iGEM 2021 — Team Sorbonne_U_Paris

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# Team:Sorbonne U Paris/Notebook

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# Team:Sorbonne U Paris/Engineering

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# Engineering

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# Team:Sorbonne U Paris/Experiments

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### Lab Work

Experiments
Notebooks
Proof of Concept
Results
Safety

# Experiments

### Cloning Protocole

Purpose :

The goal is to clone the insert we ordered into a level 0 plasmid. Once this plasmid is obtained, the insert is cloned with a promoter and a terminator to form a [...]

### Transformation

Purpose :

This operation consists in introducing an exogenous DNA sequence into the host organism that we want to genetically modify. In the Onishi protocol, we used [...]

### RNA Extraction

Purpose :

The aim of the step is simply to extract RNA coming from transformed colonies before reverse-transcription. Here we have chosen [...]

### Proof of concept

Purpose :

Here, the objective is to try and see whether the addition of our peptide can reduce mortality among Chlamydomonas reinhardtii in presence of [...]

### Trasnformed cells

Purpose :

The aim of this experiment is to assess whether the expression of our antioxidant peptide has a protective effect over cells in contact with [...]

### In vitro test

Purpose :

This step aims to assess the capacity of our antioxidant peptide to protect one protein from reactive oxygen species. We want to put together one enzyme [...]

### Irradiation

Purpose :

The aim is to submit our transformed cells that are expressing the antioxidant peptide to γ-radiations mimicking some of the space radiations. Three strains of [...]

# Notebook

## Week 1 = from the 12th to the 16th of july

* Reception of the antiox sequence.
* Assembly of plasmide pL0-294, containing our antioxidant insert, with GoldenGate method and transformation of the assembled plasmid into NEB 10-beta Competent Escherichia coli (High Efficiency), that are plated on LB-agar+Spec+Xgal plates.

## Week 2 = from the 19th to the 23th of july

* Selection of white transformed bacteria on spectinomycin plates and growth in liquid culture of LB of our selected clones.
* Extraction and purification of plasmid DNA.
* Digestion with restriction enzyme BsaI and migration on agarose gel to confirm the presence of our insert : amplification by PCR on transformed colonies and migration on agarose gel.
* Third attempt to identify if the antiox sequence is present in the plasmid : digestion by BsteII and HindIII.
* The plasmid is sent to sequencing.
* Assembly of the p1 plasmid, transformation into E. Coli and selection on ampicillin.
* PCR reaction and agarose gel electrophoresis on selected bacterial clones.
* Extraction and purification of plasmid level 1 with Nucleospin kit.
* Assembly of the pM plasmid, transformation into E. Coli and selection on spectinomycin plates.

## Week 3 = from the 26th to the 30 of july

* Assembly of the pM plasmid, transformation into E. Coli and selection on spectinomycin plates.
* Range of toxicity essay with 3 oxidant molecules (H2O2, Rose Bengale and Methyl Viologen) at different concentrations on non transformed Chlamydomonas reinhardtii.
* Revelation of dead cells by incubation with Evans Blue.
* Extraction of plasmidic DNA to recover final pM and verification by agarose gel electrophoresis after digestion with BbsI.
* Digestion of pM-156
* Linearization of pM-156 with EcoR
* First transformation of Chlamydomonas reinhardtii strain 45-33 using glass marbles with the plasmide pM-156.

## Week 4 = from the 2nd to the 6th of August

* Prepared TAP broth.
* Count of dead and alive cells within the oxidant range on non transformed Chlamydomonas strain determination of the survival rate.
* Second oxidant range on non transformed Chlamydomonas reinhardtii, dead cells are colored with Evans blue. We used doses of oxidants to try to obtain survival rates between 20 and 50%.
* Count of dead and alive cells within the second oxidant range on non transformed Chlamydomonas strain determination of the survival rate.
* Second attempt of transformation of Chlamydomonas reinhardtii strain 45-33, using Onishi protocol.

## Week 5 = from the 9th to the 13the of August

* Dilution of dry peptides we had ordered.
* Several Bradford and BCA (bicinchoninic acid) dosages were made to determine the decapeptide concentration. (LOL)
* First antioxidant test with H2O2 and Rose Bengale on non transformed Chlamydomonas reinhardtii in the presence of our 2 peptides.

## Week 6 = from the 16th to the 20th of August

* Revelation of dead cells by incubation with Evans Blue.
* Manganese toxicity range of wild-type Chlamydomonas reinhardtii.
* Transformation of Chlamydomonas reinhardtii strain 45-33 attempt 3 with Onishi protocol.
* Toxicity and characterisation assay of our 2 peptides to assess whether it’s toxic and understand why it provokes agglomeration.
* Plates patch of strain 45-33 on different conditions check its antibiotic resistance : we came to the conclusion that strain 45-33 was resistant to hygromycin, so we couldn’t use it to select our transformed algae.
* Highlight of the interaction between Evans Blue and our peptides -which forms wide aggregates
* under the microscope.

## Week 7 = from the 23th to the 27th of August

* Linearization of pM-156
* First transformation of Chlamydomonas reinhardtii strain UVM4 with Onishi protocol and spreading on hygromycin plates. Cells seemed to have not survived the electroporation process, so we had to throw them out and try again.
* Second transformation of UVM4 with Onishi protocol on hygromycin plates.

## Week 8 = from the 30th of August to the 3rd of September

* Prepared PBS buffer.
* Prepared TAP broth.
* Third transformation attempt of Chlamydomonas reinhardtii UVM4 with plasmid pM156 containing the antioxidant gene, with glass marbles.

## Week 9 = from the 6th to the 9th of of September

* First try of LDH test : we tried to characterize the activity of LDH enzyme after flowing through the Amicon tube. We wanted to use Amicon to get rid of H2O2 before putting the substrates (NADH and pyruvate) and reading the absorbance. The absorbance was too important so we modified the NADH concentration.
* Second try of LDH test : this time the absorbance was right but the enzymatic activity detected was about 0,0297 ΔAbs/min, which is quite low. We thought we had lost a lot of enzymes through the Amicon tube.
* Third try of the LDH test : we characterize the activity of LDH without Amicon tube and without any ROS to assess how the reaction takes place without any perturbation. The enzymatic activity was around 0,097 ΔAbs/min.
* Fourth try of the LDH test : we don’t need the Amicon tube because H2O2 will be diluted in the final volume of tampon and pyruvate. Incubation of LDH with H2O2 10mM reduces the activity of the enzyme just enough for us to work with.

## Week 10 = from the 13th to the 17th of September

* Selection of 10 clones that grew on hygromycin after transformation with plasmid pM-156.
* Extraction of RNAs from the transformed strain of Chlamydomonas reinhardtii UVM4.
* Electrophoresis of the total extracted RNAs on an agarose gel.

## Week 11 = from the 20th to the 24th of September

* Reverse-transcription of total RNAs extracted.
* Multiple PCR tests with primers RT-08 and RT-09 to amplify our insert and CBLP primers (housekeeping gene), with different annealing temperature and revelation with agarose gel electrophoresis.
* In vivo test on 10 clones of transformed cells UVM4 with H2O2 to see if the expression of the peptides has a protective effect.

## Week 12 = from the 27th of September to the 1st of October

* PCR of the cDNA with 2 new primers pairs : RT10/RT11 and RT12/RT13.
* Shipment of 5x10^6 cells of the clones expressing the antioxidant peptide : 2, 5, 7 and the untransformed UVM4 strain (each sample in triplicate).

## Week 13 = 4th to 8th of October

* Irradiation of Chlamydomonas reinhardtii shipped at the Belgian nuclear research center
* Preparation of 16 TAP-agar 6 wells plates.
* Collect of irradiated strain, serial dilution, spread in previously made plates and growth under light.

# Let's set the context…

The main proof we had to provide is that the undecapeptide has the same radioprotective effect as the decapeptide mentioned in our main source article, as our transformed cells will express the undecapeptide. The only difference between those two is the presence of the amino acid methionine at the beginning of the sequence.

# Did we succeed?

To achieve that evidence, we tested the two peptides in both in vitro and in vivo assays. Unfortunately, our results weren’t conclusives. We did not have time to test the antioxidant capacity of both peptides in vivo with FDA, as we lost a lot of time working with Evans blue. In vitro, we did not succeed in characterizing the activity in LDH in the presence of the MDP or MUP complexe (manganese - peptide - pyrophosphate), as the enzymatic activity wasn’t repeatable between experiments.

# Conclusion & what could be improved !

Furthermore In “MDP: A Deinococcus radiodurans Mn2+-Decapeptide Complex Protects Mice from Ionizing Radiation”, the article that we relied on to design our project, researchers tried several peptides sequences, that were less successful but still had a protective effect. With that in mind, we believe that the peptide potentializes the effect of Mn2+, so we strongly feel that the addition of only one Methionine to the sequence won’t change this effect much.

Despite the inconclusive results we remain convinced that the design of the experiments was relevant; a better preparation of the reagents and the use of a machine allowing us to carry out our experiments in one day would have made it possible to prove the effectiveness or ineffectiveness of the undecapeptide.

# Cloning results

## Construction level 0 : pL0-294 (BBa\_K3836000)

The insert we ordered from Eurofins is cloned into a recipient plasmid pAMG9121 by GoldenGate reaction. To select the plasmids that received the insert, the reaction products were transformed into Escherichia Coli NEB10b bacteria by heat shock and plated on LB plates containing X-gal and the antibiotic spectinomycin. The results can be seen in figure 1, after 24h of incubation at 37°C. A majority of white colonies confirms that most of the recipient plasmids received the insert coding for the antioxidant peptide.

![](https://static.igem.org/mediawiki/2021/8/8d/T--Sorbonne_U_Paris--Results_-_Figure_1.jpg)

# Safety

Safety is fundamental in the development of genetically modified organisms (GMOs). To reduce the risks, it is important to experiment safely and responsibly by following laboratory safety rules.

Moreover, the handling of GMOs and micro-organisms in the context of laboratory research is highly regulated in France[1]. In this perspective, we were able to develop our experimental tasks thanks to the Laboratory of Quantitative and Computational Biology (LCQB), which benefits from a convention for the use of GMOs given by the Ministry of Higher Education and Research.

## Biosafety and biosecurity

Although we have been authorized to handle GMOs, our main concern was to ensure that the modified organisms did not pose any risk to humans or the environment.

In our project, we introduced a gene from a prokaryotic organism into a eukaryotic photosynthetic organism. We chose to use a eukaryotic organism because they are non-pathogenic to humans, better controllable to avoid environmental and contamination risks.

The organisms are classified into risk groups [2] according to the danger they cause to humans and the environment (Table 1).

| Risk group | Pathogenic for humans | Hazard to workers | Spreading | Effective prophylaxis or treatment | Safety equipment |
| --- | --- | --- | --- | --- | --- |
| 1 | No | Unlikely | Unlikely | Available | Open bench work |
| 2 | Can cause human disease | Likely | Unlukily | Available | Open bench work - BSC\* for potential aerosols |
| 3 | Can cause severe human disease | Serious | possible | Usually available | BSC for aerosols - Other primary devices |
| 4 | Can cause deadly human disease | Very serious | Likely | Usually non available | Class III BSC - Positive pressure suit |

Figure 1: Safety risk groups of microorganisms. \*BSC = Biosafety cabinet (closed ventilated laboratory workspace).]

As we only used organisms belonging to risk group 1, there was no risk to the people working at the WetLab.

For cloning, we used the Escherichia coli DH5α strain. This strain is non-pathogenic and its impact on the environment is minimal. The potential risks associated with this bacterium are related to contact with the skin. We therefore handled this organism wearing gloves and a protective lab coat.

The UVM4 strain of Chlamydomonas reinhardtii is also non-pathogenic. Indeed, this microalga is a GRAS organism (Generally Recognized As Safe according to the FDA) and is therefore considered safe[3]. We still handled it while wearing gloves and a protective lab coat. However, we performed the algal cultures under a fume hood, not because of the risks involved but to protect our samples from contamination. Thanks to the laminar flow incorporated in the hood, we were able to protect our manipulations from any potential contaminants.

## Safe project design

Our project was designed so that our modified C. reinhardtii is confined in a bioregenerative life support system (BLSS), so a closed system. However, there is a risk that our alga could end up outside this closed system. If this were the case, it would end up on the International Space Station (ISS) or on Mars, so there would be no risk of environmental contamination.

As for the risk to humans, a study has shown that the peptide we use is not toxic in mice and even provides protection against radiation. Although these results have been demonstrated in human cells they have yet to be confirmed in humans. Nevertheless, these results pave the way for potential protection[4].

## Safe lab work

### General laboratory safety rules

For a laboratory to function correctly, it is necessary to respect the common spaces (tidy up and leave clean the common work stations and living spaces), to respect the common work tools, and to know how to manage one's needs in work materials (small materials, consumables, culture media, sterilized materials).

The good practices in the laboratory are as follows :

* Respect security instructions
* Know the procedures to follow in case of fire or accident
* Locate fire extinguishers and alarm buttons
* Keep corridors, stairways and halls clear
* Do not obstruct the closing of fire doors
* Leave free access to the isolation valves (gas and water) and to the electrical cut-off devices
* Close doors and windows when leaving your workplace. The last one to leave checks that the doors are closed
* Wear the individual protection equipments (IPE)
* Identify the risks – respect the labeling rules
* Work in the right place (workstation adapted to specific risks)
* Respect the procedures of the isolated worker

In case of professional risk (biological, chemical, radioactive), it is recommended to contact the professional risk prevention department by phone or by email. The Faculty of Science and Engineering also has an infirmary open every day.

### Workspace

To work in the laboratory, we had to wear appropriate clothing (long trousers, closed shoes and tied up hair) and a lab coat. To avoid contamination of our samples, we changed our gloves regularly. Then, after each experiment, the samples were disposed of in the appropriate bins and we disinfected our bench to avoid any contamination.

We worked in different spaces with specific equipment. For each room, eating and drinking is forbidden. Certain protections may be necessary depending on the danger to which the experimenter is exposed in the room.

Laundry and preparation area: Ice machine, washing machine, MilliQ water, autoclaves, scales.

The hazards associated with this room are the presence of a pressure vessel (autoclave), the risk of burns, the electromagnetic field (microwave) and corrosive chemicals. In addition, this is a noisy area. In this room, wearing a lab coat is mandatory.

Machine and storage area: Liquid nitrogen tank, centrifuges, culture chamber, cold room, -80°C freezers, plate reader, qPCR machine, PFGE (Pulse-Field Gel Electrophoresis), pipetting robot.

This room presents a biological risk, a risk of asphyxiation, and a low temperature due to the presence of dry ice and liquid nitrogen. Protection of the body, face, eyesight, hands and feet is mandatory in this room. This space is air-conditioned, so the door must remain closed.

BET area : ADN/ARN electrophoresis, gel documentation system.

The hazards associated with this room are ethidium bromide (BET), UV radiation, and the electromagnetic field (microwave). Access to this room is restricted to laboratory staff and requires the wearing of a lab coat, gloves, and face protection when using the portable UV lamp.

The safety officers left us a summary of common sense practices to have in the BET room:

* Always wear your lab coat and gloves when working in this room
* Close the BET dropper bottle well and clean if necessary. Use 1 drop/100 mL TBE/TAE liquid, not more
* Clean the UV gel box when finished with water or ethanol and put your gel in the BET dry waste bin
* After staining your gel, put staining/destaining liquids into the BET liquid waste bin
* After running a gel, cover and label the gel box you are using, noting how many times the same buffer has been reused
* Use a support tray and tissue to remove your gel to take a photo. Do not leave TBE/BET drops on the floor. Clean up spills
* Turn off the UV gel box when finished
* Respect our co-workers by keeping this room clean

For the preparation of the electrophoresis gels, we used ethidium bromide. This fluorescent marker is a DNA intercalator and allows the visualization of DNA on the gel. This product is toxic and mutagenic, which is why we only used it in a dedicated room, wearing a protective lab coat and gloves to avoid carcinogenic risks. We received explanations from the laboratory's safety officers who showed us the specific bin in which to dispose of objects contaminated with the BET. Excess and non-recyclable solutions are then disposed of by a licensed waste disposal company. We also took care not to contaminate surfaces outside this room by changing our gloves before going into another room.

### Evacuation

We have received safety instructions in case of evacuation. As soon as we hear the alarm signal :

* Exit and follow the signs
* Head to the gathering point or the secure waiting area (reserved for people with reduced mobility)
* Do not go back
* If there is smoke, get down
* Before leaving, the person in charge makes sure that the windows and doors are closed

## COVID-19 protocols

Due to the pandemic, we had to respect certain rules in order to work in a safe environment. The number of people in the lab being restricted, we limited our iGEM team members to 2 people to perform the manipulations. In order to respect this, we had set up a schedule to make sure that each day, 1 or 2 people would be present in order to make the experiments progress. We also had to sign authorisations for each member to work in the lab.

According to government guidelines, we applied the following barrier gestures:

* Wearing a mask is mandatory (except for eating and drinking)
* Wear one lab coat per person
* Wash your hands very regularly
* Cough or sneeze into your elbow
* Use single-use tissues
* Greeting without shaking hands
* Keep physical distance (1.50 m)
* Follow the directions of traffic
* Maximum 1 person in the elevator cabin

As for the work outside the laboratory, we favored remote working sessions, or in the boxes at the university library, wearing masks. We also divided the tasks into small working groups to avoid being in a confined area with all the team members.

## References

* [1] O.G.M. en milieu confiné. Ministère de l’Enseignement supérieur, de la Recherche et de l’Innovation //www.enseignementsup-recherche.gouv.fr/pid28968/o.g.m.-en-milieu-confine.html.
* [2] Use of Micro-organisms or Derivatives. https://www.esrf.fr/Infrastructure/Safety/Experiments/Biology\_Experiments/use-of-micro-organisms-or-derivatives.
* [3] Arias, C. A. D. et al. Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii. Biotechnol Prog 37, e3101 (2021).
* [4] Gupta, P. et al. MDP: A Deinococcus Mn2+sup>-Decapeptide Complex Protects Mice from Ionizing Radiation. PLOS ONE 11, e0160575 (2016).

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# Team:Sorbonne U Paris/Results

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  + [Design](https://2021.igem.org/Team%3ASorbonne_U_Paris/Design)
  + [Engineering](https://2021.igem.org/Team%3ASorbonne_U_Paris/Engineering)
  + [Perspectives](https://2021.igem.org/Team%3ASorbonne_U_Paris/Implementation)
  + [Plant Synthetic Biology](https://2021.igem.org/Team%3ASorbonne_U_Paris/Plant)
* [Lab Work](https://2021.igem.org/Team%3ASorbonne_U_Paris/Lab_Work)
  + [Experiments](https://2021.igem.org/Team%3ASorbonne_U_Paris/Experiments)
  + [Notebooks](https://2021.igem.org/Team%3ASorbonne_U_Paris/Notebooks)
  + [Proof Of Concept](https://2021.igem.org/Team%3ASorbonne_U_Paris/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3ASorbonne_U_Paris/Results)
  + [Safety](https://2021.igem.org/Team%3ASorbonne_U_Paris/Savety)
* [Part](https://2021.igem.org/Team%3ASorbonne_U_Paris/Parts)
* [Human Practices](https://2021.igem.org/Team%3ASorbonne_U_Paris/Human_Practices_Menu)
  + [IHP](https://2021.igem.org/Team%3ASorbonne_U_Paris/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3ASorbonne_U_Paris/Communication)
  + [Public Engagement](https://2021.igem.org/Team%3ASorbonne_U_Paris/Education)
  + [Inclusivity](https://2021.igem.org/Team%3ASorbonne_U_Paris/Inclusivity)
  + [Contribution](https://2021.igem.org/Team%3ASorbonne_U_Paris/Contribution)
* [Team](https://2021.igem.org/Team%3ASorbonne_U_Paris/Team)
  + [Members](https://2021.igem.org/Team%3ASorbonne_U_Paris/Members)
  + [Attribution](https://2021.igem.org/Team%3ASorbonne_U_Paris/Attributions)
  + [Collaboration](https://2021.igem.org/Team%3ASorbonne_U_Paris/Collaborations)
  + [Team bonding](https://2021.igem.org/Team%3ASorbonne_U_Paris/Partnership)
* [Award](award.html)

### Lab Work

[Experiments](https://2021.igem.org/Team%3ASorbonne_U_Paris/Experiments)
[Notebooks](https://2021.igem.org/Team%3ASorbonne_U_Paris/Notebooks)
[Proof of Concept](https://2021.igem.org/Team%3ASorbonne_U_Paris/Proof_Of_Concept)
[Results](https://2021.igem.org/Team%3ASorbonne_U_Paris/Results)
[Safety](https://2021.igem.org/Team%3ASorbonne_U_Paris/Savety)

# Cloning results

## Construction level 0 : pL0-294 (BBa\_K3836000)

The insert we ordered from Eurofins is cloned into a recipient plasmid pAMG9121 by GoldenGate reaction. To select the plasmids that received the insert, the reaction products were transformed into Escherichia Coli NEB10b bacteria by heat shock and plated on LB plates containing X-gal and the antibiotic spectinomycin. The results can be seen in figure 1, after 24h of incubation at 37°C. A majority of white colonies confirms that most of the recipient plasmids received the insert coding for the antioxidant peptide.

![](https://static.igem.org/mediawiki/2021/8/8d/T--Sorbonne_U_Paris--Results_-_Figure_1.jpg)

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IGEM Sorbonne 2021

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IGEM Sorbonne Université 2021

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IGEM Sorbonne Université 2021

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igem.sorbonne2021@gmail.com

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