Team:Sorbonne U Paris/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.
- 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:
- 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:
The following tubes are prepared:
1. Assembly of p0-atzA: atzA_B3-B4 + pAGM1287
2. Assembly of p0-atzB: atzB_B3-B4 + pAGM1287
3. Assembly of p0-atzC: atzC_B3-B4 + pAGM1287
4. Assembly of p0-COP1: COP1_B3 + piCH41258
5. Assembly of p0-TMD_HAP2: TMD_HAP2_B3 + pICH41258
6. Assembly of p0-linker+TEV_site: linker+TEV_site_B4 + pAGM1299
7. Assembly of p0-GSAT: GSAT_B4 + pAGM1299
8. Assembly of p0-NucA+SV40_NLS: NucA+SV40_NLS_B5 + pAGM1301
9. Assembly of p0-C-ter_TEV: C-ter_TEV_B5 + pAGM1301
10. Assembly of p0-N-ter_TEV: N-ter_TEV_B5 + pAGM1301
11. Negative controls (empty vector): pAGM1287 ; pICH41258 ; pAGM1299 ; pAGM1301
- 6 μL/tube of master mix are added.
- PCR thermocycler parameters:
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 kit.
Determination of plasmid concentration with nanodrop:
The concentration of the extracted DNA is measured with the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific):
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:
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:
- 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:
1. Assembly of p0-UVR8: UVR8_B3 + pICH41258
2. Negative controls (empty vector): pICH41258
PCR thermocycler parameters:
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 kit.
Determination of plasmid concentration with nanodrop:
The concentration of the extracted DNA is measured with the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific):
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:
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:
The following tubes are prepared:
1. Assembly of p1-atzA_HA: p0-pPSAD + p0-atzA + p0-3_HA + p0-tPSAD + pICH47742
2. Assembly of p1-atzB_HA: p0-pPSAD + p0-atzB + p0-3_HA + p0-tPSAD + pICH47742
3. Assembly of p1-atzC_HA: p0-pPSAD + p0-atzC + p0-3_HA + p0-tPSAD + pICH47742
4. Assembly of p1-COP1_NterTEV: p0-pPSAD + p0-COP1 + p0-GSAT + p0-C-ter_TEV + p0-tPSAD + pICH47761
5. Assembly of p1-NUCA_TEVsite: p0-pPSAD + p0-cCA + p0-TMD_HAP2 + p0-NucA_TEVsite + p0-tPSAD + pICH47742
6. Negative controls (empty vector): pICH47742 ; pICH47751; pICH47761
- PCR thermocycler parameters:
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 kit.
Determination of plasmid concentration with nanodrop:
The concentration of the extracted DNA is measured with the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific):
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:
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
2. Negative controls (empty vector): pAGM8031
- PCR thermocycler parameters:
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 kit.
Determination of plasmid concentration with nanodrop:
The concentration of the extracted DNA is measured with the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific):
