragments were assembled with pFRAMB linearised by
  inverse PCR, resulting in the final pFRAMBOISE construct.

For pFRAMBOISE-notfused, CrtY and fynAnchor-phCCD1 fragments were assembled with pFRAMBOI
linearized by inverse PCR from pFRAMBOISE-fused, resulting in the final pFRAMBOISE-notfused
construct.

##### Vectors construction

* **pFRAMB:**

  Expected size of the amplicons:

  |  |  |
  | --- | --- |
  | **Name of the amplicon** | **Expected size (pb)** |
  | Promoter Teto7 | 1008 |
  | NeoR | 1293 |
  | pCfB3034 X-3 | 4031 |

  #### [Click here for detailed results](#collapse3)

  + Promoter Teto7 was PCR amplified from the addgene plasmid #165976
    and the
    NeoR fragment was PCR amplified from IDT gblock, while pCfB3034
    X-3 was
    linearized by inverse PCR on Addgene template plasmid.
    Amplification
    products size was checked on EtBr stained agarose gel. Amplicons
    were
    further purified on gel when other amplification products were
    detected.

    **Figure 11abc shows amplification products matching
    with
    expected sizes. For Promoter Teto7, the right strip was cut
    for a
    gel purification.**
  + Promoter Teto7 and NeoR fragments were inserted into pCfB3034 X-3
    by
    InFusion. Transformants were selected on ampicillin plates.
  + 9 transformants were screened by colony PCR with primer pairs on
    the
    promoter Teto7 and the NeoR fragments (F pTet + R pTet and
    FVCNeoR + R
    NeoR ).

    **6 positive transformants (clones 4A, 4B, 4C, 4E, 5A
    and 5C)
    were detected**
  + Plasmids were extracted from the 6 clones and digested by
    restriction
    enzyme (EcoRV) to check the correctness of the assembly.

    **Figure 11d shows correct restriction maps for clone
    4E.**
  + Plasmid from clone 4E was sent to Eurofins Genomics to check the
    insert
    sequences. No mutation was detected in this clone.

  **pFRAMB from clone 4E was then chosen to
  further
  insert CrtY-phCCD1 and rtTA fragments.**

  ![](https://static.igem.org/mediawiki/2021/3/33/T--Toulouse_INSA-UPS--2021_fig11_results.png)

  ***Figure 11: pFRAMB assembly.** PCR amplicon sizes of NeoR(A),
  Promoter
  Teto7(B) and pCfB3034 X-3 linearized (C) and pFRAMB restriction profile (D)
  were
  checked with agarose electrophoresis gel and revealed with EtBr. A
  theoretical gel
  is presented on the right of each gel and the NEB 1 kb DNA ladder is on the
  right or
  the left of each of them (note that a different ladder is presented on the
  theoretical gel).*

* **pFRAMBOISE-fused:**

  Expected size of the amplicons:

  |  |  |
  | --- | --- |
  | **Name of the amplicon** | **Expected size (pb)** |
  | CrtY-phCCD1 | 2862 |
  | rtTA | 1565 |
  | pFramb | 6332 |

  #### [Click here for detailed results](#collapse4)

  + CrtY-phCCD1 and rtTA fragments were PCR amplified from IDT gblock
    while
    pFramb was linearized by inverse PCR on pFRAMB Miniprep.
    Amplification
    products size was checked on EtBr stained agarose gel. Amplicons
    were
    further purified on gel when other amplification products were
    detected.

    **Figure 12abc shows amplification products matching
    with
    expected sizes.**
  + CrtY-phCCD1 and rtTA fragments were inserted into pFRAMB by
    InFusion.
    Transformants were selected on ampicillin plates.
  + 12 transformants were screened by colony PCR with primer pairs on
    the
    CrtY-phCCD1 and rtTA fragments (F CrtY-phCCD1+ R CrtY-phCCD1 and
    F rtTA+
    R rtTA ).

    **1 positive transformant (clone C) was
    detected.**
  + Plasmid was extracted from clone C and digested by restriction
    enzymes
    (ScaI and SalI) to check the correctness of the assembly.

    **Figure 12d shows correct restriction maps for clones
    C.**
  + Plasmid from clone C was sent to Eurofins Genomics to check the
    insert
    sequence. No mutation was detected for clone C.

  **We managed the assembly of pFRAMBOISE-fused
  with the
  plasmid coming from clone C. The pFRAMBOISE-fused construction for
  production of
  β-carotene and β-ionone is ready to be integrated in the yeast
  genome**

  ![](https://static.igem.org/mediawiki/2021/e/e0/T--Toulouse_INSA-UPS--2021_fig12_results_pframboiseassembly.png)

  ***Figure 12: pFRAMBOISE-fused assembly.** PCR amplicon sizes of
  CrtY-phCCD1
  (A), rtTA (B) and pFramb linearized (C) and pFRAMBOISE-fused restriction
  profile (D) were
  checked with agarose electrophoresis gel and revealed with EtBr. A
  theoretical gel
  is presented on the right of each gel and the NEB 1 kb DNA ladder is on the
  right or
  the left of each of them (note that a different ladder is presented on the
  theoretical gel).*

* **pFramboise-notfused:**

  Expected size of the amplicons:

  |  |  |
  | --- | --- |
  | **Name of the amplicon** | **Expected size (pb)** |
  | fyn-phCCD1 | 1795 |
  | CrtY | 1796 |
  | pFramboi | 7795 |

  #### [Click here for detailed results](#collapse5)

  + CrtY and fyn-phCCD1 fragments were PCR amplified from Twist
    Bioscience
    gblock while pFRAMBOI was linearized by inverse PCR on
    pFRAMBOISE-fused
    Miniprep. Amplification products size was checked on EtBr
    stained
    agarose gel. Amplicons were further purified on gel when other
    amplification products were detected.

    **Figure 13ab shows amplification products matching
    with
    expected sizes.**
  + CrtY and phCCD1 fragments were inserted into pFRAMBOI by
    InFusion.
    Transformants were selected on ampicillin plates.
  + 5 transformants were screened by colony PCR with primer pairs on
    the CrtY
    and rtTA fragments (F CrtY+ R CrtY and F fyn-phCCD1+ R
    fyn-phCCD1 ).

    **2 positive transformants (clones 1 and 3) were
    detected.**
  + Plasmids were extracted from the two clones and digested by
    restriction
    enzymes (NotI and SalI) to check the correctness of the
    assembly.

    **Figure 13c shows correct restriction maps for clone
    3.**
  + Plasmid from clone C was sent to Eurofins Genomics to check the
    insert
    sequences. No mutation was detected for clone 3.

  **We managed the assembly of
  pFRAMBOISE-notfused with
  the plasmid coming from clone 3. The pFRAMBOISE-notfused construction
  for
  production of β-carotene and β-ionone is ready to be
  integrated in the
  yeast genome**

  ![](https://static.igem.org/mediawiki/2021/0/07/T--Toulouse_INSA-UPS--2021_fig13_results_pframboisenotfusedassembly.png)

  ***Figure 13: pFRAMBOISE-notfused assembly.** PCR amplicon sizes
  of CrtY,
  fyn-phCCD1 (A),and pFRAMBOIi linearized (B) and pFRAMBOISE-notfused
  restriction
  profile (C) were checked with agarose electrophoresis gel and revealed with
  EtBr. A
  theoretical gel is presented on the right of each gel and the NEB 1 kb DNA
  ladder is
  on the right or the left of each of them (note that a different ladder is
  presented
  on the theoretical gel).*

#### pVIOLETTE

pVIOLETTE (Fig.14) is a pCfB3040 XII-4 based vector (EasyClone-MarkerFree kit) designed to
carry:

* the LcyE gene fused to ofCCD1 with a LGS linker, for the production of both
  ε-carotene
  and α-ionone
* the inducible promoters Gal 1, driving the expression of LcyE-phCCD1
* the resistance marker NsrR to select for yeast integrants

![](https://static.igem.org/mediawiki/2021/8/82/T--Toulouse_INSA-UPS--2021_pvioletteresults.png)

***Figure 14: pVIOLETTE plasmid for production of ε-carotene and
α-ionone.** Two integrative sites flank the construction for
integration in the chromosome XII locus 4.*

##### Cloning strategy

The first cloning strategy was to directly integrate the 3 fragments LcyE, ofCCD1 and NsrR
into pCfB3040 XII-4. Unfortunately, no positive transformants were selected.
We then considered a new cloning strategy:

* First, the ofCCD1 fragment was inserted together with the NsrR fragment in the
  pCfB3040 XII-4 vector, generating the intermediate construct pVIOLET.
* Second, LcyE was assembled with pVIOLET linearised by inverse PCR, resulting in the
  final pVIOLETTE construct.

###### Vectors construction

* **pVIOLET:**

  Expected size of the amplicons:

  |  |  |
  | --- | --- |
  | **Name of the amplicon** | **Expected size (pb)** |
  | ofCCD1 | 2101 |
  | NsrR | 1196 |
  | pCfB3040 XII-4 | 3856 |

  #### [Click here for detailed results](#collapse6)

  + NsrR and ofCCD1 fragments were PCR amplified from IDT gblocks and
    pCfB3040 XII-4 was linearized by inverse PCR. Amplification
    products
    size was checked on EtBr stained agarose gel. Amplicons were
    further
    purified on gel when other amplification products were detected.

    **Figure 15abc shows amplification products matching
    with
    expected sizes.**
  + NsrR and ofCCD1 fragments were inserted into pCfB3040 XII-4 by
    InFusion.
    Transformants were selected on ampicillin plates.
  + 10 transformants were screened by colony PCR with primer pairs
    flanking
    the integration of the fragments ofCCD1 and NsrR (P seq LcyE
    CCD1 (1) +
    P reverse screen LcyE CCD1).

    **3 positive transformants (clones 8, 21 and 22) were
    detected.**
  + Plasmids were extracted from the 3 clones and digested by
    restriction
    enzymes (SacI + EcoRV) to check the correctness of the assembly.

    **Figure 15d shows correct restriction maps for clone
    8.**
  + Plasmid from clone 8 was sent to Eurofins Genomics to check the
    insert
    sequences. No mutation was detected in this clone.

  **pVIOLET from clone 8 was then chosen to
  further
  insert the LcyE fragment.**

  ![](https://static.igem.org/mediawiki/2021/7/76/T--Toulouse_INSA-UPS--2021_fig15_results_pvioletassembly.png)

  ***Figure 15: pViolet assembly.** PCR amplicon sizes of ofCCD1
  (A), NsrR
  (B),and pCfB3040 XII-4 linearized (C) and pVIOLET restriction profile (D)
  were
  checked with agarose electrophoresis gel and revealed with EtBr. A
  theoretical gel
  is presented on the right of each gel and the NEB 1 kb DNA ladder is on the
  right or
  the left of each of them (note that a different ladder is presented on the
  theoretical gel).*

* **pVIOLETTE:**

  After many unsuccessful attempts to integrate LcyE, we decided to change the homology
  region
  for InFusion. A 500 bp fragment (NewLinker) flanking the linker was designed
  together with
  new LcyE (NewLcyE) and ofCCD1 (NewofCCD1) fragments for a 3-fragment InFusion
  cloning with
  the pVio vector (pCfB3040+NsrR) from the inverse PCR of pViolet.
  *According to Takara kit handbook advice and our experiments, a GC-rich homology
  zone is
  less likely to work for inFusion cloning. We therefore advise future iGEM teams
  to have
  homology zones for InFusion that flank such sequences*

  Expected size of the amplicons:

  |  |  |
  | --- | --- |
  | **Name of the amplicon** | **Expected size (pb)** |
  | NewLcyE | 1735 |
  | NewofCCD1 | 1886 |
  | NewLinker | 498 |
  | pVIO | 7951 |

  #### [Click here for detailed results](#collapse7)

  + NewLcyE, NewofCCD1 and NewLinker fragments were PCR amplified
    from IDT
    and Twist Bioscience gblocks and pVIO was linearized by inverse
    PCR from
    pVIOLET Miniprep. Amplification products size was checked on
    EtBr
    stained agarose gel. Amplicons were further purified on gel when
    other
    amplification products were detected.

    **Figure 16abc shows amplification products matching
    with
    expected sizes.**
  + NewLcyE, NewofCCD1 and NewLinker fragments were inserted into
    pVIO by
    InFusion. Transformants were selected on ampicillin plates.
  + 12 transformants were screened by colony PCR with primer pairs
    flanking
    the integration of the fragments NewofCCD1 and NewLcyE (P seq
    LcyE CCD1
    (1) + R NsrR).

    **4 positive transformants (clones 1,2,3 and 4) were
    detected.**
  + Plasmids were extracted from the 4 clones and digested by
    restriction
    enzymes (HindIII + XbaI) to check the correctness of the
    assembly.

    **Figure 16d shows correct restriction maps for clone
    3.**
  + Plasmid from clones 1,2,3 and 4 were sent to Eurofins Genomics to
    check
    the insert sequences. No mutation was detected in clone 3,
    mutations in
    junk DNA were present for clones 1 and 2, and a 5 codons
    deletion in the
    linker sequence was detected for clone 4.

  **We managed the assembly of pVIOLETTE with
  the
  plasmid coming from clone 3. The pVIOLETTE construction for production
  of
  ε-carotene and α-ionone is ready to be integrated in the
  yeast
  genome.**

  ![](https://static.igem.org/mediawiki/2021/0/04/T--Toulouse_INSA-UPS--2021_fig16_results_pvioletteassembly.png)

  ***Figure 16: pViolette assembly.** PCR amplicon sizes of
  NewLcyE,
  NofCCD1 (A), NewLinker (B),and pVio linearized (C) and pVIOLETTE restriction
  profile
  (D) were checked with agarose electrophoresis gel and revealed with EtBr. A
  theoretical gel is presented on the right of each gel and the NEB 1 kb DNA
  ladder is
  on the right or the left of each of them (note that a different ladder is
  presented
  on the theoretical gel).*

## ***Synechococcus elongatus***

#### pCONCOMBRE

pCONCOMBRE (Fig.17) is a pAM4951 based vector designed to carry:

* the Nb-9-LOX, Cm-9-HPL and LacI genes, for the production of 3,6-nonadienal and LacI
  repressor
* the inducible promoters Trc-theoE-riboswitch, driving the expression of Nb-9-LOX and
  Cm-9-HPL
* the resistance marker SpecR to select for Cyanobacteria integrants

![](https://static.igem.org/mediawiki/2021/6/61/T--Toulouse_INSA-UPS--2021_pcomcombre.png)

***Figure 17: pCONCOMBRE plasmid design for production of
2,6-nonadienal and 2,6-nonadienol.** Two integrative sites flank the
construction for an
integration in the NSI locus.*

##### Cloning strategy

The cloning strategy is the same as for the LycoYeast plasmids, but the fragments Nb-9-LOX,
Cm-9-HPL and LacI were integrated into the pAM4951 vector in only one step.

##### Vectors construction

Expected size of the amplicons:

|  |  |
| --- | --- |
| **Name of the amplicon** | **Expected size (pb)** |
| Nb-9-LOX | 2796 |
| Cm-9-HPL | 1646 |
| LacI | 1276 |
| pAM4951 | 4021 |

#### [Click here for detailed results](#collapse8)

* Nb-9-LOX, Cm-9-HPL fragments were PCR amplified from IDT gblocks, LacI
  fragment was PCR amplified from the genome of *Syn UTEX 2973 CscB*
  strain and
  pAM4951 was linearized by inverse PCR. Amplification products size was
  checked on EtBr stained agarose gel. Amplicons were further purified on
  gel when other amplification products were detected.

  **Figure 18abc shows amplification products matching with
  expected sizes.**

   *One run of PCR colony on Synechococcus elongatus genome to
  obtain an amplified fragment is not enough, we had to purify on gel
  our LacI amplicons for 10 more cycles of PCR*
* Nb-9-LOX, Cm-9-HPL and LacI fragments were inserted into pAM4951 by
  InFusion. Transformants were selected on spectinomycin plates.
* 14 transformants were screened by colony PCR with primer pairs on the
  fragments LacI, Nb-9-LOX and Cm-9-HPL (F LacI + R LacI, F LOX + R LOX
  and F HPL and R HPL).

  **3 positive transformants (clones A2, A5 and B6) were
  detected.**
* Plasmids were extracted from the 3 clones and digested by restriction
  enzymes (EcoRI + EcoRV) to check the correctness of the assembly.

  **Figure 18d shows correct restriction maps for clone
  A5.**
* The plasmid from clone A5 was sent to Eurofins Genomics to check the
  insert sequences. No mutation was detected.

**We managed the assembly of pCONCOMBRE with the
plasmid coming from clone A5. The pCONCOMBRE construction for production of
2,6-nonadienal and 2,6-nonadienol is ready to be integrated in the
cyanobacterium genome.**

![](https://static.igem.org/mediawiki/2021/5/54/T--Toulouse_INSA-UPS--2021_fig18_results_pcomcombreassembly.png)

***Figure 18: pCONCOMBRE assembly.** PCR amplicon sizes of HPL, LOX (A),
LacI (B),and pAM4951 linearized (C) and pCONCOMBRE restriction profile (D) were
checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel
is presented on the right of each gel and the NEB 1 kb DNA ladder is on the right or
the left of each of them (note that a different ladder is presented on the
theoretical gel).*

## Conclusion

Even if some cloning required design modifications, we successfully built all of our
constructions to engineer the yeast and the cyanobacterium.

# Strain construction

---

Here we present our efforts to adapt our yeast chassis and then to introduce our
constructions in the yeast and in the cyanobacterium genomes.

### Yeast genetic engineering

##### Generate a kanMX - LycoYeast using Cre-Lox system

Integration of both pFRAMBOISE vectors in the LycoYeast genome relies on the *neoR* gene
coding for kanamycin/neomycin/G418 resistance. Unluckily, our LycoYeast is a modified yeast
strain containing several resistance markers, including the *kanMX* gene coding for
kanamycin/neomycin/G418 resistance. This *kanMX* gene is flanked by two facing loxP
sequences allowing its removal by the action of the Cre recombinase.

![](https://static.igem.org/mediawiki/2021/1/1c/T--Toulouse_INSA-UPS--2021_fig19_results.png)

***Figure 19: The loxP/Cre gene disruption and marker rescue procedure.***

Cre-Lox recombination is a site specific recombination system used to carry out genetic
modifications. The protein Cre recombinase recognizes 34 bp *loxP* sites and allows
rearrangement of the in-between genetic material. Here, we used the Cre-loxP to remove the
kanMX gene from the LycoYeast strain.

To do so, we transformed our yeast with the plasmid pSH65 which contains:

* the *cre* gene coding for the Cre recombinase, under the control of a
  galactose inducible promoter.
* a phleomycin resistance marker.

Transformants were selected on phleomycin (20 μg.mL-1) plates and further grown on
YP+Galactose medium in order to induce *cre* expression and promote *kanMX*
excision.

### Tips

It is important to know that phleomycin loses its activity at acidic pH. Thus,
the pH of the medium has to be adjusted to 8.

After 12 h of growth in presence of galactose, transformants were plated on phleomycin
plates and growing colonies were replica-plated on three media (Figure 20):

* YPDA (all clones should form colonies).
* YPDA with phleomycin 20 μg.mL-1 pH 8 (only clones containing the pSH65 plasmid
  should grow).
* YPDA with neomycin 250 μg.mL-1 (only the colonies for which the
  Cre-*loxP*
  has not worked should grow, i.e., *kanMX*+ clones)

![](https://static.igem.org/mediawiki/2021/b/b5/T--Toulouse_INSA-UPS--2021_results_fig20-yeast.png)

***Figure 20: Left: phleomycin medium. Center: no antibiotic medium. Right:
neomycin medium.** In each plate, 6 colonies from the clone B and 6
colonies from the clone D are grown.*

Unexpectedly, all colonies grew on the plate containing neomycin. We initially thought
that we had prepared the neomycin plates incorrectly. So, we conducted a test for
neomycin selectivity with three different strains: the *kanMX*+ LycoYeast and two
other strains, BY4741 and CEN-PK2-1C, that were *kanMX*-. In addition to neomycin,
we conducted the same test with YPDA medium containing G418 (500 μg.mL-1), a neomycin
analog.

![](https://static.igem.org/mediawiki/2021/c/cf/T--Toulouse_INSA-UPS--2021_fig21_yeast_results.jpg)

***Figure 21: Left: G418 dish. Right: neomycin dish.** On the G418
dish, only the resistant LycoYeast strain grows. On the contrary, on the
neomycin dish, all strains grow.*

*kanMX*+ and *kanMX*- strains both grew on neomycin plates, while only
*kanMX*+ strains grew on G418 plates, showing that the *kanMX* cassette is
specific for G418 resistance. The selection of induced transformants was then repeated
on plates containing G418.

![](https://static.igem.org/mediawiki/2021/1/1a/T--Toulouse_INSA-UPS--2021_fig22_yeast_results.jpg)

***Figure 22: Cre-lox induced transformants on YPDA medium supplemented with
G418 (500 μg.mL-1).** 3 colonies seem to have grown despite the
presence of
the antibiotic (B4, B7, D10). For these colonies, the Cre-loxP did not work
since the selection marker is still present.*

In Figure 22, we can see that most clones show G418 sensitivity profile so the kanMX
resistance gene was indeed deleted in these colonies. This kanMX- strain was named
LycoYeast-G418 and saved as glycerol stocks.

##### Integration into the *kanMX*- strain

All our constructs were designed to be integrated into the yeast genome by homologous
recombination. They must therefore be linearized by PCR. The transformation technique we
used was LiAc/ss-DNA/PEG following the TAKARA protocol (see [Protocol section](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Methods#a3)).

* PEG is necessary for the transformation since it allows the DNA to bind to
  the yeast walls.
* LiAc and heat shock promote the penetration of DNA into the cell.
* Finally, the carrier ss-DNA increases the transformation frequency but its
  function is still unclear (Kawai et al. 2010).

In addition, to improve the transformation frequency, 5 μg of DNA was added to the
transformation mix.

### Tips

We tried many protocols before successfully integrating our constructs into
the yeast genome. We then realized that the problem was the amount of DNA
template we were adding to the mix. In order to successfully integrate a large
construct into the genome of *S. cerevisiae*, large amount of DNA is necessary.
This is what we observed with our 10 kb constructs using about 5μg of DNA.

For each construction, integration of our constructs was checked by PCR (yeast DNA
extraction protocol can be found in the [protocols](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Methods)
section) with various primers pairs:

* external primers: would give a product too long to be amplified
* LA: one primer on the upstream genome region and one primer on the insert
* RA: one primer on the downstream genome region and one primer on the
  insert

The three integration sites were chosen thanks to the advice of Sara
Castano-Cerezo (see [IHP](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Human_Practices#Integrated_Human_Practices)
and [design](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Design))
because they were the few left available in the LycoYeast strain she provided
us.

* pVIOLETTE → locus 4, chromosome XII
* pFLEUR → locus 1, chromosome XII
* pFRAMBOISE (fused and not-fused) → locus 3, chromosome X

The objective is first to obtain strains containing one construct each. Then,
once the first integration is achieved, the other constructs are inserted into
the yeast genome sequentially. We started with pVIOLETTE, then pFLEUR and
finally pFRAMBOISE-fused or pFRAMBOISE-notfused.

**LycoYeast-VIOLETTE**

LycoYeast-G418 was transformed with pVIOLETTE following the TAKARA protocol

* The recombination takes place at locus 4 of chromosome XII.
* The integration of pVIOLETTE confers resistance to nourseothricin through
  the *nsrR* gene.

Transformants were selected on YPDA medium containing 250 μg/mL
nourseothricin.
About ten clones were obtained and integration was verified in all clones by PCR
(Figure 23). In Figure 23, only two clones are shown; other results are not
shown. It can be seen in Figure 23 that only clone 2 shows a correct integration
profile, i.e. a band for both LA and RA at the expected size.

![](https://static.igem.org/mediawiki/2021/a/a9/T--Toulouse_INSA-UPS--2021_fig23.violette.png)

***Figure 23: 0.8 % agarose gel stained with EtBr. Integration of
pVIOLETTE in LycoYeast-G418 at chromosomic XII locus 4 was checked
by PCR.** At the bottom left are presented the result of
genotyping of LycoYeast – VIOLETTE, clone 1 contains only the left arm
(LA) while
clone 2 contains the left and right arm (LA and RA). 4. At the
bottom right are presented the theoretical results (LA: 627 bp ; RA:
644bp).*

Integration of the pVIOLETTE insert at locus XII-4 was successful. The integrant
strain was named LycoYeast - VIOLETTE and saved as glycerol stock.

**LycoYeast–FLEUR**

LycoYeast - G418 was transformed with pFLEUR.

* The recombination takes place at locus 1 of chromosome XII.
* The integration of pFLEUR confers resistance to hygromycin through the
  *HygR* gene.

Transformants were selected on YPDA medium containing 200 μg/mL hygromycin.
About
ten clones were obtained and integration was verified on all clones by PCR
(Figure 24). In Figure 24, 3 clones are shown of which only one has the
construction (the right and left homology arms show a band at the expected
size).

![](https://static.igem.org/mediawiki/2021/2/29/T--Toulouse_INSA-UPS--fig24pfleur.png)

***Figure 24: Integration of the correct integration of pFLEUR in
LycoYeast-G418 at chromosome XII locus 1 was checked by
PCR.** At the bottom left are presented the result of
genotyping of Lycoyeast –
FLEUR, one clone has both left arm (LA) and right arm (RA) i.e. the
entire construction in the locus 1. At the bottom right are
presented the theoretical results (LA: 2,709 bp ; RA: 2,757 bp).*

Integration of the pFLEUR insert at locus XII-1 was successful. The integrant
strain was named LycoYeast - FLEUR and saved as glycerol stock.

**LycoYeast–FRAMBOISEfused**

LycoYeast-G418 was transformed with pFRAMBOISE-fused.

* The recombination takes place at locus 3 of chromosome X.
* The integration of pFRAMBOISE-fused) confers resistance to G418 through
  the *neoR* gene.

Transformants were selected on YPDA medium containing 400 μg/mL G418. About
twenty clones were obtained and integration was verified on 10 clones by PCR
(Figure 25). In Figure 25, only one correct clone is shown.

![](https://static.igem.org/mediawiki/2021/b/b9/T--Toulouse_INSA-UPS--2021_fig25pframbA.png)

***Figure 25: Integration of pFRAMBOISE-fused in LycoYeast-G418 at
chromosome X locus 3 was checked by PCR.** At the bottom left
are
presented the result of genotyping LycoYeast – FRAMBOISE(fused), one
clone has both left arm (LA) and right arm (RA) i.e. the entire
construction. At the bottom right are presented the theoretical
results (LA: 2 kbp ; RA: 2 kbp).*

Integration of the pFRAMBOISE-fused insert at locus X-3 was successful. The
integrant strain was named LycoYeast-FRAMBOISE-fused and saved as glycerol
stock.

**LycoYeast–FRAMBOISEnotfused**

LycoYeast-G418 was transformed with pFRAMBOISE-notfused.

* The recombination takes place at locus 3 of chromosome X.
* The integration of pFRAMBOISE-notfused confers resistance to this G418
  through the *neoR* gene.

Transformants were selected on YPDA medium containing 400 μg/mL G418. Only one
colony was obtained and integration was verified by PCR (Figure 26).

![](https://static.igem.org/mediawiki/2021/3/39/T--Toulouse_INSA-UPS--2021_fig26pframbB.png)

***Figure 26: Integration of pFRAMBOISE-not fused in LycoYeast-G418
at chromosome X locus 3 was checked by PCR.** At the bottom
left are presented the result of genotyping of LycoYeast – FRAMBOISE(not
fused), one clone has both left arm (LA) and right arm (RA) i.e. the
entire construction. At the bottom right are presented the
theoretical results (LA: 2 kbp ; RA: 2 kbp).*

Integration of the pFRAMBOISE-notfused insert at locus X-3 was successful. The
integrant strain was named LycoYeast-FRAMBOISE-notfused and saved as glycerol
stock.

**LycoYeast–VIOLETTE–FLEUR–FRAMBOISE**

After having successfully transformed LycoYeast-G418 with pVIOLETTE, we
sequentially transformed LycoYeast-VIOLETTE with pFLEUR, and then
pFRAMBOISE-fused or pFRAMBOISE-notfused.

The final strains containing the
three constructs are called:

* LycoYeast–VIOLETTE–FLEUR–FRAMBOIS-fused
* LycoYeast–VIOLETTE–FLEUR–FRAMBOISEnotfused

1. **LycoYeast-VIOLETTE-FLEUR:**

   Transformants were selected on YPDA medium containing 250 μg/mL
   nourseothricin and 200 μg/mL hygromycin. About twenty clones were
   obtained and integration was verified on 10 clones by PCR (Figure 27).
   In Figure 27, only one correct clone is shown.

   ![](https://static.igem.org/mediawiki/2021/c/cb/T--Toulouse_INSA-UPS--2021_fig27genotyping.png)

   ***Figure 27: Integration of pFLEUR in LycoYeast – VIOLETTE
   at chromosome XII locus 1.** At the bottom left are
   presented the result of genotyping of Lycoyeast – VIOLETTE –
   FLEUR,
   one clone has both left arm (LA) and right arm (RA) i.e. the
   entire construction in the locus 1. At the bottom right are
   presented the theoretical results (LA: 2,709 bp ; RA: 2,757
   bp).*

   Integration of the pFLEUR insert at locus XII-1 was successful. The
   integrant strain was named LycoYeast-VIOLETTE- FLEUR and saved as
   glycerol stock.

2. **LycoYeast-VIOLETE-FLEUR-FRAMBOISE-fused:**

   Transformants were selected on YPDA medium containing 250 μg/mL
   nourseothricin, 200 μgm/L hygromycin and 400 μg/mL G418. About
   twenty
   clones were obtained and integration was verified on 10 clones by PCR
   (Figure 28). In Figure 28, only one correct clone is shown.

   ![](https://static.igem.org/mediawiki/2021/b/bb/T--Toulouse_INSA-UPS--2021_fig28genotyping.png)

   ***Figure 28: Integration of pFRAMBOISE-fused in LycoYeast –
   VIOLETTE - FLEUR at chromosomic X locus 3.** At the
   bottom
   left are presented the result of genotyping of
   ELIXYeats(fused) one clone has both left arm (LA) and right
   arm (RA) i.e. the entire construction. At the bottom right
   are presented the theoretical results (LA: 2 kbp ; RA: 2
   kbp).*

   Integration of the pFRAMBOISE-fused insert at locus X-3 was successful.
   The integrant strain was named LycoYeast-VIOLETTE-FLEUR-FRAMBOISE-fused
   and saved as glycerol stock.

3. **LycoYeast-VIOLETTE-FLEUR-FRAMBOISE-not fused:**

   Transformants were selected on YPDA medium containing 250 μg/mL
   nourseothricin, 200 μgm/L hygromycin and 400 μg/mL G418. About
   twenty
   clones were obtained and integration was verified on 10 clones by PCR
   (Figure 29). In Figure 29, two clones are shown but only clone 1 has the
   correct integration profil, i.e. both left and right arms. We can see
   that clone 2 gives no bands for the LA and RA tracks but gives a band
   for the track using both primers on the yeast genome, so the construct
   is not present in its genome.

   ![](https://static.igem.org/mediawiki/2021/7/7a/T--Toulouse_INSA-UPS--2021_results_genop_notfused.png)

   ***Figure 29: Integration of pFRAMBOISE-notfused in
   LycoYeast – VIOLETTE - FLEUR at chromosome X locus
   3.** At the
   bottom left are presented the result of genotyping of
   ELIXYeast(not-fused). The clone 2 has both left arm (LA) and
   right arm (RA) i.e. the entire construction. On the
   contrary, clone 1 still has the integration insert so the
   construct is not present in clone 1. At the bottom right are
   presented the theoretical results (LA: 2 kbp ; RA: 2 kbp).*

   Integration of the pFRAMBOISE-notfused insert at locus X-3 was
   successful. The integrant strain was named
   LycoYeast-VIOLETTE-FLEUR-FRAMBOISE-fused and saved as glycerol stock.

### Success 1

The four inserts were independently integrated into the LycoYeast genome.

### Success 2

The final strain with concomitant integration of pVIOLETTE, pFLEUR and
pFRAMBOISE inserts was successfully constructed.

### Cyanobacterium genetic engineering

##### Transformation of *S. elongatus* through triparental conjugation

After successfully cloning pCONCOMBRE, it was time to use it to engineer *Syn
UTEX 2973*. Conjugal transfer of DNA between bacterial cells is a
well-described process and has been used previously to engineer cyanobacterial
species, particularly those that are not naturally competent, such as *Syn
UTEX 2973* (Elhai and Wolk 1988; Gale et al. 2019). We therefore relied
on this method to introduce pCONCOMBRE in our cyanobacterial strains.

From a practical standpoint, we followed the protocol found in the
well-illustrated video article provided by Gale et al. (2019) (for more
information, see our [Methods](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Methods)
page). The details of this technique are presented in Figure 30. In brief,
cyanobacterial cultures are incubated in presence of two different E. coli
strains called respectively the conjugal strain and the helper strain which
allow the transfer of the plasmid of interest (cargo plasmid) to the
cyanobacterium in three steps.

![](https://static.igem.org/mediawiki/2021/7/7d/T--Toulouse_INSA-UPS--2021_fig30_triparental.png)

***Figure 30: Transformation of S. elongatus through triparental
conjugation.** 1) The helper strain carries two plasmids: the
vector
to be transferred to the cyanobacteria, called cargo vector, and a
helper plasmid. The latter encodes for specific DNA-methylases that
act on the cargo vector so that it is not degraded after entry into
the cyanobacterium cellular space. (2) The conjugal plasmid carried
by the conjugal strain contains the many genes required to encode
the conjugal apparatus. In the first step of conjugation, the
conjugal plasmid is transferred from the conjugal strain to the
helper strain. (3) This helper strain is then able to transfer the
methylated cargo vector into the cyanobacterium.*

After incubating our *E. coli* strains with the cyanobacterium strains, the
conjugation mixes were plated on sterile filters placed on petri dishes
containing a mixture of BG11 and LB media. The protocol was applied to the WT
strain as well as to the two different sucrose-secreting strains CscB and CscB
SPS SPP. After 24 hours on non-selective medium, these filters were transferred
on plates containing spectinomycin, the antibiotic used to select the
integration of pCONCOMBRE. Isolated conjugants were obtained after 4 days of
incubation of these plates (Figure 31).

![](https://static.igem.org/mediawiki/2021/3/3c/T--Toulouse_INSA-UPS--2021_fig31_resultscyano.png)

***Figure 31: Evolution of the visual aspect of the triparental
conjugation.** After 24h of incubation on non-selective
medium, the
cyanobacteria cover the filter which becomes green (left). Following
the passage of these filters on a selective medium, the mutants that
have integrated the spectinomycin resistance cassette appear
isolated on the filter (center). The isolated conjugants are
restreaked successively on BG11 plates to eliminate E. coli
contamination and genotyped by PCR (right).*

### Tips

The conjugal plasmid used for triparental mating can be very large. For
example, the conjugal plasmid we used, pRL443, is 57 kb long. To transform
it into *E. coli*, do not hesitate to highly increase the quantity of
DNA you add. In our case, we went as high as 400 ng of DNA to obtain
transformants.

##### Verification of the correct integration of pCONCOMBRE

Integration of our construct was checked by PCR with various primers pairs:

* LA: one primer on the upstream genome region and one primer on the insert
* RA: one primer on the downstream genome region and one primer on the
  insert
* external primers: would give a product too long to be amplify in case of
  insert integration

Almost all the clones tested had the same profile for the different tests
performed (Figure 32). Amplification products did not however always correlate
with expected sizes:

* **insert product at 2.3 kb when expecting no signal:** It is known
  that *S. elongatus UTEX 2973* carries multiple copies of its
  chromosome (Yu et al. 2015). The conjugants were perhaps not entirely
  segregated which means that there was still at least one chromosome copy
  that did not have the insert.
* **RA additional product at 4.8 kb:** Probably corresponds to
  non-specific amplification.
* **LA product at 4 kb that was not observed for a WT strain (data not
  shown) while the expected band was supposed to be 1.5 kb:** Our
  hypothesis is that the cloning vector we used (pAM4951) was poorly
  sequenced/annotated and that there was a sequence between the LA and the
  spectinomycin resistance gene (it was noted that several primers
  designed in this region were inexplicably non-functional).

Because of discrepancies between obtained and expected genotyping profiles, we
decided to also check for the presence of all genes from our insert with
additional primers: LOX, HPL, LacI and SpecR. Figure 32 shows amplification
products for all genes of our insert.

![](https://static.igem.org/mediawiki/2021/1/1e/T--Toulouse_INSA-UPS--2021_fig32comcombre.png)

***Figure 32: Integration of pCONCOMBRE in S. elongatus at
chromosomic neutral site I (NSI)was checked by PCR.** At the
bottom
left are presented the result of genotyping of WT pCONCOMBRE clone 4
(0.8% agarose gel stained with EtBr for visualisation) and at the
bottom right are presented the theoretical results.*

Overall, it appears that we have successfully inserted pCONCOMBRE into the genome
of *S. elongatus*. The next step is to test our mutant strain!

### Tips

Efficent cell lysis ensuring optimal genomic DNA release is critical for
the success of colony PCR. The traditionals protocols used for E. coli
colony lysis proved to be inefficient in our hands. We advise future iGEM
teams to use two methodologies advised during the Phototrop Meetups ([see our
Methods page](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Methods)):

1-freezing-thawing (-80°C / 60°C cycles)

2-colony lysis in DMSO with 95°C heating for 10 minutes

## Conclusion

All our constructions have been successfully integrated in the yeast and
cyanobacterium genomes. The strains are ready to be challenged for their
capacity to produce violet scent molecules.

# Strain characterization for the production of the molecules of interest

Here we explore the functionality of our constructions. We assessed their capacity to
transform substrates from the terpene pathway and to produce the violet scent.

### Color change in the modified LycoYeasts strains

After verifying the correct integration of our constructs by PCR, our engineered LycoYeast
strains were placed on YPD plates containing the inducers with the aim to:

* Detect color changes due to the conversion of lycopenes (red) to carotenes (orange),
  as indicated on our [Design page](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Design).
* Detect smell changes due to production of ionones.

For all the strains that are supposed to produce ionones (LycoYeast-VIOLETTE,
LycoYeast–FRAMBOISEfused and LycoYeast–FRAMBOISEnotfused), the expected color change was
obvious (Figure 33).

![](https://static.igem.org/mediawiki/2021/4/47/T--Toulouse_INSA-UPS--2021_fig33_resultsprod.png)

***Figure 33: Color change in the modified Lyco Yeast strains.** The
mutants seem
to change from red (lycopene) to orange (carotene) which was the expected
result.*

For the VIOLETTE construction, it appears (upright corner of Figure 33) that without inducer,
the colonies remain red. This is coherent with the literature since PGAL1 is
supposed to be a tight promoter (Adams 1972). Conversely, for both pFRAMBOISE constructs,
the color change is observed in the presence and absence of doxycycline. Again, this is
consistent since PtetO7 is well known to suffer from significant leakage
(Tominaga et al. 2021).

**SPOILER:** motivated by this encouraging visual evidence, we smelled the Petri
plates to maybe detect a difference between the WT and induced mutant strains (knowing
this has to be safe as explained in our [Safety page](https://2021.igem.org/Team%3AToulouse_INSA-UPS/Safety)). We were
delighted to find that a characteristic ionone sweet smell could be noticed! We asked
various people from the lab to blindly smell the plates, they also recognized the ionone
aroma. We then continued the demonstration by investigating the profile of carotenoids
in the cells.

### Carotenoid profile analysis

The carotenoids contained in the cells (precursors of the ionones) were extracted using the
method described by López et al. (2020). Briefly, the yeast cells were lysed in acetone
using glass beads and the supernatant obtained after this lysis was analyzed by RP-HPLC
using a C18 column.

##### pVIOLETTE

In the VIOLETTE strains, lycopene is indeed converted into a new product with a higher
retention time upon induction (Figure 34). We did not have a standard for
ε-carotene, but
this peak had a retention time close to our β-carotene standard. Considering the yellow
color of pVIOLETTE strains, as well as the in-line following α ionone production
results, this new peak most likely corresponds to ε-carotene, the expected
precursor. This
confirms that at least the lycopene ε-cyclase activity of our fusion enzyme
LcyE-OfCCD1 is
present. As observed with the color of the strains, it appears that PGAL1 does
not have any detectable leak with our system.

![](https://static.igem.org/mediawiki/2021/1/1f/T--Toulouse_INSA-UPS--2021_fig34_chromato.png)

***Figure 34: Carotenoid analysis of the strain engineered with pVIOLETTE.**The
observed color change in the galactose-induced mutant strain LycoYeast-VIOLETTE
is confirmed in HPLC by the apparition of a new peak that most likely
corresponds to ε-carotene.*

##### pFRAMBOISE

Using the same method, we were able to detect upon doxycycline induction a peak corresponding
to the β-carotene retention time. Our Lyco Yeast pFRAMBOISE-fused and
pFRAMBOISE-notfused produce β-carotene (Figure 35)!

The previous observations on the significant leakage of PtetO7 are confirmed with
these carotenoid profiles since no significant difference between induced and non-induced
strains are observed. Two hypotheses can be formulated: either the rtTA promoter activator
is inactive, or the promoter leakage is sufficient to cause the conversion of all lycopene
present in our strains.

![](https://static.igem.org/mediawiki/2021/a/ac/T--Toulouse_INSA-UPS--2021_results_Carotenoid_analysis.png)

***Figure 35: Carotenoid analysis of the strains engineered with
pFRAMBOISE-fused
(left) and pFRAMBOISE-notfused (right)** The observed color change in the
mutant strains
LycoYeast-FRAMBOISEfused and LycoYeast-FRAMBOISEnotfused are confirmed in HPLC
by the apparition of a new peak that corresponds to β-carotene.*

### Tips

Carotenoids are sensitive to light and O2 and can degrade quite rapidly. This can be
seen for example on Figure 35 B) where the samples were left at room temperature for too
long before being injected causing the appearance of degradation products that can
disturb the analyses. It is necessary to try keeping the samples protected from light
and in the freezer (-20°C).

### Production of the molecules of interest *in vivo*

With these exciting results confirming lycopene conversion, it was finally time to test the
production of our molecules of interest *in vivo*. The molecules we aim to produce, as
all fragrance compounds, are very volatile. A common strategy to avoid losing these
molecules during the culture is to grow the engineered microorganisms in a culture medium
supplemented with an organic phase to trap the molecules of interest. This method has
particularly been applied to the production of terpenes (Chen et al. 2019; López et al.
2020). Since no other method had been described for the violet leaf aldehydes
(2E,6Z)-nonadienal and (2E,6Z)-nonadienol, the same strategy was also applied with the
mutant strains *S. elongatus*.

The most common organic solvent used for ionones is dodecane Chen et al. 2019; López et al.
2020), we therefore relied on this technique for the cultivation of all of our strains.
Briefly, yeast and cyanobacterial cultures were performed in a total volume of 20 mL
respectively in YPD supplemented with dodecane (20% v/v) for the yeast and in BG11
supplemented with dodecane (20% v/v) for the cyanobacteria. When the cultures reached the
stationary phase, samples of the organic phases of the different cultures were collected for
GC-MS analysis.

##### α-ionone

The results of GC-MS for the LycoYeast-VIOLETTE strain are presented in Figure 36. We were
delighted to observe the apparition of a peak at the same retention time as the
α-ionone standard for the induced LycoYeast-VIOLETTE strain, correlating with the
violet smell detected by human noses from the petri dishes. The mass spectra associated with
this peak matched with the one obtained with the analytical standard. The α-ionone
attribution was further confirmed by the NIST mass spectral library (National Institute of
Standards and Technology). Overall, this demonstrated the functionality of our LcyE-OfCCD1
fusion enzyme in *S. cerevisiae*.

![](https://static.igem.org/mediawiki/2021/c/c7/T--Toulouse_INSA-UPS--2021_fig37_results_LycoYeast-FRAMBOISE-notfused.png)

***Figure 36: GC-MS analysis of the dodecane layer of the LycoYeast-VIOLETTE
cultures.** α-ionone is produced in vivo by our strain when it is
induced by
galactose. On the right are presented the matching mass spectra between
the standard and the observed peak. (Note: the standard is here a little too
concentrated but it still allows to realize the attribution)*

##### β-ionone

Similarly, we were also able to detect β-ionone in the cultures of
LycoYeast-FRAMBOISE-notfused (Figure 37).

![](https://static.igem.org/mediawiki/2021/2/22/T--Toulouse_INSA-UPS--2021_fig38_results_Activity_assay_of_DBR1.png)

***Figure 37: GC-MS analysis of the dodecane layer of the
LycoYeast-FRAMBOISE-notfused cultures.** β-ionone is produced in
vivo by our
strain with or without induction with doxycycline due to promoter leakage. On
the right is presented the mass spectra of β-ionone that matched between
the standard and the observed peak (not shown).*

We unfortunately did not have time to analyze by GC-MS the cultures of
LycoYeast-FRAMBOISE-fused. This strain characterization work was indeed done during the last
three weeks of the summer with only two students still in the lab. Due to a malfunction of
the GC-MS we were supposed to use initially, we could only perform one round of analysis
which was kindly conducted by Toulouse White Biotechnology, one of our sponsors.

The LycoYeast-FRAMBOISE-fused strain was successfully constructed a few days after this
round of GC-MS analysis. We were however able to show in HPLC that at least the CrtY
activity was present (production of β-carotene) with our CrtY-PhCCD1 fusion enzyme.
Because of the odor of this strain, which is similar to the one of previous strains for
which we have detected ionones, we are confident that the fusion enzyme we have designed is
functional and can produce β-ionone.

##### Other molecules of interest

As explained before, we only were able to do one set of GC-MS analysis. Unfortunately, we
found that linalool, (2E,6Z)-nonadienal and (2E,6Z)-nonadienol were released at the same
retention time as dodecane, our solvent. It was therefore not possible to confirm that these
molecules were produced. It would be necessary to modify the temperature gradients used in
the GC-MS method, which we were unable to do in the allocated time, or to use another
extraction solvent.

### Enzymatic activity assay of DBR1

The last molecule we were interested in is dihydro-β-ionone. This molecule is produced
from β-ionone as a precursor. Since we were not sure that β-ionone was produced by
our strains when we prepared our samples for our single GC-MS analysis, we decided to try
investigating if the enzyme catalyzing this step of conversion, DBR1, was active.

To do so, we cultivated the strain LycoYeast-FLEUR and induced the expression of DBR1 by
adding copper to the culture medium in late exponential phase. After waiting for the enzymes
to be produced, the yeast cells were lysed in TRIS-HCl pH 6.5 buffer using glass beads. The
activity was studied by GC-MS using β-ionone as substrate, adapting the method
described by Zhang et al. (2018). Assays contained 50 mM TRIS-HCl (pH 6.5), 1 mM NADPH, 2 mM
dithiothreitol (DTT), 1 mM β-ionone and 500 μL of the yeast lysate in a total volume
of
1 mL at 45 °C for 1.5 h. At the beginning and end of the incubation, 200 μL of the mix
was
collected and then a liquid-liquid extraction was carried out by vortexing the sample with
200 μL of dodecane. The dodecane phase was then analyzed by GC-MS. The results are
presented
in Figure 38.

![](https://static.igem.org/mediawiki/2021/8/81/T--Toulouse_INSA-UPS--2021_fig38_results_DBR1.png)

***Figure 38: Activity assay of DBR1.** A portion of the β-ionone is
converted into dihydro- β-ionone by DBR1.*

At the initial state t=0, we can only see β-ionone which is the substrate that was added
to the reaction mix. After 1.5 hours, dihydro-β-ionone is detected in the reaction
media, thus demonstrating that the enzyme DBR1 is indeed active.

## Conclusion

The results are beyond our hopes. We managed to smell yeast actually smelling like
violet. We observed the change of color of our strain induced to produce our molecules.
Eventually, we analytically detected all three ionones. However, we have not been able
to assess the production of
linalool, (2E,6Z)-nonadienal and (2E,6Z)-nonadienol due to our concentration strategy
with dodecane and lack of time.

# Final conclusion

We engaged in this project like all iGEMers do, full of hope and great ideas. Our instructors
kept telling us that most experiments are often prone to fail. Here our main dream was to
create a violet-smelling yeast and we definitely succeeded. Our second ambition was to prove
the viability of the co-culture to sustainably produce this scent. This will require more
time and energy since the growing *Synechococcus elongatus* was a lot more challenging
than expected. Anyway, we have demonstrated that a small team of student in a limited time
can engineer a microorganism to produce a defined synthetic scent, and hence, the power and
accessibility of synthetic biology for application in the perfume industry.

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