iGEM 2024 — Team toulouse-insa-ups

![mascotte1](https://static.igem.wiki/teams/5108/general/back-top-static.png)

# iGEM Toulouse 2024 Timeline

* December 20th, 2023

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)* Team Selection *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  December 20th, 2023, the day our iGEM adventure began! To take part in this project, we first submitted an application including our iGEM project proposal, cover letter and resume in November. Some of us were called in for an interview to defend our idea and show why we were the most motivated to take part in the competition. After an unbearable wait, the answer finally arrived on that cold December day: **you are iGEM Toulouse!**

  #### December 20th, 2023
* January 18th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)* First brainstorming *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  This meeting started with explanations about finances and organization. We also divided our roles as follows: Emma was organization leader, Laura was communication leader, Léa was wet lab leader, Lou-Sahra was wiki leader, Matthieu was finance leader, Nathan was human practice leader, Rebecca was dry lab leader and Yohann was entrepreneurship leader. We all presented a project idea, including biofilm degradation, biodegradation of aromatic hydrocarbon, bioleaching, a diagnostic method using volatile organic compounds, sustainable fibers using bacteria, and a synthetic microbiota using plasmids.

  #### January 18th, 2024
* February 3rd, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)* INSA Open House day *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We participated in the "open house day" of INSA Toulouse and presented iGEM to highly motivated high schoolers and their families. We had great fun explaining how the competition works and discussing the previous Toulouse INSA-UPS team’s projects.

  ![](https://static.igem.wiki/teams/5108/notebook/jpo-afternnon-lea-lou-sahra.jpg)

  ![](https://static.igem.wiki/teams/5108/notebook/jpo1.jpg)

  ❯

  #### February 3rd, 2024
* February 8th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)* Our first financing request*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We held our first funding request meeting at INSA Toulouse, where we sought a grant of €5,000. Our finance leader successfully navigated this initial challenge, presenting the iGEM competition and leading a Q&A session. Well done, Matthieu!

  #### February 8th, 2024
* February 16th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We officially registered to the iGEM Competition*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We have officially registered for the iGEM competition! We kept on discussing fundraising. Two promising new ideas were introduced: bio-production of human milk oligosaccharides and the use of LAMP technique to detect microorganisms. We kept on discussing the idea of using insect odorant receptors for detection of volatile organic compounds.

  #### February 16th, 2024
* February 28th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)* Welcome to our two PhD advisors *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We were very pleased to meet the two PhD students who joined us as supervisors to help us with our project: Iker and Marvin! This week, we have continued brainstorming and also had our first course on cell-free methods. We learned a lot about this subject, with which none of us were familiar.

  #### February 28th, 2024
* April 4th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)* We chose our project *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  This week was THE decisive week for our project. After much deliberation and input, we voted on which project we would work on for the rest of the competition, and it was the lunar biofertilizer project that we chose to develop! From that week on, we all devoted ourselves to building our project.

  #### April 4th, 2024
* May 9th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We have a team name and logo*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  Each of us worked on its dedicated topic and we kept on researching the genes we wanted to use. We also started to think about experiments and protocols. And this week we finally all agreed on our logo and mascot for the BioMoon project!

  ![](https://static.igem.wiki/teams/5108/notebook/notebook-logo.webp)

  ![](https://static.igem.wiki/teams/5108/notebook/lupso-card.jpg)

  ❯

  #### May 9th, 2024
* May 10th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Discussing our modeling strategy*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We started brainstorming ideas for the modeling strategy, defined the tools and started learning about the use of Genome Scale Metabolic Models (GSMM), Flux Balance Analysis (FBA), and cobrapy.

  #### May 10th, 2024
* May 17th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Finding our plasmids*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  After several weeks of intensive searching, we finally identified and selected the optimal expression plasmids for our bacterial transformation experiments. We opted for the pSEVA244 and pSEVA438 plasmids. These plasmids stood out due to their compatibility with our bacterial strains and their ability to efficiently drive gene expression. With these tools in hand, we are now well-equipped to move forward with our experiments and achieve our research goals!

  ![](https://static.igem.wiki/teams/5108/notebook/lea-plasmids.jpg)

  #### May 17th, 2024
* June 3rd, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*First day in the lab*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  This was our first week in the lab! We started our journey in the lab ordering genes and biobricks, writing protocols and planning experiments. We are excited and we look forward to performing our first experiments.

  ![](https://static.igem.wiki/teams/5108/notebook/1st-day-igem.jpg)

  #### June 3rd, 2024
* June 8th, 2018

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We participated to the Toulouse Expo Science *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We participated to the “Expo Science,” an event that brings together young innovators who have undertaken projects using a scientific approach. At the event, we had the opportunity to present our iGEM project to the public, sharing our research and engaging with attendees about the scientific process and real-world applications. It was a valuable experience to showcase our work and connect with other passionate people about science and innovation.

  ![](https://static.igem.wiki/teams/5108/notebook/nathan-emma-yohann.jpg)

  ![](https://static.igem.wiki/teams/5108/notebook/nathan-pres.webp)

  ❯

  #### June 8th, 2018
* June 11th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Obtaining *P. fluorescens* metabolic model*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We obtained the first metabolic model of *P. fluorescens* and started performing simulations on it to reproduce the laboratory results reported in literature.

  #### June 11th, 2024
* June 13th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Meeting Agronutris plant experts *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We met with Agronutris to gather insights on optimal conditions for the bacteria and plants we should use, as well as the experiments necessary to test plant growth on regolith using our future engineered strain.

  #### June 13th, 2024
* June 14th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We received our first gBlocks!*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We've been impatiently waiting for the arrival of our gBlocks to start our cloning experiments, and here they are at last!

  ![](https://static.igem.wiki/teams/5108/notebook/gblocks1to1.jpg)

  #### June 14th, 2024
* June 15th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Growing our strain is fun!*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  Look at our beautiful fluorescent *Pseudomonas*!

  ![](https://static.igem.wiki/teams/5108/notebook/pf-glowing-nb.jpg)

  #### June 15th, 2024
* June 20th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Switching for *P. putida* metabolic model*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  Our experiments performed on the metabolic model of *P. fluorescens* failed due to the poor structure of the GSMM. The decision was made to use a well documented and properly structured GSMM of *P. putida*. The first experiments were conducted to verify its usability and accuracy to prove that the chosen model is optimal for our project. The modeling strategy was ultimately defined with an accurate breakdown of every step to be taken to achieve the key goals previously identified.

  #### June 20th, 2024
* June 27th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We confirmed the successful cloning of pTET promoter*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We were excited about our first cloning success and could not t wait to integrate new metabolic pathways in our strain.

  #### June 27th, 2024
* July 4th an 5th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Mini Jamboree!!*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  This was the Mini Jamboree week! On Thursday the 4th and Friday the 5th we welcomed nine teams from different European countries including Spain, Italy, Netherlands and of course France. We spent a great time together presenting our posters, our projects and visiting Toulouse. We also conducted experiments in the lab.

  ![](https://static.igem.wiki/teams/5108/notebook/minijamb1.jpg)

  #### July 4th an 5th, 2024
* July 11th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Meeting Sandra Bensmihen, plant expert*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We had the pleasure to meet Sandra Bensmihen, a plant biology researcher at LIPME. She provided us with invaluable advice on optimizing plant cultivation and developing protocols for testing plant growth.

  #### July 11th, 2024
* July 17th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We validate the equipment for plant cultivation*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  Thanks to the valuable feedback from S. Bensmihen, we have been able to optimize our growth chamber, creating a more optimal environment for our plants' development.

  ![](https://static.igem.wiki/teams/5108/notebook/plants-nb.jpg)

  #### July 17th, 2024
* July 18th, 2024

  ### *![](https://tools.igem.org/uploads/teams/5108/notebook)*Shooting our promotion video at Cité de l’espace*![](https://tools.igem.org/uploads/teams/5108/notebook)*

  We spent the morning filming scenes for our promotional video at the Cité de l'Espace in Toulouse, and we can't wait to show it to you!

  [Click here to watch our video](https://video.igem.org/w/a7bc2d06-91f6-4ff3-bbd6-6cbf1299432d)

  ![](https://static.igem.wiki/teams/5108/notebook/col-lunex3.webp)

  #### July 18th, 2024
* July 22th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*CNES is officially our sponsor!*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We were thrilled and honored to count the National Center for Space Studies among our sponsors. With a generous contribution of €12,000, they enabled us to pursue our ambitious goals and present a project of significant scale.

  #### July 22th, 2024
* July 22th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Modeling creatinine metabolization pathway*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  Growth experiments for the creatinine metabolization pathway were performed serving as a basis for lab experiments.

  #### July 22th, 2024
* August 1st, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Visiting CNES*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  As an external advisor and expert in lunar missions, Alexis Paillet warmly welcomed us to his workplace at CNES, where he gave us a tour. We had the incredible opportunity to see impressive models of lunar and Martian exploration robots and to speak with engineers who work directly with astronauts on the ISS.

  ![](https://static.igem.wiki/teams/5108/notebook/cnes1.jpg)

  #### August 1st, 2024
* August 19th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Finish independent metabolic improvement modeling*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  Results for the creatinine metabolization pathway, nitrate production pathway and oxidative stress resistance were terminated. We began the phase of combination.

  #### August 19th, 2024
* August 30th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We participated to the Space Meet-up*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We had the pleasure of attending the space meet-up organized for the first time by iGEM. We had the opportunity to present our project and learn about the exciting projects from other teams in the space village.

  #### August 30th, 2024
* September 3rd, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Meeting Catalyseur Start-Up incubator*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  To develop a strong and effective business plan for the entrepreneurship aspect of our project, we consulted with experts who provided valuable guidance. Their insights helped refine our ideas, offering us a clearer understanding of key entrepreneurial strategies, market positioning, and sustainable growth. Through these discussions, we gained a deeper perspective on how to transform our vision into a viable and successful business model.

  #### September 3rd, 2024
* September 4th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Finishing modeling*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  The last results were obtained and the modeling strategy was concluded. The report for the wiki started.

  #### September 4th, 2024
* September 4th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Meeting with Sandra Ortega, MELiSSA expert*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We had the opportunity to discuss how our project could be integrated into the space waste recycling systems planned by international space agencies, with a particular focus on the MELiSSA project. MELiSSA aims to develop a regenerative life support system that mimics the key functions of Earth's ecosystem, and we explored how our work could contribute to this groundbreaking initiative.

  #### September 4th, 2024
* September 4th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We validated creA crnA biobrick!*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We successfully engineered our *P. fluorescens* strain and made it able to grow on creatinine. We are proud of our work and we look forward to presenting it at the Jamboree !

  #### September 4th, 2024
* September 12th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*Thank you to all our sponsors*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  The Toulouse-based bioinformatics startup iMEAN was our latest sponsor, bringing our total funding to €29,295. We were thrilled that our project has attracted so many contributors, and we warmly thanked them for their support, without which BioMoon would not have come to life.

  #### September 12th, 2024
* September 24th, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We organized a workshop for middle schoolers*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We had the pleasure of welcoming a group of teenagers from Collège Antoine Carrère to our lab, where we introduced them to our project and shared the exciting world of biology. Through interactive activities and engaging presentations, we aimed to make learning about biology both educational and enjoyable, sparking their curiosity and interest in science.

  ![](https://static.igem.wiki/teams/5108/notebook/pitchounes1.png)

  #### September 24th, 2024
* October 2nd, 2024

  ### *![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*We finished wiki redaction*![](https://static.igem.wiki/teams/5108/notebook/mini-leaf.png)*

  We poured our hearts and souls into creating our wiki, dedicating countless hours to ensure it’s the best it can be.

  ![](https://static.igem.wiki/teams/5108/notebook/1727513968122.jpg)

  #### October 2nd, 2024

[Acknowledgements](https://2024.igem.wiki/toulouse-insa-ups/sponsors)

[Attributions](https://2024.igem.wiki/toulouse-insa-ups/attributions)

![mascotte1](https://static.igem.wiki/teams/5108/general/back-top-static.png)

# Engineering

* ### Content
* [Introduction](#titre1)
* [Creatinine](#titre2)
* [Nitrates](#titre3)
* [Stress](#titre4)
* [Future prospects - Biofilm](#titre5)
* [Plant experiments](#titre6)
* [Conclusion](#titre7)
* [References](#ref)

BioNoob
BioNoob

In this page, you will find a detailed description of the four different engineering cycles we put in place as well as the multiple iterations we went through over the course of the project.

## Introduction

BioMoon aimed to develop a biostimulant that would allow plant growth on the Moon using only resources available on a lunar space station. To this end, we engineered the plant growth-promoting rhizobacteria *Pseudomonas fluorescens* to be able to metabolize creatinine. Furthermore, we designed new paths that could further improve plant growth.
We put into practice the D-B-T-L cycle (Design-Build-Test-Learn) and were able to learn from our tests, redesign and rebuild the different elements of our project.

## Creatinine

#### DESIGN

The essence of our project lies in the engineering of different pathways in *P. fluorescens* to improve its metabolism to support plant needs in the adverse environment of lunar regolith. To effectively complement the project, the modeling strategy reflects this modular structure by testing each modification individually and then integrating them together alternatively to obtain meaningful insights (Figure 1).

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-1.png)

In *Pseudomonas putida*, the creatinine metabolic pathway consists of three enzymes: creatinine amidohydrolase to convert creatinine intro creatine, creatinase to convert creatine into sarcosine (while forming urea), and sarcosine oxidase to convert sarcosine into glycine (with formaldehyde and hydrogen peroxide as byproducts)[1](#ref). This amino acid can then be used as carbon and nitrogen sources. However, this metabolic pathway has not been found in *Pseudomonas fluorescens*. Thus, we aim to introduce into a plasmid three genes coding for the aforementioned enzymes: *crnA, creA* and *soxA*. Note that we are using the *soxA* gene, originating from *Bacillus subtilis*, because the enzyme is made of only one subunit contrary to the one of *Pseudomonas putida consisting* of four subunits (SoxABCD).

![](https://static.igem.wiki/teams/5108/engineering/eng-fig1.png)

**Figure 1: metabolic pathway of the creatinine degradation in *Pseudomonas putida*.**

#### BUILD

As the genes *crnA* and *creA* are present in an operon in some *Pseudomonas* species, we mimicked this by joining the two genes under the same promoter *Pm* which is inducible by m-toluic acid. The third gene, *soxA*, was under transcriptional control of another promoter, *Ptet*, inducible by anhydrotetracycline.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-2.png)

In this construct, the two inserts tap-to-tail are under the control of the same terminator, LUZ7 T50 (BBa\_K4757058)[2](#ref), which is bidirectional. The three genes were codon-optimized for *P. putida* using the Codon Optimization Tool from Integrated DNA Technologies (IDT) to lower complexity and minimize secondary structures. The three genes were introduced in a single plasmid by In-Fusion Assembly, and we designed primers with floating tails overlapping adjacent regions for each insert.

![](https://static.igem.wiki/teams/5108/engineering/eng-fig2.png)

**Figure 2: representation of the pSEVA438-Ptet-creA-crnA-soxA plasmid.**

#### TEST

We first tested the growth of our bacteria on M9 minimal medium without nitrogen source supplemented with each metabolite of the pathway individually: glycine, sarcosine, creatine, or creatinine. The goal was to test if these molecules can be used as a carbon and nitrogen source. Additionally, to further test the feasibility of our strategy, we performed *in silico*  simulations of bacterial growth, which aided in designing better informed *in vivo* experiments. See modeling [here](https://2024.igem.wiki/toulouse-insa-ups/model).

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-3.png)

After construction, our plasmids were sequenced by Sanger sequencing.
Once the constructs were validated, we repeated the growth experiments with the modified *P. fluorescens* to quantify the effect of our plasmid. Finally, SDS-PAGE was performed to assess the expression of the three enzymes introduced.

#### LEARN

After testing the growth of *P. fluorescens* on a minimal medium with creatinine, we concluded that *P. fluorescens* WT is unable to utilize creatinine as its sole carbon and nitrogen source. See results [here](https://2024.igem.wiki/toulouse-insa-ups/results). In light of the results obtained in silico, which predicted growth of the WT strain on sarcosine, we decided to also test its growth on creatine and sarcosine, the downstream products of the metabolic pathway. While no growth was observed on creatine, the bacteria were able to grow on sarcosine, thus confirming the predictions of the in silico model. See modeling [here](https://2024.igem.wiki/toulouse-insa-ups/model)

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-4.png)

#### DESIGN 2.0

Based on these findings, we decided to simplify our construct. Instead of introducing all three genes as initially planned, we opted to first clone the operon containing *creA* and *crnA* into the plasmid pSEVA438-Ptet. This allowed us to focus on the initial steps of the pathway, from creatinine degradation up to sarcosine. If necessary, the gene *soxA* could be introduced at a later stage to complete the pathway and enable the conversion of sarcosine into glycine.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-5.png)

#### BUILD 2.0

The new plasmid, without the *soxA* gene was built following this representation:

![](https://static.igem.wiki/teams/5108/engineering/eng-fig3.png)

**Figure 3: Representation of the pSEVA438-Ptet-creA-crnA plasmid.**

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-6.png)

#### TEST 2.0

After cloning and transformation of *P. fluorescens* with the pSEVA438-Ptet-creA-crnA plasmid, we conducted the same growth experiments to verify whether the engineered strain was able to grow on each of the four metabolites of the pathway: creatinine, creatine, sarcosine and glycine. *In silico*, the genes were added to the GSMM in order to simulate the growth of the new strain.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-7.png)

#### LEARN 2.0

The new round of growth tests, this time on the **engineered strain**, showed significant growth of bacteria on creatinine, creatine, or sarcosine as sole carbon and nitrogen sources. **We have successfully engineered *Pseudomonas fluorescens* to metabolize a urinary compound!**
The fact that the strain was able to grow on creatinine without cloning of the *soxA* gene further supports that there was no real need for it in our initial design.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-8.png)

#### DESIGN 3.0

With this confirmation, we envisioned a simplified pathway that only required two genes for sustainable growth on creatinine: *crnA* of the creatinine amidohydrolase, and *creA* of the creatinase.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-9.png)

#### BUILD 3.0

Because the two genes are part of the same operon, we decided to clone them into plasmid pSEVA438, under the control of the Pm promoter which is inducible by *m*-toluic acid. The new construct looked like this:

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-10.png)

![](https://static.igem.wiki/teams/5108/engineering/eng-fig4.png)

**Figure 4: Representation of the pSEVA438-creA-crnA plasmid.**

In this construct, the two inserts tap-to-tail are under the control of the same terminator, LUZ7 T50 (BBa\_K4757058), which is bidirectional. The three genes were codon-optimized for *P. putida* using the Codon Optimization Tool from Integrated DNA Technologies (IDT) to lower complexity and minimize secondary structures. The three genes were introduced in a single plasmid by In-Fusion Assembly, and we designed primers with floating tails overlapping adjacent regions for each insert.

#### TEST 3.0

The two genes were introduced in the plasmid by In-Fusion Assembly. Further experiments will be required to assay bacterial growth with this new strain.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-11.png)

## Nitrates

Like bacteria, plants need nutrients to grow. Their carbon source is gaseous CO2 and their main source of nitrogen is located in the soil[3](#ref). In a lunar space station, CO2 will be provided directly by astronauts’ respiration. However, lunar regolith is not composed of any nitrogenous compound. This is why nitrates have to be provided externally. The chosen solution is to have our *Pseudomonas fluorescens*-based biostimulant producing these nitrates by nitrifying the urea resulting from creatinine metabolization. In short, this design aims to implement nitrate-synthesis metabolic pathways into *P. fluorescens*.

#### DESIGN

Nitrification in soil is typically viewed as a two-step process, where ammonia, in our case provided by creatinine degradation, is initially oxidized to nitrite by ammonia-oxidizing bacteria (nitritation), followed by the oxidation of nitrite to nitrate by nitrite-oxidizing bacteria (nitratation).

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-1.png)

To choose the best ammonia- and nitrite-oxidizing genes, we looked into the nitrifying subsystem of the MELiSSA[4](#ref) loop, consisting of a co-culture of *Nitrosomonas europaea* ATCC® 19718 and *Nitrobacter winogradskyi* ATCC® 25391.“MELiSSA (Micro-Ecological Life Support System Alternative) is the European project of circular life support system[5](#ref). (...) The MELiSSA Project is to develop a closed system, inspired by Earth ecosystem, aiming to reproduce its main functions with highly reduced mass and volume, higher kinetics and of course under extreme safety conditions.”

We decided to select three nitrifying genes from these microorganisms and to engineer them in our *Pseudomonas fluorescens*-based biostimulant:* First, the ammonia monooxygenase (Amo, iGEM13\_DTU-Denmark BBa\_K1067003)[6](#ref) from *N. europaea*, consisting of three subunits: CAB. This enzyme catalyzes the conversion of ammonia originating from the degradation of creatinine into hydroxylamine.
* Then, the hydroxylamine dehydrogenase (Hao, iGEM13\_DTU-Denmark BBa\_K1067004)[7](#ref) from *N.europaea*. This enzyme catalyzes the conversion of hydroxylamine into nitric oxide, this compound is then spontaneously oxidized into nitrite.
* Finally, the nitrite oxidoreductase (Nxr) from *N.winogradskyi*, consisting of three subunits AXB. This enzyme catalyzes the final oxidation of nitrites into nitrates.

#### BUILD

The *amoCAB* and *hao* existing parts (iGEM13\_DTU-Denmark BBa\_K1067003 and BBa\_K1067004) and the three *nxr* subunits AXB were optimized for expression in *P. putida* (the closest host organism from *P. fluorescens* available on the sequence optimization tool)thanks to the Codon Optimization Tool from IDT. The optimized *amoCAB* and *hao* sequences were cloned in antisense orientation into the same *pSEVA438-Ptet* vector and separated by the *LUZ7 T50* bidirectional terminator (iGEM23\_Heidelberg BBa\_K4757058)[8](#ref). They are under the control of P*m* and *Ptet* promoters (BBa\_R0040)[9](#ref), respectively. The three optimized *nxrAXB* ORFs were cloned into the pSEVA244 vector under the control of the *Ptrc* promoter (iGEM20\_XMU-China BBa\_K3332038)[10](#ref). Within the same plasmid, each ORF was cloned downstream of a different *Pseudomonas sp.* RBS (BBa-K5108006[11](#ref), BBa-K5108007[12](#ref), BBa-K5108008[13](#ref)), selected to be effective in our strain. *amoCAB* and *nxrAXB* subunit ORFs were cloned in operons, as found in *N. europaea* and *N.winogradskyi*, respectively.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-2.png)

![](https://static.igem.wiki/teams/5108/engineering/eng-fig5.png)

**Figure 5: Representation of the pSEVA438-amoCAB-hao plasmid (A) and pSEVA244-nxrAXB (B).**

#### TEST AND LEARN

In order to test the nitrate pathway, in silico simulations were performed using the adapted metabolic model of *P. fluorescens*. In doing so, we identified the presence of the gene for the enzyme nitric oxide dioxygenase. This enzyme is able to catalyze the conversion of NO to NO3-, therefore simplifying the design for the engineering of this pathway. See modeling [here](https://2024.igem.wiki/toulouse-insa-ups/model).

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-4.png)

#### DESIGN 2.0

Since *P. fluorescens* is capable of catalyzing the oxidation of nitric oxide into nitrate and, we decided to not implement the *nxrAXB* genes. In addition, *hao* ORF (iGEM13\_DTU-Denmark BBa\_K1067004) and its RBS (BBa-K5108006) were eventually cloned into the pSEVA244 vector under the control of the *Ptrc* promoter (iGEM20\_XMU-China BBa\_K3332038) to simplify the construction. Optimized *amoCAB* ORFs (iGEM13\_DTU-Denmark BBa\_K1067003) and their RBS (BBa-K5108006, BBa-K5108007, BBa-K5108008) were cloned into the *pSEVA438* vector without the *LUZ7 T50* bidirectional terminator (iGEM23\_Heidelberg BBa\_K4757058).

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-5.png)

![](https://static.igem.wiki/teams/5108/engineering/eng-fig6.png)

**Figure 6: Representation of the pSEVA438-amoCAB plasmid.**

![](https://static.igem.wiki/teams/5108/engineering/eng-fig7.png)

**Figure 7: Representation of the pSEVA244-hao plasmid.**

## STRESS

#### DESIGN

Another key aspect of our project is the survival of our bacteria to the hostile environment of the Moon. Our initial hypothesis was that our bacteria will be exposed to two major sources of stress.
On the one hand, lunar regolith will impact the bacteria's growth in different ways, the most notable one being osmotic pressure due to solubilization of ions when water is added. Other causes are not well characterized, which is why we decided to focus our efforts on the global stress response to increase our chances of success.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-1.png)

On the other hand, the creatinine metabolization pathway that we implemented leads to the production of H2O2, which can be toxic for the cell at high concentration. This can be added to the oxidative stress caused by regolith itself. Therefore, we aimed at engineering an efficient H2O2 detoxification pathway.

#### BUILD

Firstly, we designed a construct to overproduce the KatB enzyme, a native catalase to *P. fluorescens*, whose function is to decompose hydrogen peroxide into water and oxygen. Overexpression of KatB would promote degradation of H2O2 produced by the creatinine metabolism. We amplified by PCR the *katB* gene from the bacterial genome and cloned it into the pSEVA 244 vector, under control of the promoter *Ptrc* which is inducible with IPTG.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-2.png)

![](https://static.igem.wiki/teams/5108/engineering/eng-fig8.png)

**Figure 8: Representation of the pSEVA244-katB plasmid.**

The second construct consisted of overexpressing two global stress response regulators : Hfq, a small chaperone protein involved in the regulations of mRNA, and RpoS, a specific polymerase subunit. The two genes were cloned on the same transcription cassette, using the bidirectional terminator TerLuz7 (insert registry reference), and cloned into the vector that we previously designed : pSEVA438 -Ptet. *Hfq* was under control of the promoter Pm, inducible with m-toluic acid and *rpoS* under control of the promoter Ptet, inducible with tetracycline.

![](https://static.igem.wiki/teams/5108/engineering/eng-fig9.png)

**Figure 9: Representation of the pSEVA438-Ptet-hfq-rpoS plasmid.**

#### TEST

The plasmids were cloned via In-fusion Assembly and separately transformed into *P. fluorescens* SBW25, creating two new strains, each one tackling a different source of stress.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-3.png)

After transformation, we noticed that none of the two strains was able to effectively produce the proteins of interest.

#### LEARN

Sequencing of the pSEVA438-Ptet-hfq-rpoS plasmid revealed multiple mutations, suggesting that either there had been an error during the cloning process or during proliferation.

 Sequencing of the pSEVA244-katB plasmid validated the correct sequence. However, no protein expression in the soluble fraction was detected. Doing additional research we found that KatB is organized in a homotetramer, which may complicate its production as a soluble form.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-4.png)

A sequence alignment also revealed the presence of a peptide signal, possibly changing the location of the enzyme into the periplasm, a paper released on KatB from *Pseudomonas aeruginosa*[14](#ref) (an homologous protein of our *P. fluorescens* catalase) suggests that the enzyme is in the periplasm.

In the meantime we assessed that *P. fluorescens* growth was not fully inhibited on regolith and could slowly grow on creatinine, despite the toxic byproducts. Therefore, our stress module may improve survival but is not absolutely necessary.

#### DESIGN 2.0

We decided to redo all the steps of the pSEVA438-Ptet-hfq-rpos cloning, in case the mutations occurred somewhere along the process. As for the overexpression of KatB, testing the different hypotheses was not possible within the timeframe of the project.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-5.png)

## Future prospects - Biofilm

Moon regolith’s low water retention limits plant growth. Biofilm provides a potential solution to this problem by enhancing water-holding capacity and improving regolith’s suitability for cultivation. Therefore, we developed a strategy to optimize biofilm formation in *Pseudomonas fluorescens*.

#### DESIGN

##### Introduction

Biofilm formation involves several steps: initial reversible and irreversible attachment to a surface, biofilm maturation, and finally, dispersion[15](#ref). This transition allows bacteria to shift from a free-swimming planktonic form to a sessile, biofilm-forming state. The extracellular polymeric substances (EPSs), which compose the biofilm, enhance resistance to antibiotics and environmental stress[16](#ref). Biofilm from soil organisms can alter soil properties, such as water retention, pore size distribution, surface tension, and viscosity[17, 18](#ref), — key factors in environments like regolith, which poorly retains water. Notably, *Pseudomonas fluorescens* is capable of biofilm formation, making it relevant to our project[19](#ref).

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-1.png)

##### Pathway description

At the molecular level, c-di-GMP is a key bacterial second messenger regulating biofilm formation. In *Pseudomonas*, the Wsp (wrinkly spreader phenotype) signaling pathway is a c-di-GMP-dependent mechanism for biofilm production[20](#ref). The membrane protein WspA is activated by surface growth or pressure. This causes WspE to phosphorylate itself. Phosphorylated WspE then adds phosphate groups to WspF and WspR. Phosphorylated WspR increases c-di-GMP production, which promotes biofilm formation. Meanwhile, phosphorylated WspF inactivates WspA, reducing WspE activity and lowering c-di-GMP levels to decrease biofilm production. Therefore, inhibiting the transcription of *wspF* could increase intracellular c-di-GMP levels, thereby promoting biofilm formation.

#### BUILD

Elevated levels of c-di-GMP in biofilm drive bacteria to rapidly consume large amounts of energy for EPS production, resulting in a reduced metabolic state[21](#ref). To address this, we chose to employ the CRISPR interference method, which allows for gene knockdown rather than complete knockout[22](#ref), enabling targeted gene suppression after reaching the desired biomass. This strategy has also been successfully validated in *Pseudomonas aeruginosa*[23](#ref). We will use it to prevent the expression of *wspF*.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-2.png)

Two components are needed: a dead Cas9 (dCas9) and a guide RNA (sgRNA). The dCas9 binds to the sgRNA, which is complementary to a target DNA sequence near the gene's promoter, using a PAM (Protospacer Adjacent Motif) sequence. This binding sterically prevents the promoter from being activated or directly blocking the transcription elongation. It is important to note that the gene is not deleted from the genome since Cas9 is dead; this inactivation is then reversible. To apply this method, two plasmids are required: one for the dCas9 and another for the sgRNA.

The protein dCas9 we used is from *Streptococcus pasteurianus*, and it proved to be efficient in *P. fluorescens*[24](#ref). We will test two different sgRNA, one targeting the supposed promoter sequence and one at the beginning of the coding sequence of *wspF*. As a control to validate the approach, we will use an mScarlettI plasmid as the gene target and another plasmid containing a designed sgRNA. This control will allow us to quantify the efficiency of the CRISPRi method by fluorescence.

#### FUTURE TEST

Two parameters need to be assessed: water retention and biofilm formation. First, water retention tests will be conducted on fertile soil and regolith without bacteria to show regolith's poor water retention.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-3.png)

After establishing conditions for biofilm overexpression, we will compare water retention between raw regolith and regolith inoculated with *P. fluorescens* (wild-type and engineered strains). We expect that engineered bacteria will enhance biofilm formation and, thus, water retention. Biofilm formation assays will use crystal violet dye, which binds to negatively charged molecules, such as acidic polysaccharides and nucleic acids of the extracellular polymeric substance of the biofilm[25](#ref). This test will be performed on wild-type *P. fluorescens* and engineered strains with sgRNAs targeting *wspF*.

## Plant experiments

The aim of the tests on plants was to provide proof-of-concept experiments showing that plants can survive on regolith, while exhibiting severe signs of stress and are slowed down in their development. Inoculating *P. fluorescens* should help plants grow stronger and live longer.

There are many problems to keep in mind when designing plant biology experiments. Here, we summarized the main issues we encountered, how we built a set-up to do our tests, and what we learned from our results.

#### DESIGN

##### *Arabidopsis thaliana*, a model plant

The first problem we encountered when designing our project was time. We knew that it would take weeks to have any meaningful results when it came to the different conditions we wanted to test. We needed to choose a plant that could grow fast, survive well, and still provide valuable results. We found that in most peer-reviewed articles dealing with plant assays, the model plant used was *Arabidopsis thaliana*. This plant, generally considered as a weed, has a short life cycle (6 weeks, from germination to maturation of the first seeds) and its genotype and phenotype are well characterized[26](#ref). Discussions with various plant biologists also encouraged us to choose *Arabidopsis thaliana* for our experiments.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-1.png)

##### How to obtain significant results

Another issue we anticipated thanks to our extensive bibliographic research was the need for multiple replicates. Assays on plants are extremely variable from one subject to another and it is of the utmost importance to multiplex assays in order to obtain exploitable results. In early May, we had the chance to visit the LIPME (Laboratoire des Interactions Plantes-Microbes-Environnement). During this visit, we gained valuable insight about how to grow our replicates in the limited space of our lab that is neither designed nor equipped for this. Their precious advice coupled with bibliographic research[27](#ref)made us opt for a microplate set-up.

When deciding the number of seeds to plant for each condition to be tested, we had to choose a number high enough to limit the impact of variability in our data analysis, while staying reasonable because of time and space issues. It was essential to ensure the significance and the relevance of the statistical models we wanted to use. Thanks to an online sample size calculator[28](#ref), we estimated that 6 plants per condition was the minimum to get relevant results. To fill the 24-well microplates we chose, we planted 12 plants per condition.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-1.png)

##### Sterilizing seeds

The next challenge we faced was the need to sterilize the *Arabidopsis thaliana* seeds that were donated to us by Sandra Belshiem, a researcher at the LIPME. Because we would add the bacterium *Pseudomonas fluorescens* in the plant medium to test its impact on growth, it was necessary to start from sterile material, to be sure that the impact we would observe would come from our bacterium only, and not from differences in the microbiomes of the seeds. The [protocol](https://2024.igem.wiki/toulouse-insa-ups/protocol#plant) we ended up deciding on included one ethanol wash, one bleach wash and several physiological-water washes to remove any trace of bleach.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-1.png)

##### A controlled environment

Finally, we designed a setup to control the environmental parameters. The purpose was to limit bias and standardize conditions as much as possible.
Firstly, we wanted the plants to grow in a closed place where it would be easy to control temperature, humidity and light. Because the experiments were conducted over the Summer, it was important to ensure that the temperature will not rise too much, which can be deadly for plants.

To be sure to start with only viable seeds we planned to make them germinate before planting only the germinated seedlings, after one week.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-1.png)

#### BUILD

We decided to place the plants in a room, where the temperature always stays between 21 and 23°C and humidity varies only between 55 and 70 % (controlled with a thermometer and hygrometer). We used a Greenception GC 4 Led Module in order to provide optimal UV lighting for the plants, along with an automatic time–switch in 10-14 hour cycles. The lighting system was hung one meter above the plants.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-2.png)

The seeds were sterilized, germinated, and then planted in 6-well microplates. They were watered everyday, once the lighting had gone off.
During the building of our set-up we were lucky enough to receive support from Antoine Berger, a researcher at TBI, our host-lab, who has done extensive work in plant biology before. In 2017, he published an article[29](#ref) with a plate cultivation method on which we adapted our own.
Here is a schematic of the experimental set-up of the growth system:

![](https://static.igem.wiki/teams/5108/engineering/eng-fig10.png)

Figure 10: Set-up of the plant experiments.

![](https://static.igem.wiki/teams/5108/engineering/eng-fig11.png)

Figure 11: Representation of planted well.

#### TEST

Three conditions were tested during our first round of experiments on plants: *A. thaliana* growing in commercial grade soil, *A.thaliana* growing in lunar regolith simulant and *A.thaliana* growing in lunar regolith simulant inoculated with *Pseudomonas fluorescens* SBW25.

After 14 days, there was a significant difference between the plants on fertile ground and those on regolith. In the latter, plants were clearly smaller and less [developed](https://2024.igem.wiki/toulouse-insa-ups/results#titre3). Most importantly, there was a clear difference between the inoculated plants on regolith and the non-inoculated. This means that *P. fluorescens* helps the plant grow stronger and live longer.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-3.png)

#### LEARN

We were able to familiarize ourselves with ImageJ[30](#ref) (version 1.0) which we used to extract data on the color and surface area of the different plants, with R (version 4.4.1 and the package grDevices, version 4.4.1) which we used to process all our data, and with the watering protocol.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-4.png)

#### DESIGN 2.0

Having constructed our first engineered strain, we wanted to test the effect it would have on plant growth. This meant that we had the opportunity to change our experimental set-up. For starters, we decided to move the plants outside of the room with the perfectly controlled humidity and temperature. In addition, we decided to change the photoperiod of plants to 16 hours of light and 8 hours of darkness.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-5.png)

#### BUILD 2.0

The new set-up consisted of a glass aquarium filled with 5 centimeters of water. The plants were inside with a non-sealed lid that allowed air to flow in and out of the aquarium. This allowed humidity to stay between 70 and 80 %. In the new room, temperatures fluctuated between 25 and 30 °C.

Because we were constrained by the space available in the aquarium and we had more conditions to consider, we decided to switch to 24-well microplates. This time, the wells did not have holes at the bottom to aid retain water in the wells.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-6.png)

![](https://static.igem.wiki/teams/5108/engineering/plant-3.jpg)

Figure 12: Second set-up of the plant experiments.

#### TEST 2.0

In order to test our engineered bacteria, several changes needed to be made to the medium. First, we had to add antibiotics, to avoid our strain to lose the plasmid. Secondly, we needed the presence of m-toluic acid, the inducer, to ensure transcription of our genes. Finally, we added creatinine to observe whether the new metabolic pathway had an effect on plant growth. The three components were mixed together and the plants were watered with this mix every three [days](https://2024.igem.wiki/toulouse-insa-ups/protocol#plant).

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-7.png)

Four conditions were tested. We tested the growth of plants on regolith inoculated with the engineered strain with pSEVA438-Ptet-creA-crnA. Secondly, we used a *P. fluorescens* strain transformed with the pSEVA438-Ptet empty plasmid, also resistant to the antibiotic. To attest the influence of the antibiotic, inducer and creatinine, the third condition consisted of plants on non-inoculated regolith treated with the mix. Finally, we did the same control of plants in non-inoculated regolith without the mix-treatment. See results [here](https://2024.igem.wiki/toulouse-insa-ups/results)

#### LEARN 2.0

After 9 days, almost all the plants without bacteria being treated with the mix were dead. In fact, Streptomycin (the antibiotic chosen as a selection marker) was detrimental to plants. In contrast, the plants treated with the mix and inoculated with the antibiotic-resistant bacteria showed significantly higher survival rates than the non-inoculated ones. We concluded that the effect of the bacteria was beneficial, probably because the bacteria was inactivating the antibiotic. In conclusion, any further testing of our engineered bacteria on plants would require chromosomal integration of our genes of interest, thus eliminating the need for the antibiotic.

![](https://static.igem.wiki/teams/5108/engineering/cycledesign-8.png)

## Conclusion

During this process, we learnt that *P. fluorescens* did promote plant growth on regolith. Furthermore, we were able to engineer the bacteria so that it could grow using only a urine-compound, creatinine. Finally, we determined that, for our engineered strain to support plant-growth on the Moon, there would need to be chromosomal integration of our target genes.

![](https://static.igem.wiki/teams/5108/lupso/lupso-astronaut2.webp)

References
*![plus](https://static.igem.wiki/teams/5108/general/plus-large-svgrepo-com.png)*
*![minus](https://static.igem.wiki/teams/5108/general/minus-svgrepo-com.png)*

1. Tsuru, D., Oka, I., & Yoshimoto, T. (1976b). Creatinine Decomposing Enzymes inPseudomonas putida. Agricultural And Biological Chemistry, 40(5), 1011‑1018. [Click](https://doi.org/10.1186/1752-0509-7-74)
2. Part:BBa\_K4757999 [Click](https://parts.igem.org/Part%3ABBa_K4757999)
3. Fredes, I., Moreno, S., Díaz, F. P., & Gutiérrez, R. A. (2018). Nitrate signaling and the control of Arabidopsis growth and development. Current Opinion In Plant Biology, 47, 112‑118.  [Click](https://doi.org/10.1016/j.pbi.2018.10.004%20)
4. Nitrification. (s. d.). Melissa Foundation  [Click](https://www.melissafoundation.org/page/nitrification)
5. Melissa Foundation. (s. d.).  [Click](https://www.melissafoundation)
6. Part:BBa\_K1067003  [Click](https://parts.igem.org/Part%3ABBa_K1067003%20)
7. Part:BBa\_K1067004  [Click](https://parts.igem.org/Part%3ABBa_K1067004)
8. Part:BBa\_K4757999  [Click](https://parts.igem.org/Part%3ABBa_K4757999%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20)
9. Part:BBa\_R0040  [Click](https://parts.igem.org/Part%3ABBa_R0040%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20%20)
10. Part:BBa\_K3332038  [Click](https://parts.igem.org/Part%3ABBa_K3332038)
11. Part:BBa\_K5108006
     [Click](https://parts.igem.org/cgi/partsdb/part_info.cgi?part_name=BBa_K5108006                            )
12. Part:BBa\_K5108007
     [Click](https://parts.igem.org/cgi/partsdb/part_info.cgi?part_name=BBa_K5108007                            )
13. Part:BBa\_K5108008
     [Click](https://parts.igem.org/cgi/partsdb/part_info.cgi?part_name=BBa_K5108008)
14. Panmanee, W., & Hassett, D. J. (2009). Differential roles of OxyR-controlled antioxidant enzymes alkyl hydroperoxide reductase (AhpCF) and catalase (KatB) in the protection ofPseudomonas aeruginosaagainst hydrogen peroxide in biofilm vs. planktonic culture. FEMS Microbiology Letters, 295(2), 238‑244.  [Click](https://doi.org/10.1111/j.1574-6968.2009.01605.x%20)
15. Rather, M. A., Gupta, K., & Mandal, M. (2021). Microbial biofilm : formation, architecture, antibiotic resistance, and control strategies. Brazilian Journal Of Microbiology, 52(4), 1701‑1718.  [Click](https://doi.org/10.1007/s42770-021-00624-x%20)
16. Baum, M. M., Kainović, A., O’Keeffe, T., Pandita, R., McDonald, K., Wu, S., & Webster, P. (2009). Characterization of structures in biofilms formed by a Pseudomonas fluorescens isolated from soil. BMC Microbiology, 9(1), 103.  [Click](https://doi.org/10.1186/1471-2180-9-103%20)
17. Zheng, W., Zeng, S., Bais, H., LaManna, J. M., Hussey, D. S., Jacobson, D. L., & Jin, Y. (2018). Plant Growth‐Promoting Rhizobacteria (PGPR) Reduce Evaporation and Increase Soil Water Retention. Water Resources Research, 54(5), 3673‑3687.  [Click](https://doi.org/10.1029/2018wr022656%20)
18. Rosenzweig, R., Shavit, U., & Furman, A. (2011b). Water Retention Curves of Biofilm‐Affected Soils using Xanthan as an Analogue. Soil Science Society Of America Journal, 76(1), 61‑69  [Click](https://doi.org/10.2136/sssaj2011.0155)
19. O’Toole, G. A., & Kolter, R. (1998). Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways : a genetic analysis. Molecular Microbiology, 28(3), 449‑461.  [Click](https://doi.org/10.1046/j.1365-2958.1998.00797.x%20)
20. Francis, V. I., Stevenson, E. C., & Porter, S. L. (2017b). Two-component systems required for virulence in Pseudomonas aeruginosa. FEMS Microbiology Letters, 364(11).  [Click](https://doi.org/10.1093/femsle/fnx104)
21. Lichtenberg, M., Kragh, K. N., Fritz, B., Kirkegaard, J. B., Tolker-Nielsen, T., & Bjarnsholt, T. (2022). Cyclic-di-GMP signaling controls metabolic activity in Pseudomonas aeruginosa. Cell Reports, 41(3), 111515  [Click](https://doi.org/10.1016/j.celrep.2022.111515)
22. Zhang, R., Xu, W., Shao, S., & Wang, Q. (2021). Gene Silencing Through CRISPR Interference in Bacteria : Current Advances and Future Prospects. Frontiers In Microbiology, 12.  [Click](https://doi.org/10.3389/fmicb.2021.635227%20)
23. O’Neal, L., Baraquet, C., Suo, Z., Dreifus, J. E., Peng, Y., Raivio, T. L., Wozniak, D. J., Harwood, C. S., & Parsek, M. R. (2022). The Wsp system ofPseudomonas aeruginosalinks surface sensing and cell envelope stress. Proceedings Of The National Academy Of Sciences, 119(18).  [Click](https://doi.org/10.1073/pnas.2117633119%20)
24. Tan, S. Z., Reisch, C. R., & Prather, K. L. J. (2018). A Robust CRISPR Interference Gene Repression System in Pseudomonas. Journal Of Bacteriology, 200(7).  [Click](https://doi.org/10.1128/jb.00575-17)
25. Preparation for endoscopy. (2011). Dans Elsevier eBooks (p. 52‑83).  [Click](https://doi.org/10.1016/b978-0-7020-3128-1.00002-x)
26. Meinke, D. W. (1998). Arabidopsis thaliana: A Model Plant for Genome Analysis. Science, 282(5389), 662–682.  [Click](10.1126/science.282.5389.662%20)
27. Anna Lisa-Paul (2022). Plants grown in Apollo lunar regolith present stress-associated transcriptomes that inform prospects for lunar exploration. Nature, 382(2022).  Click
28. Schoenfeld DA. Sample-size formula for the proportional-hazards regression model. Biometrics 1983;39:499-503.  [Click](https://pubmed.ncbi.nlm.nih.gov/6354290/%20)
29. Pierre Piétracq (2017). Metabolite profiling of non‐sterile rhizosphere soil. The Plant Journal, 92(1): 147–162.  Click
30. Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA  [Click](https://imagej.net/ij/%2C%201997-2018.)

[Results](https://2024.igem.wiki/toulouse-insa-ups/results)

[Project Description](https://2024.igem.wiki/toulouse-insa-ups/description)

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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](https://parts.igem.org/Main_Page).

## Inspirations

---

* [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)

{% endblock %}

![mascotte1](https://static.igem.wiki/teams/5108/general/back-top-static.png)

# Results

* ### Content
* [Engineering a *Pseudomonas fluorescens* strain that can grow on creatinine](#titre1)
* [Engineering a *Pseudomonas fluorescens* strain that can cope with oxidative stresses](#titre2)
* [Testing the biostimulant properties of *Pseudomonas fluorescens* for plants on regolith](#titre3)
* [Conclusion](#titre4)
* [References](#ref)

BioNoob
BioNoob

This section presents our wet lab achievements. Our main goal was to engineer the bacterium *Pseudomonas fluorescens* - our BioMoon biostimulant - so that it could be grown from a yet non-recycled substrate in a space base. After successfully completing this task, we considered improving the bacterial capacity to facilitate plant growth on lunar regolith. We eventually demonstrated the ability of our strain to act as a plant biostimulant on regolith.

## Engineering a *Pseudomonas fluorescens* strain that can grow on creatinine

#### The wild-type strain *Pseudomonas fluorescens* SBW25 does not grow on creatinine

##### Objectives

The pathway for **creatinine degradation** is well characterized in some *Pseudomonas* bacteria, e.g., *P. putida* [1](#ref). However, creatinine metabolization by our strain of interest *P. fluorescens* has not been investigated so far. Our objective was to assess whether the wild-type strain *P. fluorescens* was able to metabolize creatinine and the downstream compounds of the pathway.

##### Method

We studied growth of *P. fluorescens* on each compound of the metabolic pathway separately, namely creatinine, creatine, sarcosine, and glycine on a M9 minimal medium deprived of NH4Cl (i.e., no additional nitrogen source). The growth was monitored by measuring OD at 600 nm in a 96-well microplate with a microplate reader.

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

##### Hypothesis

*In silico* simulations obtained from the GSMM (Genome-Scale Metabolic Model) of *P. fluorescens* helped us make predictions about bacterial growth under different [compounds](https://2024.igem.wiki/toulouse-insa-ups/model#titre5). The modeling predicted that glycine supports growth of the wild-type (WT) *P. fluorescens* strain. Sarcosine should also be usable as a substrate. Moreover, the presence of a sarcosine oxidase gene, catalizing the sarcosine degradation into glycine, was uncovered in the genome of *P. fluorescens*. However, the model predicts that growth on creatinine and creatine is not possible in the WT strain.

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

##### Results

We experimentally tested these hypotheses and found that *P. fluorescens* SBW25 was only able to grow on sarcosine as a sole source of carbon and nitrogen (56 mM) with a maximal growth rate of 0.16 ± 0.01 h–1 (mean ± standard deviation, from 9 biological replicates), but not on creatinine (44 mM), creatine (38 mM), nor glycine (67 mM) (Figure 1).

![](https://static.igem.wiki/teams/5108/results/fig1-resu.jpg)

**Figure 1: growth of the wild-type *Pseudomonas fluorescens* SBW25 strain on M9 minimal medium complemented with creatinine, creatine, sarcosine or glycine.**Measurement of the OD at 600 nm every 30 minutes during 24 hours. The mean value of the biological and technical triplicates for each condition is shown (n=9). The growth rate on sarcosine is 0.158 ± 0.008 h–1 (n=9).

To quantify the degradation of sarcosine by the WT *P. fluorescens* strain, we monitored **sarcosine uptake** using Nuclear Magnetic Resonance (NMR) spectroscopy (500 MHz NMR spectrometer Bruker). NMR analysis was conducted on the supernatants collected during the exponential growth phase of the bacteria. A **sarcosine uptake rate of 5.2 mmol.h⁻¹.gDW⁻¹** was obtained (Fig. 2), while the measured growth rate under this condition was 0.19 h⁻¹ (n=1). The cultures used for this analysis were grown in Erlenmeyer flasks, providing more effective aeration and agitation compared to microplate experiments, which may explain the higher growth rate.

![](https://static.igem.wiki/teams/5108/results/fig2-resu-2.png)

**Figure 2: uptake of sarcosine by the wild-type *Pseudomonas fluorescens* SBW25 strain on M9 minimal medium complemented with sarcosine.**Samples of supernatants were taken every hour during the exponential phase and sarcosine concentration was analyzed by 500 MHz NMR spectrometry. Simulation of sarcosine uptake (dashed line) was done with Physiofit. The shaded area indicates the 95% confidence interval.

To validate the predictability of our metabolic model, we used the experimentally measured sarcosine uptake rate as an input parameter and the model predicted a growth rate of 0.20 h⁻¹, which is in perfect agreement with the experimentally observed rate of 0.19 h⁻¹. These results strongly suggest that the **sarcosine degradation pathway is functioning optimally in the WT strain**, and that our whole-cell model is able to give **accurate predictions** about metabolic fluxes.

The results from Figure 1 also demonstrate that *P. fluorescens* is unable to utilize glycine as a sole carbon and nitrogen source *in vivo*. However, *P. putida* is able to degrade creatinine following the creatinine metabolic pathway which ends with glycine conversion and provides a source of carbon and nitrogen[2, 3](#ref) (Figure 3). The obtained results from *P. fluorescens* are different, except for the sarcosine oxidase (SoxA) which catalyzes the conversion of sarcosine into glycine with co-production of formaldehyde and H2O2:

![](https://static.igem.wiki/teams/5108/results/fig3-resu.png)

**Figure 3: metabolic pathway of the creatinine degradation in *Pseudomonas putida*.**

It is important to note that, although *P. fluorescens* cannot grow on creatinine, creatine, or glycine as sole carbon and nitrogen sources, it may still be capable of utilizing creatinine or glycine as nitrogen sources. To explore this hypothesis, we supplemented the M9 minimal medium with sarcosine, creatine, or creatinine along with citrate, which served as primary carbon source. Citrate, a key intermediate in the TCA cycle, allows bacteria to produce energy and biosynthetic precursors. In addition, modeling simulations demonstrated that citrate can be co-consumed with [creatinine](https://2024.igem.wiki/toulouse-insa-ups/model#titre4).

This was experimentally demonstrated. The WT strain significantly grew on creatinine supplemented with citrate (Figure 4, left), exhibiting a clear exponential phase. In contrast, no growth was observed when citrate was provided along with creatine (Figure 4, center). Bacterial growth was also observed on glycine and citrate (0.28 ± 0.01 h⁻¹ (n=9), Figure 4, right), with a growth curve slightly delayed compared to that of creatinine and citrate (0.29 ± 0.01 h⁻¹ (n=9), Figure 4, left).

These findings suggest that *P. fluorescens* is **unable** to use **creatinine**, creatine, or glycine **as sole carbon and nitrogen sources**. The WT strain **can utilize creatinine** and glycine only **as nitrogen sources when citrate is provided as the primary carbon source**, but not creatine.

![](https://static.igem.wiki/teams/5108/results/fig4-resu-4.jpg)

**Figure 4: growth of the wild-type strain *Pseudomonas fluorescens* SBW25 on M9 minimal medium complemented with creatinine, creatine or glycine along with citrate.** Measurement of the OD at 600 nm every 30 minutes during 20 hours. The mean value of the biological and technical triplicates for each condition is shown (n=9). The growth rate on citrate and creatinine is 0.289 ± 0.003 h-1 (n=9). The growth rate on citrate and glycine is 0.282 ± 0.009 h-1 (n=9).

##### New questions arise

Why didn’t bacteria grow on citrate plus creatine?
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![](https://static.igem.wiki/teams/5108/results/lupso-question.png)

Further experiments will be needed to explain why the WT strain does not grow in the presence of citrate and creatine. Bacteria could either be unable to effectively metabolize creatine as primary nitrogen source, or creatine diffusion/transport across the bacterial membrane may be limiting.

Why didn’t bacteria grow on glycine alone?
*![plus](https://static.igem.wiki/teams/5108/general/plus-large-svgrepo-com.png)*
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![](https://static.igem.wiki/teams/5108/results/lupso-question.png)

According to the creatinine metabolic pathway in *P. putida*[4](#ref) and *in silico* results, sarcosine is degraded into glycine and formaldehyde (Figure 3). We have demonstrated that glycine cannot be used as a carbon and nitrogen source but only as a nitrogen source (Figure 1 and 4). As *P. fluorescens* can grow on sarcosine, we suggest that **formaldehyde**, a by-product of sarcosine degradation was further converted into formic acid, which was then used **as carbon source**, while glycine was used as nitrogen source. We tested this hypothesis *in* [*silico*](https://2024.igem.wiki/toulouse-insa-ups/model#titre5).

Formaldehyde was indeed observed to have a large impact on *P. fluorescens* growth rate. To verify the in silico predictions, we cultured bacteria on M9 minimal medium supplemented with 10 mM formate and 67 mM glycine. *P. fluorescens* **was not able to grow under this condition**. Further experiments are required to understand how sarcosine is metabolized by *P. fluorescens* and what the influence of the reaction products is on growth.

How to make *P. fluorescens* grow on creatine or creatinine?

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![](https://static.igem.wiki/teams/5108/results/lupso-question.png)

To enable *P. fluorescens* to grow on creatine or creatinine as sole carbon and nitrogen sources, we proposed to **introduce the creatinine metabolization pathway from *P. putida*** into *P. fluorescens*. The genes ***creA*** and ***crnA*** were cloned in *P. fluorescens* to convert creatinine into creatine and then into sarcosine. Simulation results from our **in silico model showed that *P. fluorescens* expressing CreA and CrnA enzymes should be able to grow on creatinine alone**. This prompted us to engineer a strain with the *creA* and *crnA* genes.

#### Construction of the plasmid pSEVA438-Ptet cloned with the operon *creA-crnA* and *soxA* genes

##### Objectives

The initial design aimed at cloning the genes *creA, crnA,* and *soxA* into pSEVA438-Ptet vector to obtain the pSEVA438-Ptet-creA-crnA-soxA plasmid for transformation of *P. fluorescens*.

##### Method

The strategy to construct the pSEVA438-Ptet-creA-crnA-soxA plasmid was to perform a three-step cloning through In-Fusion Assembly, transformation into *E. coli Stellar Competent Cells*, vector isolation and linearisation by PCR (Fig. 5). The first cloning step introduced the *Ptet* promoter from the pHD\_SS9\_PLtetO1\_TLt0\_eutC-GFP plasmid into the pSEVA438 vector. Then, the *creA-crnA* operon (BBa\_K5108009) was cloned. Finally, the bidirectional terminator LUZ7 T50 (BBa\_K4757058) and the *soxA* gene were cloned. The final construct was transformed into *P. fluorescens*.

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

We experimentally tested these hypotheses and found that *P. fluorescens* SBW25 was only able to grow on sarcosine as a sole source of carbon and nitrogen (56 mM) with a maximal growth rate of 0.16 ± 0.01 h–1 (mean ± standard deviation, from 9 biological replicates), but not on creatinine (44 mM), creatine (38 mM), nor glycine (67 mM) (Figure 1).

![](https://static.igem.wiki/teams/5108/results/fig5-resu.png)

**Figure 5: schematic of the cloning strategy for pSEVA438-Ptet-creA-crnA plasmid.**Measurement of the OD at 600 nm every 30 minutes during 24 hours. The mean value of the biological and technical triplicates for each condition is shown (n=9). The growth rate on sarcosine is 0.158 ± 0.008 h-1 (n=9).

##### Results

We **succeeded in cloning the Ptet promoter into pSEVA438** and the **creA-crnA operon** (BBa\_K5108009) into the pSEVA438-Ptet backbone, as shown in Figure 6. The construct was confirmed by Sanger sequencing (Genewyz, Germany). However, we failed to introduce the bidirectional terminator LUZ7 T50 (BBa\_K4757058) and the *soxA* gene into the pSEVA438-Ptet-creA-crnA plasmid. As we demonstrated that *P. fluorescens* could grow on sarcosine, the *soxA* gene should be unnecessary for bacteria to grow on creatinine. This is why we decided to directly transform *P. fluorescens* with the pSEVA438-Ptet-creA-crnA plasmid.

![](https://static.igem.wiki/teams/5108/results/fig6-resu.png)

**Figure 6: restriction digest of pSEVA438-Ptet-creA-crnA plasmid.**The plasmid was digested with EcoRI and HindIII separately or in combination. The expected (left) and experimental (right) digestion patterns are shown.

#### The engineered strain *Pseudomonas fluorescens* SBW25 can express creatinine amidohydrolase (CrnA) and creatinase (CreA)

##### Objectives

The objective was to verify the overproduction of the creatinine amidohydrolase (CrnA) and creatinase (CreA) in *P. fluorescens* by activation of the P*m* promoter with the inducer *m*-toluic acid.

##### Method

The pSEVA438-MBPeGFP plasmid, originally used in *P. putida* KT2440, was employed as positive control of P*m* promoter's inducibility in *P. fluorescens*. This construct encodes the maltose-binding protein (MBP) fused to eGFP (enhanced Green Fluorescent Protein) under the control of the P*m* promoter and is issued from Vogeleer et al. (2024)[5](#ref). We cultured the pSEVA438-MBPeGFP- and pSEVA438-Ptet-creA-crnA-transformed *P. fluorescens* in M9 minimal medium supplemented with glucose (28 mM), with or without 0.5 mM of *m*-toluic acid inducer. After incubation, a whole-protein extraction was performed for each strain to assess the level of expression, as well as the solubility of our proteins.

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

##### Hypothesis

We expected that MBPeGFP, creatinine amidohydrolase (CrnA) and creatinase (CreA) expression should be activated in the presence of *m*-toluic acid. These proteins should be soluble with an expected molecular weight of 69 kDa, 28 kDa and 46 kDa, respectively.

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

##### Results

The obtained SDS-PAGE is presented in Figure 7.

![](https://static.igem.wiki/teams/5108/results/fig7-resu.png)

**Figure 7 : SDS-PAGE of soluble and insoluble protein fractions from cultures of *Pseudomonas fluorescens* transformed with pSEVA438-MBPeGFP or pSEVA438-Ptet-creA-crnA.** *P. fluorescens* was cultured with or without the inducer *m*-toluic acid. Arrows indicate the expected size of MBPeGFP, creatinine amidohydrolase (CrnA) and creatinase (CreA).

Both soluble and insoluble fractions contained MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. Although transcriptional leakage was clearly observed without the inducer, MBPeGFP was overproduced when the P*m* promoter was activated with 0.5 mM of m-toluic acid, **confirming the functionality of the P*m* promoter in *P. fluorescens***. The presence of insoluble MBPeGFP can be caused by its overexpression leading to protein aggregation.
SDS-PAGE analysis of the cell lysate derived from *P. fluorescens* transformed with pSEVA438-Ptet-creA-crnA revealed a **clearly visible band at the expected size of CreA** in both soluble and insoluble fractions when its expression is induced. As expected based on the leaky expression of MBPeGFP, CreA is also produced in lower quantities without the inducer in the soluble protein fraction. In contrast, there is **no visible band at the expected size of CrnA**, suggesting that the *crnA* gene is not or poorly expressed.

##### New questions arise

Why is CrnA expressed at lower yield than CreA?
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Both genes are expressed from an operon system, i.e., from a single transcription cassette. In such genetic systems, the expression level of the downstream gene, here *crnA*, can be lower than that of the upstream gene, *creA*.

#### The engineered strain *Pseudomonas fluorescens* SBW25 expressing creatinine amidohydrolase (CrnA) and creatinase (CreA) grows on creatinine

##### Objectives

Our goal was to implement the metabolic pathway of creatinine degradation in *P. fluorescens* and to assay the growth of the engineered strain on creatinine, creatine, or sarcosine.

##### Method

We studied the growth dynamics of *P. fluorescens* on each compound of the metabolic pathway of creatinine degradation into sarcosine separately, namely creatinine, creatine and sarcosine. The M9 minimal medium did not contain NH4 Cl and growth was monitored by measuring OD at 600 nm in a 96-well microplate with a microplate reader. Production of CreA and CrnA was induced during the pre-culture to avoid a too long lag phase during growth curve measurements.

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

##### Hypothesis

We expected growth of our engineered strain on creatine after induction with *m*-toluic acid. We did not expect growth on creatinine even after induction with *m*-toluic acid because of the absence of CrnA on the SDS-PAGE (Figure 7). Bacteria transformed with the empty plasmid should not grow either (negative control).

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

##### Results

Growth curves of three different strains of *Pseudomonas fluorescens* were monitored on M9 minimal medium supplemented with creatinine, creatine, or sarcosine as sole carbon and nitrogen sources:

* the wild-type strain (negative control),
* a strain transformed with the empty plasmid pSEVA438-pTET (negative control),
* a strain transformed with the plasmid pSEVA438-pTET creA-crnA (engineered strain).

![](https://static.igem.wiki/teams/5108/results/fig8-resu.png)

**Figure 8: Growth of the engineered *Pseudomonas fluorescens* SBW25 strain with creA-crnA operon on minimal medium supplemented with creatinine, creatine, or sarcosine.** Measurement of the OD at 600 nm every 30 minutes. The mean value of the technical replicates for each condition is shown (n ≥ 3). Growth curves of the WT strain and the strain transformed with the empty plasmid pSEVA438-Ptet are also shown.

No growth was observed in either the WT or the negative control strains when cultured with **creatinine**, confirming that our strain *P. fluorescens SBW25* does not naturally metabolize this compound. In contrast, the **engineered strain** harboring the *creA* and *crnA* genes **exhibited clear growth on creatinine**, demonstrating that **the introduced metabolic pathway is functional** with a growth rate of 0.06 h⁻¹ (Figure 9).

![](https://static.igem.wiki/teams/5108/results/fig9-resu.jpg)

**Figure 9: Growth rate of the engineered and the wild-type strains of *Pseudomonas fluorescens* SBW25 on minimal medium supplemented with creatinine, creatine, or sarcosine.** Measurement of the OD at 600 nm every 30 minutes. The mean value of the biological and technical replicates for each condition is shown (n ≥ 3). The error bars represent the standard deviation.

In analogy with the results obtained with creatinine, neither the WT nor the negative control strains grew on creatine. However, the strain engineered with creA-crnA operon showed moderate growth, confirming that the **transformed bacteria could also metabolize creatine**. The same growth has been observed for growth on creatine as a sole source of carbon and nitrogen.

However, the growth rate on sarcosine (0.10 h⁻¹) was higher than on creatinine, indicating that the pathway for creatinine degradation may not be as efficient as for sarcosine. This suggests potential possibilities to optimize the metabolic pathway, as differences in growth rates point to bottlenecks or suboptimal expression of the enzymes involved in creatinine degradation.

Using **sarcosine** as a substrate, all three strains could grow, as expected. Growth rates (Figure 9) of the strains transformed with the empty plasmid (0.11 h⁻¹) or with the pSEVA438-Ptet-creA-crnA plasmid (0.10 h⁻¹) were lower than that of the WT strain (0.15 h⁻¹), indicating that plasmid introduction had an adverse effect on the growth rate. This could be explained by a metabolic burden on the bacteria caused by the production of CreA and CrnA , along with the cost of maintaining antibiotic resistance.

Overall, these results clearly demonstrate that **transformation of *P. fluorescens* with the plasmid pSEVA438-pTET-creA-crnA enabled the bacteria to grow on creatinine** and creatine, whereas the WT and negative control strains could not utilize these nutrients. This validates our engineering approach and the constructed DNA [creatinine](https://2024.igem.wiki/toulouse-insa-ups/parts) parts.

##### New questions arise

How can we further optimize the growth of bacteria on creatinine?
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The slower growth rates on creatinine compared to sarcosine suggests room for further optimization to improve the pathway efficiency. For instance, we suggest to increase the activity of creatinase (CreA).

CrnA, the first enzyme in the creatinine pathway (Figure 3), catalyzes the hydrolysis of creatinine into creatine with a catalytic efficiency (kcat/Km​) of 1.48 s⁻¹·mM⁻¹. This high efficiency makes CrnA[6](#ref) highly effective in processing creatinine. In contrast, CreA[7](#ref), which catalyzes the conversion of creatine to urea and sarcosine, has a much lower catalytic efficiency of 0.02 s⁻¹·mM⁻¹, mainly due to its low catalytic constant kcat (0.246 s⁻¹). This significant difference creates a bottleneck in the pathway, where creatine accumulates more quickly than it can be metabolized by CreA.

The genetic organization as an operon has a notable effect on the expression levels of the enzymes. CreA, located upstream of CrnA in the operon, is expressed at higher levels, as demonstrated by SDS-PAGE analysis (Figure 7). This difference in expression compensates for the lower catalytic efficiency of CreA compared to CrnA, helping to balance the overall metabolic process. This likely explains why in spite of the poor expression of CreA, the construction successfully allowed creatinine consumption.

To further optimize the pathway, we propose redesigning the genetic organization of our construct (Figure 5). Currently, both *creA* and *crnA* are expressed from the same operon, which limits our control over the individual expression levels of these enzymes. By placing *creA* and *crnA* under the control of their own distinct promoters and terminators, we could independently regulate the expression levels of each enzyme.

To better understand the pathway and optimize the genetic construct, it would be crucial to gain more insights into the uptake fluxes of each substrate (sarcosine, creatine and creatinine) in the transformed bacteria. By quantifying how efficiently sarcosine, creatine, and creatinine are imported into the cells, we could identify potential bottlenecks that affect growth rates, which could further inform us to redesign the metabolic pathway.

#### NMR Analysis of Creatinine, Creatine, and Sarcosine Uptake and Degradation in engineered strain *Pseudomonas fluorescens* SBW25

##### Objectives

The objective of these experiments was to monitor and quantify the degradation of sarcosine, creatine, and creatinine by *Pseudomonas fluorescens* transformed with the creA-crnA plasmid, and to compare the experimental growth and degradation rates to those predicted by our in silico metabolic model.

##### Method

Supernatants from bacterial cultures were collected during the exponential growth phase and analyzed using 500 MHz NMR spectroscopy to monitor the concentrations of sarcosine, creatine, and creatinine over time. Growth rates and uptake rates were calculated using Physiofit[8](#ref).

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

##### Hypothesis

We expected that *Pseudomonas fluorescens* carrying the creA-crnA plasmid efficiently degrades sarcosine, creatine, and creatinine. Moreover, we expected the uptake flux to be similar in every condition.

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

##### Results

###### Sarcosine degradation:

As shown in Figure 10, the experimental data reveals a clear decrease in sarcosine concentration over time, which can be attributed to the uptake and subsequent degradation by the cells. A **sarcosine uptake rate of 3.6 mmol.h⁻¹.gDW⁻¹** was obtained and the measured growth rate under this condition was 0.10 h⁻¹ (n=1). The lower growth rate observed in the transformed *Pseudomonas fluorescens* strain on sarcosine (0.10 h⁻¹) compared to the wild-type (0.19 h⁻¹) in the same condition (growth in Erlenmeyer flasks) can likely be attributed to the metabolic burden imposed by the plasmid carrying the *creA-crnA* genes.

![](https://static.igem.wiki/teams/5108/results/fig-resu10.png)

**Figure 10: Uptake of sarcosine by the transformed pSEVA438-Ptet-creA-crnA *Pseudomonas fluorescens* SBW25 strain on M9 minimal medium complemented with sarcosine.** Samples of supernatants were taken every hour during the exponential phase and sarcosine concentration was analyzed by 500 MHz NMR spectroscopy. Simulation of sarcosine uptake (dashed line) was done with Physiofit. The shaded area indicates the 95% confidence interval.

To validate the predictability of our metabolic model, we used the experimentally measured sarcosine uptake rate as an input parameter and the model predicted a growth rate of 0.11 h⁻¹, which is in **perfect agreement with the experimentally observed rate**.

###### Creatine degradation:

The degradation of creatine as the only substrate was also monitored by NMR (Figure 11). The experimental data fit well with the simulated kinetics, further validating our metabolic model for creatine degradation. A **creatine uptake rate of 2.05 mmol.h⁻¹.gDW⁻¹** was obtained and the measured growth rate under this condition was 0.05 h⁻¹ (n=1). To validate the predictability of our metabolic model, we also used the experimentally measured creatine uptake rate as an input parameter and **the model predicted** a growth rate of 0.08 h⁻¹, which **remains in agreement** with the experimentally observed rate.

![](https://static.igem.wiki/teams/5108/results/fig-resu-11.png)

**Figure 11: Uptake of creatine by the transformed strain creA-crnA *Pseudomonas fluorescens* SBW25 on M9 minimal medium supplemented with creatine.** Samples of supernatants were taken every hour during the exponential phase and creatine concentration was analyzed by 500 MHz NMR spectroscopy. Simulation of creatine uptake (dashed line) was done with Physiofit. The shaded area indicates the 95% confidence interval.

###### Creatinine Degradation:

Lastly, the degradation of creatinine was measured (Figure 12). Although the uptake rate of creatinine was higher than that of sarcosine and creatine, the observed growth rate was, in fact, lower. This indicates a lack of optimisation of the creatinine degradation, which may be due to a low expression of the gene *crnA*, as discussed above. A **creatinine uptake rate of 3.58 mmol.h⁻¹.gDW⁻¹** was obtained and the measured growth rate under this condition was 0.03 h⁻¹ (n=1). To validate the predictability of our metabolic model, we also used the experimentally measured creatinine uptake rate as an input parameter and the model predicted a growth rate of 0.14 h⁻¹, **which is higher in comparison to the experimentally observed rate**.

![](https://static.igem.wiki/teams/5108/results/fig12-resu-12.png)

**Figure 12: Uptake of creatinine by the transformed strain *creA-crnA Pseudomonas fluorescens* SBW25 on M9 minimal medium supplemented with creatinine.** Samples of supernatants were taken every hour during the exponential phase and creatinine concentration was analyzed by 500 MHz NMR spectroscopy. Simulation of creatinine uptake (dashed line) was done with Physiofit. The shaded area indicates the 95% confidence interval.

###### Conclusion:

These results demonstrate that ***Pseudomonas fluorescens*** carrying the *creA-crnA* plasmid can degrade sarcosine, creatine, and creatinine, and utilize each of them as a sole source of carbon and nitrogen. As summarized in Table 1, the experimental data from NMR were in good agreement with the simulations for sarcosine and creatine uptake, validating our model and overall approach.

**Table 1: Measured and predicted growth and uptake rates for sarcosine, creatine and creatinine.** The experimental measured growth rates were obtained in Erlenmeyer flasks (n=1). The predicted values for the growth rate were calculated using the GSMM based on the experimentally measured uptake rates. The predicted uptake rates, on the other hand, were calculated from the experimentally observed growth rates.

![](https://static.igem.wiki/teams/5108/results/tab-resu.png)

##### New questions arise

Since a new metabolic pathway has been introduced, what could be the consequences in terms of co-product production?
*![plus](https://static.igem.wiki/teams/5108/general/plus-large-svgrepo-com.png)*
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![](https://static.igem.wiki/teams/5108/results/lupso-question.png)

Take a look at the Figure 3 for a better understanding.

The 3 co-products of the creatinine degradation pathway are:* Urea, produced during the conversion of creatine to sarcosine, is naturally degraded by *P. fluorescens*[9](#ref). This presents an opportunity to exploit the ammonia released during urea degradation for the production of nitrate — a critical nutrient for plants. This strategy has been integrated in the design of a **nitrate production module** as a perspective to enhance plant growth.
* **Hydrogen peroxide**, another by-product, is known to induce oxidative stress in bacteria, potentially inhibiting growth. To mitigate this, *P. fluorescens* could be engineered to become more tolerant to oxidative stress by incorporating stress-resistance genes. We designed a **stress resistance module** to help bacteria cope with elevated H₂O₂ levels.
* Formaldehyde, a toxic by-product, may pose a growth challenge. However, according to Dr. Charles Cockell, with whom we met on the 30th of May, formaldehyde may be absorbed by regolith, reducing its impact on bacterial survival. Therefore, formaldehyde toxicity might not be a significant issue.

## Engineering a *Pseudomonas fluorescens* strain that can cope with oxidative stresses

To go further in optimizing the project, we wanted to reduce oxidative stress-related damages by overexpressing the catalase (KatB) that degrades H2O2 which is produced during glycine synthesis from sarcosine. H2O2 generates superoxide radicals, which can damage cellular components, including DNA, proteins, and lipids. In addition, we knew that regolith is a hostile environment for both [plants](#titre3) and bacteria. To help *P. fluorescens* overcome the stress it could be exposed to, we intended to stimulate the global stress response by overexpressing the native stress factors *hfq* and *rpoS*.

#### Oxidative stress has an impact on bacterial growth

##### Objectives

The first objective was to observe how *P. fluorescens* reacted to oxidative stress.

##### Method

We monitored growth of *Pseudomonas fluorescens* during the exponential phase on M9 minimal medium supplemented with sarcosine (56 mM) under varying concentrations of paraquat, a molecule known to induce oxidative stress.

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

##### Hypothesis

We expected *Pseudomonas fluorescens* to be sensitive and to stop growing at high paraquat concentrations.

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

##### Results

As shown in Figure 13.A, growth of ***Pseudomonas fluorescens* was affected by paraquat** in a concentration dependent manner, with a strong inhibition from 1 mM.

Calculated growth rate dropped from 0.16 h⁻¹ at 0 mM of paraquat (control) down to 0.03 h⁻¹ at 10 mM (Figure 13.B), following the reduction of biomass measured at OD600. This result indicates that the WT strain is significantly affected by oxidative stress. Interestingly, bacteria still exhibited measurable growth at 0.1 mM paraquat, suggesting some natural ability to cope with moderate levels of oxidative stress, likely through the activation of **detoxification mechanisms** mediated by catalases.

![](https://static.igem.wiki/teams/5108/results/fig13-resu-13.png)
![](https://static.igem.wiki/teams/5108/results/fig13b-resu-13b.png)

**Figure 13 : (A) Growth of *Pseudomonas fluorescens* SBW25 under different concentrations of paraquat.** OD measurements every 10 minutes were taken over 30 hours. The medium is a minimal medium (M9) supplemented with glucose. 40 µL of paraquat was added at 7.5 hours at different concentrations of 0, 0.1, 1, and 10 mM paraquat. For 0 mM of paraquat, distilled water was added. Data points represent the mean ± standard deviation for each condition (n = 3). **(B) Specific growth rates of *Pseudomonas fluorescens* SBW25 under different concentrations of paraquat.** Growth rates were calculated for each condition (0, 0.1, 1, and 10 mM paraquat) with error bars representing the standard deviation (n = 3).

##### New questions arise

How can we lessen oxidative damages?
*![plus](https://static.igem.wiki/teams/5108/general/plus-large-svgrepo-com.png)*
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![](https://static.igem.wiki/teams/5108/results/lupso-question.png)

To mitigate the oxidative stress caused by H₂O₂ during creatinine degradation, an effective strategy would be **to enhance the bacterial strain’s resistance** to radical oxidative stress. *P. fluorescens* could be engineered to overexpress the *katB* gene which encodes a catalase, an enzyme responsible for the degradation of hydrogen peroxide (H₂O₂).

#### Construction of the plasmid pSEVA244 cloned with the gene *katB* and transformation of *Pseudomonas fluorescens* SBW25

##### Objectives

The aim was to clone the gene *katB* into the plasmid pSEVA244 for transformation of *P. fluorescens*.

##### Method

*katB* was amplified from *P. fluorescens* genomic DNA via PCR. pSEVA244 served as a backbone for cloning of *katB* by In-fusion prior transformation of *E. coli Stellar Competent Cells*.

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

##### Hypothesis

Transformation of *P. fluorescens* with pSEVA244-katB was expected to yield overexpression of the KatB protein.

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

![](https://static.igem.wiki/teams/5108/results/fig14-resu.png)

**Figure 14: Representation of the pSEVA244-katB plasmid.**

##### Results

After transformation in *E. coli Stellar Competent Cells* and isolation, the plasmid was digested using the enzymes EcoRI and HindIII, which validated the cloning (Figure 15). **The correct sequence was validated by Sanger sequencing**.
![](https://static.igem.wiki/teams/5108/results/fig15-resu.png)

**Figure 15: Restriction digest of pSEVA244-KatB plasmid.**The plasmid was digested with EcoRI and HindIII separately or in combination. The expected (left) and experimental (right) digestion patterns are shown.

#### Attempts to overexpress a catalase in *Pseudomonas fluorescens* SBW25

##### Objectives

The objective was to verify the overproduction of the catalase KatB in *P. fluorescens* by activation of the *Ptrc* promoter with the inducer IPTG.

##### Method

The pSEVA438-MBPeGFP plasmid was employed again as positive control for heterologous protein expression in *P. fluorescens* SBW25. The pSEVA438-MBPeGFP- and pSEVA244-katB-transformed *P. fluorescens* SBW25 strains were cultured in M9 minimal medium supplemented with glucose (28 mM), with or without 0.5 mM of *m*-toluic acid or IPTG (1 mM). After incubation, a whole-protein extraction was performed for each strain to assess the level of expression, as well as the solubility of our proteins..

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

##### Hypothesis

We expected that MBPeGFP and KatB were activated in the presence of *m*-toluic acid and IPTG, respectively. These proteins should be soluble with an expected molecular weight of 69 kDa and 57 kDa, respectively.

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

##### Results

The obtained SDS-PAGE is presented in Figure 16. Both soluble and insoluble fractions contained inducible MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. SDS-PAGE analysis of the cell lysate derived from *P. fluorescens* transformed with pSEVA244-katB revealed a faint but **visible band at the expected size of KatB** in both soluble and insoluble fractions when its expression was induced (Figure 16).
.
![](https://static.igem.wiki/teams/5108/results/fig16-resu.png)

**Figure 16: SDS-PAGE of soluble and insoluble protein fractions from cultures of *Pseudomonas fluorescens* transformed with pSEVA244-katB.***P. fluorescens* was cultured with or without the inducer IPTG. Arrows indicate the expected size of catalase KatB.

##### New questions arise

Why was KatB found in the insoluble fraction and how could we remedy this problem?
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The presence of proteins in the insoluble fraction suggests that the ​​conditions of production (e.g., inducer concentration and temperature) are not optimal for overexpressing KatB in the soluble form. After aligning the sequence of KatB with database PFAM, we noticed the presence of a peptide signal, suggesting the targeting of KatB in the periplasm. In our lysis protocol, we could not differentiate periplasmic proteins from cytoplasmic ones. To solve this problem, we could change the KatB production conditions and our lysis protocol to purify exclusively periplasmic proteins.

Can over-expression of KatB help bacterial survival at high concentrations of H2O2?

*![plus](https://static.igem.wiki/teams/5108/general/plus-large-svgrepo-com.png)*
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If the protein is indeed located in the membrane, it could help our *P. fluorescens* strain metabolizing creatinine to survive the increasing concentrations of H2O2. In order to prove that the engineered bacterium could better survive certain conditions, the oxidative impact test should be repeated, as well as a viability assay on regolith. Plant growth should also be assayed in the presence of *P. fluorescens* pSEVA244-KatB. We expect plants will live longer after overexpression of the catalase.

Can we envision other solutions to improve stress resistance?
*![plus](https://static.igem.wiki/teams/5108/general/plus-large-svgrepo-com.png)*
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![](https://static.igem.wiki/teams/5108/results/lupso-question.png)

To mitigate stresses, an effective strategy could be to enhance the bacterial strain’s resistance to general stress. *P. fluorescens* could be engineered to overexpress the *hfq* and *rpoS* native genes which encodes respectively for Hfq, a chaperon protein, and RpoS, a transcriptional factor involved in the stress response. We performed preliminary experiments in this direction but we failed to construct the expression plasmid. Likewise, we intended in the beginning of the project to improve the biofilm production of *P. fluorescens* to counter the poor water retention of regolith, but we encountered cloning problems.

## Testing the biostimulant properties of *Pseudomonas fluorescens* for plants on regolith

In this section, we describe our experiments with plants.

We addressed three questions:* How do plants grow on regolith?
* Does *P. fluorescens* have a positive effect on plant growth on regolith?
* Do our modifications enabling creatinine consumption by *P. fluorescens* impact the growth of plants?

#### Plants grow poorly on regolith

##### Objectives

This first experiment aimed to observe the negative effect of lunar-simulant regolith on plant growth. It was an important step to ensure that our setup to grow plants was effective.

##### Method

We monitored the growth of plants on fertile ground or regolith (LSP-2 Lunar South Pole Simulant). *Arabidopsis thaliana* Col-0 was grown for 22 days after germination, watered every day with 3 mL of osmotic water. Pictures of plants were taken during the process.

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

##### Hypothesis

Based on literature[10](#ref), we expected that the regolith condition would result in a strong development delay compared to the soil condition.

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

##### Results

On regular potting soil, we obtained **green and healthy plants** which grew vigorously (Figure 17). The plant on our lunar regolith simulant showed a **slower development and a browner color**. The smaller size and area of the rosette is due to the lack of nutrients present in the regolith. Under starving conditions, the leaves tend to be smaller[11](#ref) and the color browner. The change in color is due to the production of anthocyanins, a pigment indicative of abiotic stresses. Because the lightning, humidity, and temperature were designed to be as optimal as possible for the plant, the production of anthocyanins could be explained by the lack of nutrients on regolith[12](#ref).

![](https://static.igem.wiki/teams/5108/results/fig17-resu-17.png)

**Figure 17: Pictures of *Arabidopsis thaliana* Col-0 taken 22 days after germination, watered every day with 3 mL of osmotic water, grown on LSP-2 Lunar South Pole Simulant (A) or on potting soil (B).**

This experiment confirmed that **regolith is an unfavorable environment for plant growth**. The use of a biostimulant to improve the development and limit stress is relevant.

#### *Pseudomonas fluorescens* WT increases the fraction of healthy leaves and plant hue on regolith

##### Objectives

The objective of this second experiment was to observe if *P. fluorescens* WT could relieve the negative effect of lunar-simulant regolith on plant growth.

##### Method

We monitored the growth of plants under three different conditions by varying the soil (fertile ground or regolith), and the presence or not of the WT strain on the regolith.

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

This experiment also allowed us to define which metrics to use to quantify the growth of plants:* **Proportion of healthy leaves**
  The proportion of healthy leaves were counted every day and used to analyze the plant’s survival.
* **Plant area**
  We determined the plant area for each plant. Pictures were taken every day and analyzed using ImageJ 2.7.0 version software. To calculate the plant area, the contour of each plant was cut out precisely on the image and the area was measured with the Measure tool in the Analyze tab.
* **Color hue**
  We determined the color for each plant. To measure the color, we used the Color\_Histogram-2.7.0.jar plugin from ImageJ to extract the mean value of Red, Green and Blue pixels present in each pixel of the cropped plant. Using the rgb2hsv command from the grDevices package on R Studio, we converted RGB space (Red/Green/Blue) to HSV space (Hue/Saturation/Value). Only the Hue value was used in our analysis, which correlates with chlorophyll content and is suitable to estimate the stress[13](#ref).

![](https://static.igem.wiki/teams/5108/results/fig18-resu.png)

**Figure 18: Images of plants and color analysis with Image J and R.**  The plants were first cropped using polygon selection. An RGB histogram was then drawn for each plant image with the Color Histogram tool in the Analyze tab. The R, G, and B mean values obtained were used to calculate the Hue on R with the rgb2hsv command.

##### Hypothesis

Based on literature[14](#ref), we hypothesized that the presence of *P. fluorescens* should improve the growth and/or health of the plants on regolith.

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

##### Results

Images of plants at different stages are shown for the tested conditions in Figure 19. Addition of *P. fluorescens* on regolith **moderately increased the number of leaves per plant** after 14 days compared to the control without bacteria.

![](https://static.igem.wiki/teams/5108/results/fig19-resu.png)

**Figure 19: Pictures of *Arabidopsis thaliana* Col-0 taken after one day (left) and 14 days (right) after transfer on potting soil or on our LSP-2 Lunar South Pole Simulant.**

To collect more quantitative information about the impact of stress on the phenotype of plants, we also analyzed the proportion of healthy leaves per plant, the rosette area, and the plant hue. While the percentage of healthy leaves per plant decreased to 40 % after 8 days in the absence of bacteria on regolith, it remained higher than 80 % for plants growing on soil or on a regolith containing bacteria (Figure 20).

![](https://static.igem.wiki/teams/5108/results/fig20-resu.png)

**Figure 20: Proportion of healthy leaves per plant over 14 days.**The mean and standard deviation (across 6 different plants) values are shown. The corresponding density diagram at day 14 is also displayed (right panel) for three different conditions: Soil + No bacteria + Water, Regolith + No bacteria + Water, and Regolith + WT + Water. A Wicoxon-Mann-Whitney with a Benjamini & Hochberg correction analysis was performed. \*\* p-value = 0.01.

Therefore, **the addition of *Pseudomonas fluorescens* WT on regolith results in plants that wilts significantly less than on regolith only**. Furthermore, after 14 days, plants grown on regolith without bacteria exhibited the browner color and the smallest size (Figure 21), two characteristics of stress.

![](https://static.igem.wiki/teams/5108/results/unnamed21.png)

**Figure 21: Assessment of the stress level for plants as defined by the plant area (Y axis) and the hue (color) (X axis) on day 1 and day 14.** Plant area and the hue value were measured from pictures taken on day 1 and day 14. A Kernel density is represented and gives the distribution of densities.

From these results, we conclude that ***P. fluorescens* improves the growth and health of the plants on regolith**.

#### Transformed *P. fluorescens* may have a protecting role on plant when grown on regolith

##### Objectives

This last experiment aimed to examine whether (i) our modification of *P. fluorescens* affects plant growth and (ii) the use of chemical agents to maintain the plasmid in the strain and to trigger the creatinine pathway expression could have some impact too.

##### Method

We monitored the growth of plants under four different conditions by varying the bacteria (no bacteria, the strains transformed with the empty plasmid or *creA-crnA* plasmid), and the watering solution (water or a mix composed of creatinine 44 mM and streptomycin (1 µg/mL)).

![](https://static.igem.wiki/teams/5108/results/lupso-crazyscientist-good.png)

**Table reporting the tested conditions:**

| Condition | Comment |
| --- | --- |
| Regolith + No bacteria + Water | Negative control |
| Regolith + No bacteria + Mix | Negative control for the “Mix” condition. This experiment showed us the impact of the mix alone on plant growth. |
| Regolith + Empty plasmid + Mix | Negative control for the “*creA-crnA*” condition. This experiment showed us the impact of bacteria in the presence of creatinine on plant growth. |
| Regolith + *creA-crnA* plasmid + Mix | This experiment showed us the impact of bacteria in the presence of creatinine that was metabolized to sustain bacterial growth on plant growth. |

##### Hypothesis

It is possible that the implementation of the creatinine pathway in *P. fluorescens* results in oxidative stress because of the production of H2O2 associated with glycine biosynthesis. Likewise, streptomycin and creatinine addition could have some impact too. Another scenario could be that the wild-type strain inoculated with water only and the bacteria transformed with the empty plasmid could have a lower positive effect since both strains are not able to metabolize creatinine, hence to grow.

![](https://static.igem.wiki/teams/5108/results/lupso-opportunities.png)

##### Results

After 8 days on regolith, it appeared that the four conditions led to different phenotypes (Figure 22). This required investigating each measured parameter separately (healthy leaves, plant surface, and hue).
.

![](https://static.igem.wiki/teams/5108/results/fig22-rsu.png)

**Figure 22: Pictures of *Arabidopsis thaliana* Col-0 taken after one day (left) and 8 days (right) after transfer on our LSP-2 Lunar South Pole Simulant.**The four tested conditions are indicated.

The **proportion of healthy leaves per plant was clearly lower without *P. fluorescens*** and in the presence of creatinine and streptomycin mix (Figure 23), pointing to a negative effect of one or both compounds. Notably, a clear positive effect of bacteria can be seen, leading to >60 % of healthy leaves after 9 days. The **strain with the functional creatinine pathway did not protect more than the strain with the empty plasmid**. This suggests a possible detoxification of the soil through the degradation of streptomycin by the cells.

![](https://static.igem.wiki/teams/5108/results/fig23-resu.png)

**Figure 23: Proportion of healthy leaves per plant on different days for three different conditions : Soil + No bacteria + Water, Regolith + No bacteria + Water, and Regolith + WT + Water.**Symbols are mean values with appended standard deviation across 12 replicates. The corresponding density diagram at day 8 is also displayed (right panel). A Wicoxon test was performed. \*\* p-value = 0.01.

To strengthen these observations, we carried out a survival analysis by using the **Kaplan-Meier estimator** (Figure 24). This allowed us to estimate the probability to reach an event in function of time. Here, the event of interest we considered is the time an individual reached less than 45 % of healthy leaves. The survival curves indicated that after 8 days, the probability to have less than 45 % of healthy leaves was between 0.8 and 0.9 for all conditions, except the No bacteria+Mix condition that led to a probability of 0.25 (Figure 24A). This indicates that the probability to have a healthy plant is a lot lower without bacteria and with the mix.

![](https://static.igem.wiki/teams/5108/results/fig24-resu.png)

**Figure 24: Proportion of healthy leaves per plant on days for three different treatments : Soil + No bacteria + Water, Regolith + No bacteria + Water and Regolith + WT + Water.** A) Survival curve made with a Kaplan-Meier estimator, log-rank p < 0.001. B) Verification of the Cox proportional hazards model assumption. C) Cox proportional hazard model.

We coupled this analysis with a Cox proportional Hazard model (Figure 24C), which allowed us to evaluate simultaneously the effect of several factors on survival. The results are given by the exp(coef), which corresponds to the Hazard Ratio (HR). HR gives an estimation of how often a condition reaches more than a half (55 %) of wilted leaves over time. Here, the conditions No bacteria+Mix, the Empty plasmid+Mix and the *creA-crnA* plasmid+Mix were compared to the control No bacteria+Water.

For interpretation:* HR = 1: No difference in the wilting compared to the control group over time.
* HR < 1: Reduction of the risk it gets 55 % of wilted leaves compared to the control group over time.
* HR > 1: Increased risk of getting 55 % of wilted leaves compared to the group control over time.

For the **No bacteria+Mix**, we obtained an HR=8, which means that this condition **gives a high risk of getting 55 % of wilted leaves compared to the No bacteria+Water condition**. For the bacteria conditions, the HR < 1 indicates that transformation with either the empty plasmid or *creA-crnA* plasmid reduces the risk of getting 55 % of wilted leaves compared to the No bacteria condition. This result suggests a **negative effect of streptomycin**.

Because the Cox proportional hazards model makes several assumptions, we needed to validate them for each of the covariable sets (Figure 24B). We proved the validity of our model by reaching p-values < 0.05 for each covariable.

The second metric used was the **plant hue** (Figure 25). The hue works like a binary metric: plants are either stressed (hue < 0.15) or not (hue > 0.15). For this study, dead plants have been arbitrarily set to a value of -0.025.
The negative control No bacteria+Water may have led to a high production of anthocyanins, hence the purple color (Figure 25, day 8). The three other conditions presented a more pronounced yellowing. With this color analysis, it is impossible to say whether yellowing or purpuling is a stronger stress mark. After 8 days, every condition showed signs of stress (mean hued < 0.15) on regolith. Notably, the conditions with bacteria transformed with either the empty-plasmid or *creA-crnA* displayed less stress than the condition No bacteria+Mix, with an averaged hue value at 0.14, corroborating the results described above.

![](https://static.igem.wiki/teams/5108/results/fig25-resu.png)

**Figure 25: Plant hue on day 1 and day 8 for four different conditions, as indicated.** Each symbol represents a plant and the color was assigned to match the global tonality of this plant. The mean and standard deviation (across 12 different plants) values are shown. For each treatment n>10, the hue value for dead plants was arbitrarily set to negative value.

To get a complete response of the plan phenotype to stress exposure, we combined the hue metric with plant surface (*i.e.*, the third metric used in this analysis) (Figure 26). This provided a better discrimination of each plant state. At day 1, the hue indicates an absence of stress with some variability in the plant area. At day 8, the profiles are a lot more varied, indicating specific responses. The condition No bacteria+Mix clearly led to death. Condition No bacteria+Water resulted in a strong stress and lesser plant development. Conditions *creA-crnA*-plasmid+Mix and especially empy-plasmid+Mix resulted in greener hue and larger plant area.

![](https://static.igem.wiki/teams/5108/results/unnamed26-26.png)

**Figure 26: Assessment of the stress level for plants as defined by the plant area (Y axis) and the hue (color) (X axis) on day 1 and day 8.**  Plant area and the hue value were measured from pictures taken on day 1 and day 8. A Kernel density is represented and gives the distribution of densities.

These results demonstrate that our engineered strain of ***P. fluorescens* is endowed with protective properties for the plant development on regolith**. Further improvements will be necessary to take full advantage of the implemented creatinine pathway as it seems to create some more stress to plants than the strain transformed with the empty plasmid.

##### New questions arise

Why did the Regolith+No bacteria+Mix condition decrease the chances of survival of plants?
*![plus](https://static.igem.wiki/teams/5108/general/plus-large-svgrepo-com.png)*
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![](https://static.igem.wiki/teams/5108/results/lupso-question.png)

In order to keep the plasmid into the bacteria, Streptomycin was present in the Mix. However, studies demonstrated the toxicity of Streptomycin for plants. The plasmid pSEVA438 contains the aadA gene coding an adenylyltransferase enzyme that O-adenylates the position 3″ of streptomycin and deactivates its antimicrobial activity. Therefore, bacteria transformed with this plasmid may have a protecting role on plants by inactivating streptomycin.

Why did the plants inoculated with bacteria engineered with creA-crnA plasmid show stress signs (yellowing/purpuling and smaller rosette area)?
*![plus](https://static.igem.wiki/teams/5108/general/plus-large-svgrepo-com.png)*
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![](https://static.igem.wiki/teams/5108/results/lupso-question.png)

During the metabolization of creatinine, H2O2 is produced by bacteria. If H₂O₂ is released into the medium, it would trigger the plant stress response and would explain the observed sign of stress compared to the strain transformed with the empty plasmid.

##### New potential research

To mitigate the oxidative stress caused by the release of H₂O₂ during creatinine degradation, an effective strategy would be to enhance the bacterial strain’s resistance to Reactive Oxygen Species. Overexpressing the catalase gene *katB* in *P. fluorescens* would be a way to degrade H₂O₂ and avoid its adverse effect on plant growth.

In order to avoid having to add Streptomycin to preserve the plasmid in the bacteria, chromosomal integration of *creA-crnA* should be considered.

## Conclusion

In this BioMoon project, we choose to optimize the properties of *Pseudomonas fluorescens* as a **biostimulant** for growth in poor soil conditions like the lunar regolith.

Our main goal was to engineer the strain so that it could utilize non-recycled resources in a space base. We identified creatinine as a high-value compound unconsidered so far in spatial programs and that could serve as both carbon and nitrogen source. We demonstrated that *Pseudomonas fluorescens* is not able to naturally grow from creatinine. A **synthetic creatinine pathway was successfully implemented** in the bacterium and the properties of the resulting *Pseudomonas fluorescens* were thoroughly characterized both ***in silico* and in wet lab experiments**.

We set up a home-made system to ensure efficient and reproducible plant assays. We qualitatively and quantitatively analyzed the **deleterious effect of regolith** on plant growth, as well as the **positive impact of *Pseudomonas fluorescens***. We managed to observe that our strain engineered with the creatinine pathway has kept most of its protective properties. We discussed how it could be optimized further to provide stronger stress resistance. Several strategies to reduce *Pseudomonas fluorescens* oxidative stress have been proposed and are currently in progress.

This work paves the way to **future developments** in using synthetic biology for the production of efficient biostimulants, both on Earth and in space. Please visit our [entrepreneurship](https://2024.igem.wiki/toulouse-insa-ups/entrepreneurship) page to learn more about it.

References
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1. Tsuru, D., Oka, I., & Yoshimoto, T. (1976). Creatinine Decomposing Enzymes in *Pseudomonas putida*. Agricultural And Biological Chemistry, 40(5), 1011‑1018.  [Click](https://doi.org/10.1080/00021369.1976.10862151)
2. Tsuru, D., Oka, I., & Yoshimoto, T. (1976). Creatinine Decomposing Enzymes in Pseudomonas putida. Agricultural And Biological Chemistry, 40(5), 1011‑1018.  [Click](https://doi.org/10.1080/00021369.1976.10862151)
3. Cloning, Expression and Purification of *Pseudomonas putida* ATCC12633 Creatinase. (2017, 1 décembre). PubMed.  [Click](https://pubmed.ncbi.nlm.nih.gov/29090065/)
4. Tsuru, D., Oka, I., & Yoshimoto, T. (1976). Creatinine Decomposing Enzymes in *Pseudomonas putida*. Agricultural And Biological Chemistry, 40(5), 1011‑1018.  [Click](https://doi.org/10.1080/00021369.1976.10862151)
5. Vogeleer, P., Millard, P., Arbulú, A. O., Pflüger-Grau, K., Kremling, A., & Létisse, F. (2023). Metabolic impact of heterologous protein production in *Pseudomonas putida* : Insights into carbon and energy flux control. Metabolic Engineering, 81, 26‑37.  [Click](https://doi.org/10.1016/j.ymben.2023.10.005)
6. Information on EC 3.5.2.10 - creatininase - BRENDA Enzyme Database. (s. d.).  [Click](https://www.brenda-enzymes.org/enzyme.php?ecno=3.5.2.10)
7. Information on EC 3.5.3.3 - creatinase - BRENDA Enzyme Database. (s. d.).  [Click](https://www.brenda-enzymes.org/enzyme.php?ecno=3.5.3.3)
8. PhysioFit | Anaconda.org. (s. d.).  [Click](https://anaconda.org/bioconda/physiofit)
9. Information on EC 3.5.1.5 - urease - BRENDA Enzyme Database. (s. d.).  [Click](https://www.brenda-enzymes.org/enzyme.php?ecno=3.5.1.5#ORGANISM)
10. Paul, A., Elardo, S. M., & Ferl, R. (2022). Plants grown in Apollo lunar regolith present stress-associated transcriptomes that inform prospects for lunar exploration. Communications Biology, 5(1)  [Click](https://doi.org/10.1038/s42003-022-03334-8)
11. Kolář, J., & Seňková, J. (2008). Reduction of mineral nutrient availability accelerates flowering of Arabidopsis thaliana. Journal Of Plant Physiology, 165(15), 1601‑1609.  [Click](https://doi.org/10.1016/j.jplph.2007.11.010)
12. Diaz, C., Saliba-Colombani, V., Loudet, O., Belluomo, P., Moreau, L., Daniel-Vedele, F., Morot-Gaudry, J., & Masclaux-Daubresse, C. (2005). Leaf Yellowing and Anthocyanin Accumulation are Two Genetically Independent Strategies in Response to Nitrogen Limitation in Arabidopsis thaliana. Plant And Cell Physiology, 47(1), 74‑83.  [Click](https://doi.org/10.1093/pcp/pci225)
13. Information on EC 3.5.3.3 - creatinase - BRENDA Enzyme Database. (s. d.-b).  [Click](https://www.brenda-enzymes.org/enzyme.php?ecno=3.5.3.3)
14. Paul, A., Elardo, S. M., & Ferl, R. (2022). Plants grown in Apollo lunar regolith present stress-associated transcriptomes that inform prospects for lunar exploration. Communications Biology, 5(1).  [Click](https://doi.org/10.1038/s42003-022-03334-8)

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