iGEM 2020 — Team Sorbonne_U_Paris

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

# Team:Sorbonne U Paris/Notebook

The Chlamy Cleaner

Team

[Members](https://2020.igem.org/Team%3ASorbonne_U_Paris/Members)
[Attributions](https://2020.igem.org/Team%3ASorbonne_U_Paris/Attributions)
[Inclusion](https://2020.igem.org/Team%3ASorbonne_U_Paris/Inclusion)

Project

[Description](https://2020.igem.org/Team%3ASorbonne_U_Paris/Description)
[Design](https://2020.igem.org/Team%3ASorbonne_U_Paris/Design)
[Engineering](https://2020.igem.org/Team%3ASorbonne_U_Paris/Engineering)
[Implementation](https://2020.igem.org/Team%3ASorbonne_U_Paris/Implementation)
[Plant](https://2020.igem.org/Team%3ASorbonne_U_Paris/Plant)

Lab

[Basic parts](https://2020.igem.org/Team%3ASorbonne_U_Paris/Basic-parts)
[Composite parts](https://2020.igem.org/Team%3ASorbonne_U_Paris/Composite-parts)
[Experiments](https://2020.igem.org/Team%3ASorbonne_U_Paris/Experiments)
[Results](https://2020.igem.org/Team%3ASorbonne_U_Paris/Results)
[Notebook](https://2020.igem.org/Team%3ASorbonne_U_Paris/Notebook)
[Safety](https://2020.igem.org/Team%3ASorbonne_U_Paris/Safety)
[Contribution](https://2020.igem.org/Team%3ASorbonne_U_Paris/Contribution)

HP

[Human practices](https://2020.igem.org/Team%3ASorbonne_U_Paris/Human_Practices)
[Science and communication](https://2020.igem.org/Team%3ASorbonne_U_Paris/Education)
[Collaborations](https://2020.igem.org/Team%3ASorbonne_U_Paris/Collaborations)
[Partnership](https://2020.igem.org/Team%3ASorbonne_U_Paris/Partnership)

Acknowledgements

[Sponsors](https://2020.igem.org/Team%3ASorbonne_U_Paris/Sponsors)

Competition

[Medal and Awards](https://2020.igem.org/Team%3ASorbonne_U_Paris/Medal_and_Awards)

☰

Notebook

# Notebook

#### **Monday 3rd of August 2020:**

**Gabriel Chemin

‍**
Preparation of stock solutions of atrazine and cyanuric acid at
**1 g/L**
:

- Dissolve 10 mg of Atrazine powder  (MERCK®) in 10 ml of  DMSO 100%.

- Dissolve 10 mg of Cyanuric acid powder  (MERCK®) in 10 ml of milli-Q H2O.

Store the solutions at -20°C.

‍
**Caution: Work under a fume hood during the preparation of stock solution in order to avoid any inhalation of atrazine and cyanuric acid.**

#### **Tuesday 4th of August 2020:**

**Antonin Chevenier

Gabriel Chemin**

##### Toxicity tests for atrazine and cyanuric acid in plates:

**Caution: Work under a biosafety cabinet to avoid any contamination.

-**
Prepare 3 solutions of atrazine and cyanuric acid diluted to 1/10, 1/100 and 1/1000 from the stock solutions (1 g/L).

- Prepare 2 plates 24 wells “tissue culture treated” and add 1 mL of Chlamydomonas D66 culture concentrated to 4.106 cells/mL.

- Add atrazine and cyanuric acid at different concentrations in each well as mentioned below. Don’t forget the negative control with DMSO for the test with atrazine.

![](https://static.igem.org/mediawiki/2020/a/aa/T--Sorbonne_U_Paris--Notebook0.png)
![](https://static.igem.org/mediawiki/2020/d/d2/T--Sorbonne_U_Paris--Notebook01.png)

- Seal the plates with tape to prevent evaporation during incubation.

- Incubate at 25°C under constant light.

#### **Tuesday 11th of August 2020:**

**Antonin Chevenier**

##### Resuspension of DNA fragments synthetized by Twist Biosciences:

The plate delivered by Twist Biosciences contains varying amounts of lyophilized DNA:

![](https://static.igem.org/mediawiki/2020/e/ee/T--Sorbonne_U_Paris--Notebook1.png)

- Centrify the plate for 1 min at 5000 x g.

- DNA fragments are resuspended with elution buffer (5 mM Tris HCl pH 8,5) to get a final concentration of 50 ng/µL or 25 ng/µL (total resolubilization of the DNA may take time, wait at least 30 min).

- Aliquot 10 µL of each solution in the plate, store it at -20 °C and use the rest as a working solution in a 1,5 mL Eppendorf tube.

#### **Wednesday 12th of August 2020:**

**Antonin Chevenier**

##### Digestion/Ligation reaction to assemble our constructs into level 0 plasmid:

We used an excel table provided on the supplementary material from
*“Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. Crozet P, et al. ACS Synth Biol. 2018”.*
A master mix is prepared for 15 reactions:

![](https://static.igem.org/mediawiki/2020/a/ad/T--Sorbonne_U_Paris--Notebook2.png)

The following tubes are prepared:

1. Assembly of p0-atzA: atzA\_B3-B4 + pAGM1287

![](https://static.igem.org/mediawiki/2020/2/22/T--Sorbonne_U_Paris--Notebook3.png)

2. Assembly of p0-atzB: atzB\_B3-B4 + pAGM1287

![](https://static.igem.org/mediawiki/2020/b/bd/T--Sorbonne_U_Paris--Notebook4.png)

3. Assembly of p0-atzC: atzC\_B3-B4 + pAGM1287

![](https://static.igem.org/mediawiki/2020/d/d1/T--Sorbonne_U_Paris--Notebook5.png)

4. Assembly of p0-COP1: COP1\_B3 + piCH41258

![](https://static.igem.org/mediawiki/2020/2/2c/T--Sorbonne_U_Paris--Notebook6.png)

5. Assembly of p0-TMD\_HAP2: TMD\_HAP2\_B3 + pICH41258

![](https://static.igem.org/mediawiki/2020/7/7d/T--Sorbonne_U_Paris--Notebook7.png)

6. Assembly of p0-linker+TEV\_site: linker+TEV\_site\_B4 + pAGM1299

![](https://static.igem.org/mediawiki/2020/a/a2/T--Sorbonne_U_Paris--Notebook8.png)

7. Assembly of p0-GSAT: GSAT\_B4 + pAGM1299

![](https://static.igem.org/mediawiki/2020/5/5b/T--Sorbonne_U_Paris--Notebook9.png)

8. Assembly of p0-NucA+SV40\_NLS: NucA+SV40\_NLS\_B5 + pAGM1301

![](https://static.igem.org/mediawiki/2020/4/4a/T--Sorbonne_U_Paris--Notebook10.png)

9. Assembly of p0-C-ter\_TEV: C-ter\_TEV\_B5 + pAGM1301

![](https://static.igem.org/mediawiki/2020/a/ab/T--Sorbonne_U_Paris--Notebook11.png)

10. Assembly of p0-N-ter\_TEV: N-ter\_TEV\_B5 + pAGM1301

![](https://static.igem.org/mediawiki/2020/e/e1/T--Sorbonne_U_Paris--Notebook12.png)

11. Negative controls (empty vector): pAGM1287 ; pICH41258 ; pAGM1299 ; pAGM1301

![](https://static.igem.org/mediawiki/2020/7/75/T--Sorbonne_U_Paris--Notebook13.png)

- 6 μL/tube of master mix are added.

- PCR thermocycler parameters:

![](https://static.igem.org/mediawiki/2020/6/6e/T--Sorbonne_U_Paris--Notebook14.png)

##### Transformation samples of competent cells:

The following tubes are prepared:

1. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**atzA\_B3-B4**
and
**pAGM1287**
2. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**atzB\_B3-B4**
and
**pAGM1287**
3. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**atzC\_B3-B4**
and
**pAGM1287**
4. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**COP1\_B3**
and
**piCH41258**
5.
25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**TMD\_HAP2\_B3**
and
**pICH41258**
6.
25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**linker+TEV\_site\_B4**
and
**pAGM1299**
7.
25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**NucA+SV40\_NLS\_B5**
and
**pAGM1301**
8.
25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**GSAT\_B4**
and
**pAGM1299**
9.
25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**NucA+SV40\_NLS\_B5**
and
**pAGM1301**
10. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**C-ter\_TEV\_B5**
and
**pAGM1301**
11.
25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**N-ter\_TEV\_B5**
and
**pAGM1301**
12. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**pAGM1287**
13. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**pAGM1287**
14. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**pICH41258**
15. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**pAGM1301**
16. 25 µL of NEB10 bacteria alone

- Add DNA to bacteria and mix gently by pipetting up and down.

- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.

- Add 700 µLof LB and incubate 1h at 37°C.

- Centrifuge 2 min at 4,000 g, discard surpernatant (keep only 100 µL), resuspend the bacteria.

- Spread bacteria on a LB/ Spectinomycin (50 µg/mL) Petri dish and incubate at 37°C overnight.

#### **Thursday 13th of August 2020:**

**Antonin Chevenier**

##### Results of the transformation:

- For the plates transformed with samples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11: mostly all colonies were white with few blue colonies → the digestion/ligation reaction was very efficient.

- For the plates transformed with samples 12, 13, 14, 15: all colonies were blues → the alpha complementation is working and all plasmids vectors do not recircularize after digestion.

- For the plate transformed with sample 16: there were no colonies → spectinomycin is efficient.

##### Transplanting colonies into liquid medium:

Pick 2 white colonies of each transformation plate and incubate them in LB/ Spectinomycin (50 µg/mL) medium at 37°C overnight.

#### **Friday 14th of August 2020:**

**Antonin Chevenier**

##### Plasmid extraction:

Extract plasmid DNA from each clone of tubes 1 to 11 using MACHEREY‑NAGEL
[NucleoSpin® Plasmid](https://www.mn-net.com/media/pdf/45/51/02/Instruction-NucleoSpin-Plasmid.pdf)
kit.

##### Determination of plasmid concentration with nanodrop:

The concentration of the extracted DNA is measured with the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific):

![](https://static.igem.org/mediawiki/2020/4/49/T--Sorbonne_U_Paris--Notebook15.png)

#### **Monday 17th of August 2020:**

**Antonin Chevenier**

##### Sequencing of the extracted plasmids:

- Add 2 µL of primer forward (GG40) or reverse (GG41) at 10 µM. These primers hybridize to sequences located upstream and downstream of the insert on the recipient plasmid.

- Add DNA and water:

![](https://static.igem.org/mediawiki/2020/2/2f/T--Sorbonne_U_Paris--Notebook16.png)

#### **Friday 21st of August 2020:**

**Antonin Chevenier

Pierre Foucault**

##### Analysis of sequencies:

The sequencing results show us that the sequences are indeed those expected for:

- p0-atzA C1

- p0-atzB C1

- p0-atzC C1 and C2

- p0-COP1 C1

- p0-TMD\_HAP2 C1 and C2

- p0-linker+TEVsite C1 and C2

- p0NucA+SV40\_NLS C1

- p0-GSAT C1 and C2

- p0-Cter\_TEV C1 and C2

- p0-Nter\_TEV C1 and C2

For each part synthesized at this point, we have at least one clone containing the correct sequence in the recombinant plasmid. Only the part coding for UVR8 is missing. This is due to a delay in the synthesis at the supplier compared to the delivery of others.

#### **Monday 31st of August 2020:**

**Pierre Foucault**

##### Resuspension of UVR8 DNA fragments synthetized by Twist Biosciences:

The plate delivered by Twist Biosciences contains varying amounts of lyophilized DNA:

![](https://static.igem.org/mediawiki/2020/d/d6/T--Sorbonne_U_Paris--Notebook17.png)

- Centrify the plate for 1 min at 5000 x g

- DNA fragments are resuspended with elution buffer (5 mM Tris HCl pH 8,5) to get a final concentration of 50 ng/µL or 20 ng/µL (total resolubilization of the DNA may take time, wait at least 30 min).

- Store at -20°C.

#### **Tuesday 1st of September 2020:**

**Antonin Chevenier**

##### Digestion/Ligation reaction to assemble our constructs into level 0 plasmid:

Master mix is prepared for 2 reactions:

![](https://static.igem.org/mediawiki/2020/5/5a/T--Sorbonne_U_Paris--Notebook33.png)

1. Assembly of p0-UVR8: UVR8\_B3 + pICH41258

![](https://static.igem.org/mediawiki/2020/9/92/T--Sorbonne_U_Paris--Notebook18.png)

2. Negative controls (empty vector): pICH41258

![](https://static.igem.org/mediawiki/2020/9/95/T--Sorbonne_U_Paris--Notebook32.png)

PCR thermocycler parameters:

![](https://static.igem.org/mediawiki/2020/6/6e/T--Sorbonne_U_Paris--Notebook14.png)

##### Transformation of competent cells with p0-UVR8\_B3:

The following tube is prepared:

‍

1. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction with
**UVR8\_B3**
and
**pICH41258.**
2. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**pICH41258.**
3. 25 µL of NEB10 bacteria alone.

- Add DNA to bacteria and mix gently by pipetting up and down.

- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.

- Add 700 µLof LB and incubate 1h at 37°C.

- Centrifuge 2 min at 4,000 g, discard supernatant (keep only 100 µL), resuspend the bacteria.

- Spread bacteria on a LB/Spectinomycin (50 µg/mL) Petri dish and incubate at 37°C overnight.

#### **Wednesday 2nd of September 2020:**

**Antonin Chevenier**

##### Results of the transformation:

- For the plates transformed with samples 1: mostly all colonies were white with few blue colonies → the digestion/ligation reaction was very efficient.

- For the plates transformed with sample 2: all colonies were blues → the alpha complementation is working and all plasmids vectors do not recircularize after digestion.

- For the plate transformed with sample 3: there were no colonies → spectinomycin is efficient.

##### Transplanting colonies into liquid medium:

Pick 2 white colonies of each transformation plate and incubate them in LB/ Spectinomycin (50 µg/mL) medium at 37°C overnight.

#### **Thursday 3rd of September 2020:**

**Thomas Champcommunal**

##### Plasmid extraction:

Extract plasmid DNA from each clone of tubes 1 to 11 using MACHEREY‑NAGEL
[NucleoSpin® Plasmid](https://www.mn-net.com/media/pdf/45/51/02/Instruction-NucleoSpin-Plasmid.pdf)
kit.

##### Determination of plasmid concentration with nanodrop:

The concentration of the extracted DNA is measured with the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific):

![](https://static.igem.org/mediawiki/2020/b/bc/T--Sorbonne_U_Paris--Notebook25.png)

#### **Friday 4th of September 2020:**

**Antonin Chevenier**

##### Sequencing of the extracted plasmids:

- Add 2 µL of primer forward (GG40) or reverse (GG41) at 10 µM. These primers hybridize to sequences located upstream and downstream of the insert on the recipient plasmid.

- Add DNA and water:

![](https://static.igem.org/mediawiki/2020/c/cc/T--Sorbonne_U_Paris--Notebook26.png)

#### **Monday 7th of September 2020:**

**Pierre Foucault**

##### Analysis of sequencing of p0-UVR8

p0-UVR8 sequencing showed that the sequence inserted into the vector did not correspond to that expected. We suspect that this is due to the difficulties encountered by the supplier for the synthesis of the two fragments of UVR8.

#### **Tuesday 8th of September 2020:**

**Antonin Chevenier**

##### Digestion/Ligation reaction to assemble our constructs into level 1 plasmid:

Master mix is prepared for 8 reactions:

![](https://static.igem.org/mediawiki/2020/f/fd/T--Sorbonne_U_Paris--Notebook31.png)

The following tubes are prepared:

1. Assembly of p1-atzA\_HA: p0-pPSAD + p0-atzA + p0-3\_HA + p0-tPSAD + pICH47742

![](https://static.igem.org/mediawiki/2020/3/3f/T--Sorbonne_U_Paris--Notebook19.png)

2. Assembly of p1-atzB\_HA: p0-pPSAD + p0-atzB + p0-3\_HA + p0-tPSAD + pICH47742

![](https://static.igem.org/mediawiki/2020/0/01/T--Sorbonne_U_Paris--Notebook20.png)

3. Assembly of p1-atzC\_HA: p0-pPSAD + p0-atzC + p0-3\_HA + p0-tPSAD + pICH47742

![](https://static.igem.org/mediawiki/2020/4/49/T--Sorbonne_U_Paris--Notebook21.png)

4. Assembly of p1-COP1\_NterTEV: p0-pPSAD + p0-COP1 + p0-GSAT + p0-C-ter\_TEV + p0-tPSAD + pICH47761

![](https://static.igem.org/mediawiki/2020/2/2e/T--Sorbonne_U_Paris--Notebook22.png)

5. Assembly of p1-NUCA\_TEVsite: p0-pPSAD + p0-cCA + p0-TMD\_HAP2 + p0-NucA\_TEVsite + p0-tPSAD + pICH47742

![](https://static.igem.org/mediawiki/2020/8/88/T--Sorbonne_U_Paris--Notebook23.png)

6. Negative controls (empty vector): pICH47742 ; pICH47751; pICH47761

![](https://static.igem.org/mediawiki/2020/8/82/T--Sorbonne_U_Paris--Notebook29.png)

- PCR thermocycler parameters:

![](https://static.igem.org/mediawiki/2020/6/6e/T--Sorbonne_U_Paris--Notebook14.png)

#### **Wednesday 9th of September 2020:**

**Antonin Chevenier**

##### Transformation of competent cells with level 1:

The following tubes are prepared:

4. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**p1-atzA\_HA**
5.
25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**p1-atzB\_HA**
6. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**p1-atzC\_HA**
7. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**p1-COP1\_NterTEV**
8.
25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**p1-NUCA\_TEVsite**
9. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**pICH47751**
10.
25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**pICH47761**
11.
25 µL of NEB10 bacteria alone.

- Add DNA to bacteria and mix gently by pipetting up and down.

- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.

- Add 700 µLof LB and incubate 1h at 37°C.

- Centrifuge 2 min at 4,000 g, discard supernatant (keep only 100 µL), resuspend the bacteria.

- Spread bacteria on a LB/Ampicillin (50 µg/mL) Petri dish and incubate at 37°C overnight.

#### **Thursday 10th of September 2020:**

**Antonin Chevenier**

##### Results of the transformation:

- For the plates transformed with samples 1, 2, 3, 4, 5: mostly all colonies were white with few blue colonies → the digestion/ligation reaction was very efficient.

- For the plates transformed with samples 5, 6, 7: all colonies were blues → the alpha complementation is working and all plasmids vectors do not recircularize after digestion.

- For the plate transformed with sample 16: there were no colonies → ampicillin is efficient.

##### Transplanting colonies into liquid medium:

Pick 2 white colonies of each transformation plate and incubate them in LB/ampicillin (50 µg/mL) medium at 37°C overnight.

#### **Friday 11th of September 2020:**

**Antonin Chevenier**

##### Plasmid extraction:

Extract plasmid DNA from each clone of tubes 1 to 8 using MACHEREY‑NAGEL
[NucleoSpin® Plasmid](https://www.mn-net.com/media/pdf/45/51/02/Instruction-NucleoSpin-Plasmid.pdf)
kit.

##### Determination of plasmid concentration with nanodrop:

The concentration of the extracted DNA is measured with the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific):

![](https://static.igem.org/mediawiki/2020/2/21/T--Sorbonne_U_Paris--Notebook24.png)

#### **Monday 21th of September 2020:**

**Pierre Foucault**

##### Growth tests with the Algem® photobioreactor:

Prepare two erlenmeyer containing 400 mL of Chlamydomonas D66 culture with 1.106 cells/mL. One test will be led with no atrazine added in the culture medium as a control and the other with a concentration of atrazine of 77 µg/L.

- Add 30,8 µL of the stock solution of atrazine in DMSO (1 g/L) in order to get a final concentration of 77 µg/L in the culture media.

Set the environmental parameters at pH=7 and T=28°C and set the measure of the absorbance at 740 nm.

#### **Monday 28th of September 2020:**

**Antonin Chevenier**

##### Growth tests with the Algem® photobioreactor:

Prepare two erlenmeyer containing 400 mL of Chlamydomonas D66 culture with 1.106 cells/mL. One test  with a concentration of atrazine of 250 µg/L and the other with a concentration of 500 µg/L.

- Add 100 µL in order to get a final concentration of 250 µg/L in the culture media,

- Add 200 µL in order to get a final concentration of 77 µg/L in the culture media.

Set the environmental parameters at pH=7 and T=28°C and set the measure of the absorbance at 740 nm.

##### Ligation of multigenic plasmid pM-BSR\_atzA-HA\_atzB-HA\_atzC-HA:

A master mix is prepared for 2 reactions:

![](https://static.igem.org/mediawiki/2020/0/07/T--Sorbonne_U_Paris--Notebook30.png)

The following tubes are prepared:

1. Assembly of pM-BSR\_atzA-HA\_atzB-HA\_atzC-HA\_HA: p1-BSR + p1-atzA + p1-atzB + p1-atzC + pICH50900 + pAGM8031

![](https://static.igem.org/mediawiki/2020/f/f7/T--Sorbonne_U_Paris--Notebook27.png)

2. Negative controls (empty vector): pAGM8031

![](https://static.igem.org/mediawiki/2020/f/f6/T--Sorbonne_U_Paris--Notebook28.png)

- PCR thermocycler parameters:

![](https://static.igem.org/mediawiki/2020/6/6e/T--Sorbonne_U_Paris--Notebook14.png)

##### Transformation of competent cells with level M:

The following tubes are prepared:

1. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**pM-BSR\_atzA-HA\_atzB-HA\_atzC-HA\_HA**
2. 25 µL of NEB10 bacteria + 4 µL of reaction mix from digestion/ligation reaction containing
**pAGM8031**
3.
25 µL of NEB10 bacteria alone.

- Add DNA to bacteria and mix gently by pipetting up and down.

- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.

- Add 700 µLof LB and incubate 1h at 37°C.

- Centrifuge 2 min at 4,000 g, discard supernatant (keep only 100 µL), resuspend the bacteria.

- Spread bacteria on a LB/Spectinomycin (50 µg/mL) Petri dish and incubate at 37°C overnight.

#### **Tuesday 29th of September 2020:**

**Antonin Chevenier**

##### Results of the transformation:

- For the plates transformed with samples 1: mostly all colonies were white with few blue colonies → the digestion/ligation reaction was very efficient.

- For the plates transformed with samples 2: all colonies were blue → the alpha complementation is working and all plasmids vectors do not recircularize after digestion.

- For the plate transformed with sample 3: there were no colonies → spectinomycin is efficient.

##### Transplanting colonies into liquid medium:

Pick 2 white colonies of each transformation plate and incubate them in LB/spectinomycin (50 µg/mL) medium at 37°C overnight.

#### **Tuesday 29th of September 2020:**

**Thomas Chamcommunal**

##### Plasmid extraction:

Extract plasmid DNA from each clone of tubes 1 to 8 using MACHEREY‑NAGEL
[NucleoSpin® Plasmid](https://www.mn-net.com/media/pdf/45/51/02/Instruction-NucleoSpin-Plasmid.pdf)
kit.

##### Determination of plasmid concentration with nanodrop:

The concentration of the extracted DNA is measured with the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific):

![](https://static.igem.org/mediawiki/2020/1/1d/T--Sorbonne_U_Paris--Notebook34.png)

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# Team:Sorbonne U Paris/Engineering

The Chlamy Cleaner

Team

[Members](https://2020.igem.org/Team%3ASorbonne_U_Paris/Members)
[Attributions](https://2020.igem.org/Team%3ASorbonne_U_Paris/Attributions)
[Inclusion](https://2020.igem.org/Team%3ASorbonne_U_Paris/Inclusion)

Project

[Description](https://2020.igem.org/Team%3ASorbonne_U_Paris/Description)
[Design](https://2020.igem.org/Team%3ASorbonne_U_Paris/Design)
[Engineering](https://2020.igem.org/Team%3ASorbonne_U_Paris/Engineering)
[Implementation](https://2020.igem.org/Team%3ASorbonne_U_Paris/Implementation)
[Plant](https://2020.igem.org/Team%3ASorbonne_U_Paris/Plant)

Lab

[Basic parts](https://2020.igem.org/Team%3ASorbonne_U_Paris/Basic-parts)
[Composite parts](https://2020.igem.org/Team%3ASorbonne_U_Paris/Composite-parts)
[Experiments](https://2020.igem.org/Team%3ASorbonne_U_Paris/Experiments)
[Results](https://2020.igem.org/Team%3ASorbonne_U_Paris/Results)
[Notebook](https://2020.igem.org/Team%3ASorbonne_U_Paris/Notebook)
[Safety](https://2020.igem.org/Team%3ASorbonne_U_Paris/Safety)
[Contribution](https://2020.igem.org/Team%3ASorbonne_U_Paris/Contribution)

HP

[Human practices](https://2020.igem.org/Team%3ASorbonne_U_Paris/Human_Practices)
[Science and communication](https://2020.igem.org/Team%3ASorbonne_U_Paris/Education)
[Collaborations](https://2020.igem.org/Team%3ASorbonne_U_Paris/Collaborations)
[Partnership](https://2020.igem.org/Team%3ASorbonne_U_Paris/Partnership)

Acknowledgements

[Sponsors](https://2020.igem.org/Team%3ASorbonne_U_Paris/Sponsors)

Competition

[Medal and Awards](https://2020.igem.org/Team%3ASorbonne_U_Paris/Medal_and_Awards)

☰

Design

# Engineering

To drive our project, we were inspired by physicist Richard Feynman’s famous quote “What I cannot create, I do not understand”. We implemented this thinking through the use of a fundamental pipeline:

Research → Imagine → Design → Build → Test → Learn → Improve

### Research

Our project entitled “The Chlamy Cleaner” consists in making the photosynthetic green microalgae named
*Chlamydomonas reinhardtii*
efficient for bioremediation of hazardous compounds found in the Seine river water. This model organism is well characterized and easy to cultivate. It made sense to use it since a MoClo toolkit has been adapted to Chlamy, making it suitable for synthetic biology projects. The widespread use of that microalgae in various bioremediation strategies was also an advantage.

Next, we established a list of interesting polluants to target. We learned from an exhaustive search that several classes of hazardous compounds can be found in the water environment : pesticides, antibiotics, polycyclic aromatic hydrocarbon (PAH), synthetic hormones... Since atrazine, an herbicide of the triazine class, is one of the most broadly found and a degradation pathway has been described, we chose to target it.

We also wanted to develop a kill switch system suitable for
*Chlamydomonas*. While checking the literature about what had been done or not, we came across the system developed by the iGEM team in Munich 2013 for their
[PhyscoFilter project](https://2013.igem.org/Team%3ATU-Munich)
where they had developed an innovative system in
*Physcomitrella patens*
moss. We got inspiration from their concept in order to adapt it to
*Chlamydomonas reinhardtii.*

### Imagine

We imagined an engineered microalgae able to express a part of the atrazine degradation pathway found in the bacterium
*Pseudomonas sp. ADP*
strain (de Souza et al. 1996; de Souza et al. 1998). Because of the experimental difficulty to introduce a whole new pathway in a host organism, we limited ourselves to the first 3 genes: atzA, B and C which metabolize atrazine into cyanuric acid, a less toxic compound.

To prevent any environmental pollution due to an accidental spread of our engineered *Chlamydomonas reinhardtii* strain, we also imagined a kill switch device, based on a UV light sensible programmed cell death. This safeguard relies on the release of a nuclease anchored on the plasma membrane induced by COP1 and URV8 dimerisation. It will allow the assembly of the N-ter and C-ter of the TEV protease, addressing the nuclease to the nucleus. Thus, a UV light highpass filter would have to be implemented in our bioremediation filter.

### Design

According to the MoClo guidelines, each gene was designed as level 0 plasmids containing strong promoters and implemented in level 1 transcriptional unit plasmids. Finally, multigenic level M plasmids were assembled.

For more information see the page [Design.](https://2020.igem.org/Team%3ASorbonne_U_Paris/Design)

### Build

We carried out the experiments in the laboratory since August in order to :

- Synthesize our new originals parts

- Adapt them to MoClo standard for
*Chlamydomonas reinhardtii

-*
Clone them in level 0 plasmids

- Construct our transcriptional units in  level 1 plasmids

- Construct our multigenic level M plasmids for transfection to
*Chlamydomonas reinhardtii*

### Test

We could not test the efficiency of our genetic designs due to safety measures. Still, we had planned some tests to ensure the expression and functionality of our parts.

##### Gene experssion:

Human Influenza hemagglutinin tag (HA-tag) was introduced in the atzA, atzB and atzC C-terminal coding region in order to confirm the expression of our transgenes. With these elements, the expressed protein would be detectable by separation on a SDS PAGE coupled with immunodetection:

- Fusion protein atrazine chlorohydrolase (atzA) - HA - tag has a theoretical molecular weight of 55,99 kDa (505 amino acids) and pI of 6,05.

- Fusion protein hydroxyde-chloro-atrazine ethylaminohydrolase (atzB) - HA - tag has a theoretical molecular weight of 55,39 kDa (512 amino acids) and pI of 5,39.

- Fusion protein N-isopropylammelide isopropyl amidohydrolase (atzC) - HA - tag has a theoretical molecular weight of 48,45 kDa (512 amino acids) and pI of 4,85.

As the molecular weights of atzA and atzB are similar, bidimensional electrophoresis (using isoelectric point) followed by a western-blot is well suited to detect their expression.

##### Functionality of our parts:

Toxicity assays like those we performed on the wild type microalgae to test the efficiency of the implemented degradation pathway would have measured the effect of atrazine exposure on the growth of the engineered
*Chlamydomonas reinhardtii*
strain. If an increase of the tolerated concentration of atrazine compared to the wild type
*Chlamydomonas reinhardtii*
D66 strain is observed with our modified strain, we could conclude that the enzymes are well expressed and functional.

Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) could have been used to assess precisely the potential decrease of atrazine levels in the medium due to our modified microalgae.

UV light exposure of our tinkered strains would have been an easy way to determine the functionality of our kill-switch. A simple 740nm OD lecture would confirm it.

### Learn

The new covid-related safety measures limited our access to the lab. We were not able to test the expression, functionality and efficiency of our parts, still we imagined what we could have learned from various scenarii.

1)
**Protein detection:**
since the molecular weight of parts atzA and atzB are very close, we may not be able to confirm their respective expression by SDS-page electrophoresis and immodetecion of the HA-tags. Separation by two-dimensional electrophoresis would allow us to separate atzA and atzB according to their isoelectric point. We could also change the design of our part and use distincts tags for immunodetection.

2)
**Efficiency of atrazine degradation:**
growth of our engineered microalgae on high atrazine concentrations over 250µg/L may not be observed. Underexpression of our transgene could be an explanation.

3)
**Kill-switch efficiency:**
growth of some microalgae clones may still be observed. A default in plasma membrane anchoring or an absence of interaction between URV8 and COP1 could be an explanation.

### Improve

We faced problems during the synthesis of the UVR8 sequence by our supplier. This difficulty may be linked with the coding sequence high GC content adapted to
*Chlamydomonas reinhardtii*
(which has a high GC content genome).

To face this problem, we wanted to extract UVR8 cDNA from
*Chlamydomonas reinhardtii*
wild type strain by conducting RT-PCR experiments on total RNA extracts. Sequence UVR8 insertion into the level 0 plasmid requires insertion of enzyme BbsI fusion and restriction sites into this plasmid. In order to do that, we planned to use specific primers containing flanking nucleotides at both 3’ and 5’ ends. Gene domestication is possible if the Cr-UVR8 nucleotide sequence contains BsaI and BbsI restriction sites. It could be done via site-directed mutagenesis on the amplicon that would make silent mutations insertion. We first considered using the At-UVR8 coding sequence of
*Arabidopsis thaliana*
to express the UVR8-CterTEV fusion protein. Finally, using the Cr-UVR8 sequence would not be a problem insofar as Cr-UVR8 and At-COP1 interaction has been demonstrated (Tilbrook et al., 2016).

The huge malleability potential provided by the modular cloning allows us to consider new transcriptional units construction including other exogenous expression reporter parts.

Transgene quantification and localization could be carried out by fluorescence and confocal microscopy experiments. It would have been useful to use a fluorescent C-terminal tag to mark our proteins like GFP (Green Fluorescent protein), YFP (Yellow Fluorescent Protein) or mCherry (Red Fluorescent Protein) to conduct these microscopy experiments. It would be interesting to check if the NucA-TEVsite part is well expressed at the plasma membrane.

References:

- de Souza ML, Sadowsky MJ, Wackett LP. Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization. J Bacteriol. (1996).

- de Souza ML, Wackett LP, Sadowsky MJ. The atzABC genes encoding atrazine catabolism are located on a self-transmissible plasmid in Pseudomonas sp. strain ADP. Appl Environ Microbiol. (1998).

- Tilbrook K, Dubois M, Crocco CD, Yin R, Chappuis R, Allorent G, Schmid-Siegert E, Goldschmidt-Clermont M, Ulm R. UV-B Perception and Acclimation in Chlamydomonas reinhardtii. Plant Cell. (2016).

# Follow us !

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Contact us: mail
igem.sorbonne2020@gmail.com

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

# Team:Sorbonne U Paris/Experiments

The Chlamy Cleaner

Team

[Members](https://2020.igem.org/Team%3ASorbonne_U_Paris/Members)
[Attributions](https://2020.igem.org/Team%3ASorbonne_U_Paris/Attributions)
[Inclusion](https://2020.igem.org/Team%3ASorbonne_U_Paris/Inclusion)

Project

[Description](https://2020.igem.org/Team%3ASorbonne_U_Paris/Description)
[Design](https://2020.igem.org/Team%3ASorbonne_U_Paris/Design)
[Engineering](https://2020.igem.org/Team%3ASorbonne_U_Paris/Engineering)
[Implementation](https://2020.igem.org/Team%3ASorbonne_U_Paris/Implementation)
[Plant](https://2020.igem.org/Team%3ASorbonne_U_Paris/Plant)

Lab

[Basic parts](https://2020.igem.org/Team%3ASorbonne_U_Paris/Basic-parts)
[Composite parts](https://2020.igem.org/Team%3ASorbonne_U_Paris/Composite-parts)
[Experiments](https://2020.igem.org/Team%3ASorbonne_U_Paris/Experiments)
[Results](https://2020.igem.org/Team%3ASorbonne_U_Paris/Results)
[Notebook](https://2020.igem.org/Team%3ASorbonne_U_Paris/Notebook)
[Safety](https://2020.igem.org/Team%3ASorbonne_U_Paris/Safety)
[Contribution](https://2020.igem.org/Team%3ASorbonne_U_Paris/Contribution)

HP

[Human practices](https://2020.igem.org/Team%3ASorbonne_U_Paris/Human_Practices)
[Science and communication](https://2020.igem.org/Team%3ASorbonne_U_Paris/Education)
[Collaborations](https://2020.igem.org/Team%3ASorbonne_U_Paris/Collaborations)
[Partnership](https://2020.igem.org/Team%3ASorbonne_U_Paris/Partnership)

Acknowledgements

[Sponsors](https://2020.igem.org/Team%3ASorbonne_U_Paris/Sponsors)

Competition

[Medal and Awards](https://2020.igem.org/Team%3ASorbonne_U_Paris/Medal_and_Awards)

☰

Experiments

# Experiments

### **Design *in silico* :**

All
*in silico*
sequence designs and analyses were performed with Serial Cloner and Geneious prime.

### **MoClo Assembly Conditions:**

The Modular Cloning (MoClo, Weber et al, 2009) provides a quick and reliable method to assemble basic parts such as promoters, CDSs or terminators. The MoClo relies on the Golden Gate cloning strategy (Engler et al, 2008).

##### Materials :

To make our MoClo Assembly protocols, we used an excel file provided on the supplementary material from: " Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. Crozet P, et al. ACS Synth Biol. 2018". For each reaction this table enabled us to calculate the appropriate volumes of DNA based on the DNA concentrations of the used solutions, as well as as the amount of water to reach the final volume (20 µL).

Master mix (for 2 ligations):

![](https://static.igem.org/mediawiki/2020/c/c8/T--Sorbonne_U_Paris--tab1.png)

\*The choice of the restriction enzyme depends on the level of the construction wanted. For level 0 and level M constructions BbsI-HF must be used, while for level 1 construction BsaI-HF must be used.

The reaction is performed in a thermocycler to allow several cycles of digestion (at37°C) and ligation (at16°C). The program used includes a final digestion to remove background (37°C) and the denaturation of the enzymes (with a step at 65°C for 20 min). The samples are then cooled to 16°C until they are taken out of the thermocycler.

![](https://static.igem.org/mediawiki/2020/3/3b/T--Sorbonne_U_Paris--tab2.png)

### **Transformation of bacteria:**

Chemically competent E. coli DH10β was used for heat shock transformation.

##### Materials :

- NEB 10-beta Competent
*E. coli

-*
Digestion/Ligation products (assay or negative control)

- LB medium (Sigma)

- Ice bucket

- LB + X-Gal + antibiotics\* solid medium

- Shaking incubator with modular temperature

\*The antibiotic depends on the construction wanted, for level 0 and level M the media contains spectinomycin at 50μg/mL, for level 1 the media contains ampicillin at 50μg/mL.

##### Methods:

1. Take competent bacteria out of -80°C and thaw on ice.

2. Mix 4 μL of Digestion/Ligation products with 25 μL of E. coli.

3. Incubate the competent cell/DNA mixture on ice (4°C) for 10 min

4. Heat shock each transformation tube by placing the tube at 42°C for 45 seconds.

5. Put the tubes back on ice (4°C) for 2 min.

6. Add 700 μL of LB medium (without antibiotic) and incubate 1h at 37°C with shaking (400rpm).

7. Spin for 2 min at 4.000 rcf to pellet the cells.

8. Discard the supernatant, leaving only about 100 μL of media.

9. Resuspend cells in the remaining medium.

10. Spread 100 μL on a culture plate containing LB + X-gal (50 μg/mL) + antibiotic\* (see above) (50 μg/mL).

11. Incubate plates at 37°C overnight.

### **Transplanting a colony into a liquid medium:**

Transfer micro-organisms from agar to liquid medium for growth.

##### Materials :

- LB/Antibiotic (50 μg/mL)/X-gal (50 μg/mL) liquid medium

- Culture plates with transformed bacteria

##### Methods:

1. Collect a white clone of interest using a tip (white clones correspond to recombinant clones whereas blue clones correspond to clones with empty vectors).

2. Drop the tip in 2 mL of liquid medium in a 15mL Falcon tube.

3. Incubate overnight at 37°C or at 30°C if the plasmid is bigger than 10kb.

4. Harvest bacterial culture and pellet by centrifuging at 4.000 rcf for 2 min.

5. Discard supernatant.

Minipreps were carried out according to the manufacturer's instructions.

### **Plasmid extraction:**

The plasmids were extracted from the recombinant clones using the MACHEREY‑NAGEL NucleoSpin® Plasmid kit. The plasmid DNA is extracted based on the alkaline lysis of the bacterial cells. The DNA is purified by means of a silica membrane which selectively retains DNA.

##### Materials :

- Ozyme ZymoPURE™ Plasmid Miniprep Kit

- Liquid culture of bacteria

##### Methods:

1. Harvest bacterial culture and pellet by centrifuging at 4.000 g for 2 min.

2. Discard supernatant.

3. Minipreps were carried out according to the manufacturer's instructions.

### **DNA concentration measurement:**

The concentration of the extracted DNA is measured with the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific).

##### Materials:

- NanoDrop 2000 spectrophotometer (ThermoFisher Scientific)

- Lint - free laboratory wipe

- Highly pure MiliQ water

- Elution buffer from the Plasmid Kit

- Samples

##### Methods:

DNA concentration measurement was performed according to the NanoDrop 2000 spectrophotometer protocol.

1. Make the blank with the buffer used to extract the DNA

2. Pipet 1 to 2 μL of sample directly onto the measurement pedestal\*.

\*Between measurements: wipe the sample off of both the upper and lower pedestals with a clean, dry lint.

When the measurement is complete, raise the sampling arm and wipe the sample off of both the upper and lower pedestals using a dry, lint-free laboratory wipe, pipet 2μL of water to clean any DNA remaining on the pedestals, and dry it with lint-free wipe.

### **Quality Control of generated DNAs:**

All plasmids were controlled by differential restriction to check the size of the insert.

##### Materials :

- BsaI-HF, BbsI-HF, EcoRI (NEB)

- Purified DNACut

- Smart buffer 10X(NEB)

- Sterile Water

##### Methods:

1. Add 100-200ng of DNA.

2. Add water to a final volume of 8.7μL.

3. Prepare the mix for all reactions

For one reaction :

![](https://static.igem.org/mediawiki/2020/1/13/T--Sorbonne_U_Paris--Tab3.png)

4. Dispatch 1.3 μL per tube.

5. Incubate 1 hour at 37°C

### **Preparation of DNA samples for sequencing:**

Preparation of DNA samples for sequencing were carried out according to the manufacturer’s instructions (Eurofins) : add 2µL of primer at 10µM and then 15µL of plasmid DNA concentrated at 50-100 ng.µL-1.

### **DNA gel electrophoresis:**

After the quality control digestion, the DNA fragments are separated according to their size on an agarose gel. The size of the DNA fragment is evaluated in comparison with a DNA size marker.

##### Materials :

- Agarose powder (Sigma)

- TAE buffer 0.5X (Tris-acetate 40mM, EDTA 1mM, pH 7.8 from Sigma)

- 1 kb DNA ladder (NEB)

- Gel tank

- Comb

- Microwave oven

- Scales

- Ethidium bromide 10 mg.mL-1

##### Methods:

1. Preparation of the 1% agarose gel in TAE buffer:

1.1 Dissolve 0.5g of agarose powder in 50 mL of TAE buffer 0.5X by heating the solution in the microwave. Heat for 30 seconds and swirl the flask until the powder is completely dissolved and the solution is clear.

1.2 Cool the solution (not for too long or it will solidify) on the table or you can allow hot agarose to cool in a water bath set at 60°C for 10min.

1.3 Add 1 μL of Ethidium bromide (EtBr) solution (final concentration:0.5 μg/mL) to the cooled gel.

1.4 Cast the solution on an electrophoresis gel mold, insert the comb and let the gel solidify at room temperature.

2. Gel migration:

2.1 Remove the comb.

2.2 Immerse the gel with 0.5X TAE migration buffer. Cover the gel with 2-3 mm of buffer for best results.

2.3 Load 3 µL of the DNA ladder on a well.

2.4 Load 15 µL DNA samples with the corresponding amount of DNA loading dye (DNA loading dye 1X) Run the gel for 30 minutes at 100V.

3. Gel revelation: At the end of the electrophoresis migration, collect the gel and take a picture of it using a gel reader (GelDocBiorad) under UV light. Adjust the brightness if necessary.

##### Caution with hot agarose!

Wear protective gloves, lab coat and eyes protection when handling an extremely hot agarose solution and heat the solution in several short intervals—do not let the solution boil for long periods as it may boil out of the flask or cause a loss to water vapor.

##### Caution with Ethidium Bromide (EtBr)!

Ethidium Bromide is a sensitive fluorescent dye used to detect nucleic acids in agarose gels. It is a probable mutagen and probable carcinogen. Always wear gloves, lab coat and eyes protection when working with ethidium bromide. Wipe the area with a damp cloth after working with ethidium bromide. Also, while wearing gloves after handling ethidium bromide, be careful not to touch and thereby contaminate other surfaces. Discard the gloves and pipet tips in appropriate trash.

### **Culture of Chlamydomonas:**

##### Materials :

- Tris-Acetate-Phosphate (TAP) medium 65

- Agar (1.6 % m/V),

Bold’s minimal media supplemented with 36 gL-1 NaCl

### **Toxicity test :**

The toxicity test is an important part of our project. Indeed, we designed this test in order to be able to screen our genetically modified algae. It should allow us to determine whether an algae colony expresses our plasmid or not. To do so, we have to determine the natural resistance of
*Chlamydomonas reinhardtii*
for each pollutant. We want to experiment by testing wild type culture in different concentrations of these pollutants. Once we determine the deadly concentration for the cells, we will be able to make some culture medium with a high toxicity level for each pollutant. After this, only the transformed cells which express the specific degradation enzymes will be able to grow on the different mediums. Currently, we tested two pollutants, the atrazine and the cyanuric acid. We would like to assess whether the cyanuric acid is toxic for
*Chlamydomonas reinhardtii*
since it does not appear to influence the growth of the cells. So we have to determine whether the concentrations were too low or the cells are resistant.

##### Materials:

- Laminar flow hood

- Algem Pro Photobioreactor System

- 24 x 1mL well plates

- 1L erlenmeyer flask

- TAP medium (https://www.chlamycollection.org/methods/media-recipes/)

- DMSO

- Pollutant (Atrazine/Cyanuric acid)

-
*Chlamydomonas reinhardtii*
strain
D66

##### Methods:

1. Put Chlamydomonas reinhardtii in exponential phase (2 millions cells per mL) in TAP (Tris-Acetate Phosphate) medium.

2. Prepare stock solutions for atrazine and cyanuric acid (1g/L). These pollutants have to be diluted into DMSO. The growth ability of the wild-type microalgae was assessed by colorimetry and a toxicity threshold was determined for every pollutant (minimum concentration to inhibit the growth of
*Chlamydomonas reinhardtii*
strain
D66).

3. Put 1mL of culture into some 24 1mL well plates with a 1% DMSO control and growing concentrations of each pollutant (0, 1, 2, 5, 10, 25, 50, 75, 100, 250, 500, 1000, 1100, 1250 and 1500 µg/L).

4. At each time point (0h, 6h, 12h, 24h, 48h, 72h, 96h) take a photograph of each plate.

We also performed a toxicity test with the Algem Pro Photobioreactor System. This hightech bioreactor allows us to record in real time the growth of our microalgae with reproducible optical density (OD) mesure while monitoring environmental parameters (pH and temperature). The Algem also enables us to verify our plate experiment on a larger scale (from 1mL wells to 1L standard erlenmeyer flask). Each flask is mixed along the experiment with an automatic hand-swirling like mouvement.

1. Put some
*Chlamydomonas reinhardtii*
strain D66 cells at exponential phase (1 millions cells/mL) with 400mL TAP medium into a 1L erlenmeyer flask.

2. Try several atrazine concentrations ( 0, 77, 250 and 500 µg/L).

3. Collect real time data for 50h. Set the environmental parameters at pH=7 and T =28°C. Measure the OD at 740nm (absorbance peak of Chlorophyll a pigment).

# Follow us !

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Contact us: mail
igem.sorbonne2020@gmail.com

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

# Team:Sorbonne U Paris/Results

The Chlamy Cleaner

Team

[Members](https://2020.igem.org/Team%3ASorbonne_U_Paris/Members)
[Attributions](https://2020.igem.org/Team%3ASorbonne_U_Paris/Attributions)
[Inclusion](https://2020.igem.org/Team%3ASorbonne_U_Paris/Inclusion)

Project

[Description](https://2020.igem.org/Team%3ASorbonne_U_Paris/Description)
[Design](https://2020.igem.org/Team%3ASorbonne_U_Paris/Design)
[Engineering](https://2020.igem.org/Team%3ASorbonne_U_Paris/Engineering)
[Implementation](https://2020.igem.org/Team%3ASorbonne_U_Paris/Implementation)
[Plant](https://2020.igem.org/Team%3ASorbonne_U_Paris/Plant)

Lab

[Basic parts](https://2020.igem.org/Team%3ASorbonne_U_Paris/Basic-parts)
[Composite parts](https://2020.igem.org/Team%3ASorbonne_U_Paris/Composite-parts)
[Experiments](https://2020.igem.org/Team%3ASorbonne_U_Paris/Experiments)
[Results](https://2020.igem.org/Team%3ASorbonne_U_Paris/Results)
[Notebook](https://2020.igem.org/Team%3ASorbonne_U_Paris/Notebook)
[Safety](https://2020.igem.org/Team%3ASorbonne_U_Paris/Safety)
[Contribution](https://2020.igem.org/Team%3ASorbonne_U_Paris/Contribution)

HP

[Human practices](https://2020.igem.org/Team%3ASorbonne_U_Paris/Human_Practices)
[Science and communication](https://2020.igem.org/Team%3ASorbonne_U_Paris/Education)
[Collaborations](https://2020.igem.org/Team%3ASorbonne_U_Paris/Collaborations)
[Partnership](https://2020.igem.org/Team%3ASorbonne_U_Paris/Partnership)

Acknowledgements

[Sponsors](https://2020.igem.org/Team%3ASorbonne_U_Paris/Sponsors)

Competition

[Medal and Awards](https://2020.igem.org/Team%3ASorbonne_U_Paris/Medal_and_Awards)

☰

Results

# Results

#### **Overview:**

- Analysed the pollutant diversity and abundance in the Seine river

- Synthesized 10 level 0 and level 1 plasmids (only URV8 part missing)

- Assembled the atrazine degradation pathway multigenic plasmid

- Assessed the effect of atrazine and cyanuric acid on wild type
*Chlamydomonas reinhardtii*
by plates and photobioreactor toxicity assays

- Demonstrated the lesser toxicity of cyanuric acid on wild type
*Chlamydomonas reinhardtii*

#### **Chemical analysis of Seine water:**

Our IGEM project, The ChlamyCleaner, aims to provide a new biological filter to reduce the environmental exposure to micropollutants in the Seine, the river that crosses the city of Paris. To gain insight into the pollutant diversity and abundance in the Seine, we sent collected samples of seine water from four different places. One in Herblay-sur-Seine (1) near the “Seine-Aval” wastewater treatment plant, the largest in the Ile-de-France region, one at the Seine-Marne confluence in Vitry-sur-Seine (2), another on the quay Saint-Bernard (3) bordering the campus of Sorbonne Université in the center of Paris and the last sample was provided to us by the METIS laboratory (CNRS UMR 7619) and was sampled from the Orgeval river (4) near a heavily agricultural area in the Seine et Marne department, in the east of Paris.

![](https://static.igem.org/mediawiki/2020/3/3b/T--Sorbonne_U_Paris--Mapprelevement.png)

Satellite view of the Paris region with the different sampling locations indicated by a red cross.

The samples were analysed in the METIS laboratory (Organic contaminant departement) where they performed a chemical analysis by liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Here is the list of the pollutants detected:

![](https://static.igem.org/mediawiki/2020/a/ae/T--Sorbonne_U_Paris--Listcomp.png)

LOQ stands for limits of quantification, the lowest concentration of pollutant detectable with LC MS/MS. 17 different pollutants were detected. Our targeted pollutant, atrazine, was detected in all the samples. Two products of atrazine degradation, DEA (diethyl-atrazine) and DIA (desisopropyl-atrazine) were also detected in the samples, with an even larger concentration for DEA. These findings validate the significant impact that our project can achieve.

#### **Parts cloning:**

As of october 16th 2020, by using the Moclo tool kit for
*Chlamydomonas Reinhardtii*
developed by Pierre Crozet and Stephane Lemaire, we were able to clone most of the Level 0 parts of the degradation genes of atrazine and for our kill-switch system, except for the UVR8 part. We believe we will be able to complete the UVR8 cloning, to create our Level 1 plasmids as well as the level M in the near future. We systematically checked the sequence for each Level 0 plasmid we cloned, by sending samples to Eurofin Genomics for Sanger sequencing.

We were able to finalize our genetic construct related to the atrazine degradation pathway into cyanuric acid. All the level 1 plasmids were completed and cloned into a blasticidin and spectinomycin resistant level M multigenic plasmid.

Unfortunately, due to new covid-related safety norms, we were not able to transform our
*Chlamydomonas reinhardtii*
strain with our level M plasmid. We could not test neither its efficiency nor the increased resistance of our engineered microalgae to atrazine exposure.

#### **Measure of the resistance of *Chlamydomonas* to atrazine and cyanuric acid:**

In order to assess the atrazine degradation ability of our engineered
*Chlamydomonas*
strain, we needed to characterize the basal toxicity of atrazine on the
*Chlamydomonas reinhardtii*
D66 strain. We also needed to demonstrate the lesser toxicity of the final product coming from the degradation of atrazine, the cyanuric acid, on our microalgae. We performed two types of toxicity tests: exposure to atrazine and cyanuric acid on plate (TAP medium, 96H) and monitored exposure to test a wide range of pollutant concentration. The Algem® photobioreactor (Algenuity®) allowed us to record more parameters (pH, Temperature) and perform the toxicity test with a larger volume (from 1mL to 1L).

The first toxicity test performed was the exposure to atrazine. We tested a range of concentrations from 0 to 1500 µg/L in 1mL TAPmedium 24 wells plates. At T=0, the
*Chlamydomonas*
cells
were in exponential phase (5 millions cells/mL). Simultaneously, we ran the experiment with cyanuric acid and the same concentration range of polluant.

![](https://static.igem.org/mediawiki/2020/7/7e/T--Sorbonne_U_Paris--Expatrazine.png)

![](https://static.igem.org/mediawiki/2020/3/3d/T--Sorbonne_U_Paris--Expcyanuric.png)

These experiments show that exposure to DMSO did not alter the growth of
*Chlamydomonas reinhardtii*
strain D66. Thus, any observed effect will be caused by the pollutant in the other wells. Exposure to atrazine alters the growth of
*Chlamydomonas reinhardtii*
strain D66 at the minimal concentration of 250µg/L in TAP medium.

Cyanuric acid did not seem to alter the growth of
*Chlamydomonas reinhardtii*
strain D66 in TAP medium, even at the maximum exposure levels.

These results provide two keypoints for the future of our project:

- It strengthens our strategy : the atrazine degradation into cyanuric acid is sufficient to hamper its toxic effect.

- We were able to determine a threshold value to test the efficiency of our genetically engineered microalgae. Growing on high atrazine concentrations will be required.

The third toxicity test performed was the exposure to atrazine in the Algem® photobioreactor system. We tested a range of concentration from 0 to 500 µg/L in 400mL TAP medium in a 1L erlenmeyer flask. At t=0, the
*Chlamydomonas*
concentration was 1 million cells/mL. Environmental parameters were set : pH=7 and T=28°C and were stable during the experiment.

![](https://static.igem.org/mediawiki/2020/e/ef/T--Sorbonne_U_Paris--CourbeAlgem.png)

Growth curve of
*Chlamydomonas reinhardtii D66*
strain D66 at various atrazine concentrations:

Green: 0 µg/L ; yellow: 77 µg/L ; orange: 250 µg/L ; red: 500 µg/L.

These results do not fit completely with the previous ones. All the atrazine condition hampers the microalgae growth. However, the polluant starts to hinder it at 77µg/L, below the threshold of 250 established earlier. It was expected since the Algem has more precise data.

All
*Chlamydomonas reinhardtii*
cultures were made in a rich medium (TAP), the growth of the microalgae was not nutrient-limited. This medium is not representative of the final environment of our filter: the Seine river, a nutrient low environment. Therefore, we wanted to run our experiment in a minimum medium (HSM) or even better, in sterilized Seine water. It was not possible due to the new covid-related safety norms which cut our lab access.

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