Difference between revisions of "Team:Sorbonne U Paris/Notebook"

Resuspension of HiBiT-B2 and HiBiT-B5 DNA

- Oligos are resuspended in water to get a final concentration of 100 pmol/μL:

1. HiBit-B5-Forward
2. HiBit-B5-Reverse
3. H3. HiBit-B2-Forward
4. HiBit-B2-Reverse

- A dilution (x1000) is done to get a final concentration of 100 nM : 2 μL of oligos + 2 mL of water for each sample.
- Oligos are hybridized :

1. 20 μL of HiBit-B5-Forward + 20 μL of HiBit-B5-Reverse are mixed and incubated 10 min at 50°C
2. 20 μL of HiBit-B2-Forward + 20 μL of HiBit-B2-Reverse are mixed and incubated 10 min at 50°C

Digestion/Ligation reaction to assemble HiBiT-B2 and HiBiT-B5 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 5 reactions :

- The following tubes are prepared :

1. Assembly of p0-191 : HiBit-B2 + pAGM1276
2. Negative control : pAGM1276 (empty vector)

1. Assembly of p0-192 : HiBit-B5 + pAGM1301
2. Negative control : pAGM1301 (empty vector)

- 6 μL/tube of master mix are added.
- PCR thermocycler parameters :

Transformation samples of competent cells with p0-191 and p0-192

-The following tubes are prepared :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with HiBiT-B2 and pAGM1276
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with HiBiT-B5 and pAGM1301
3. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pAGM1276 alone (negative control)
4. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pAGM1301 alone (negative control)
5. 25 µL of JM109 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 surnageant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/ Spectinomycin (50 µg/mL) Petri dish and incubate at 37°C overnight.

Results of the transformation with p0-191 and p0-192

For plates transformed with samples 1 and 2, all colonies were white but 7/236 on plate 1 and 6/98 on plate 2.
→ the digestion/ligation reaction was very efficient.
For plates transformed with samples 3 and 4, all colonies were blue.
→ the alpha complementation is working.
→ the white/blue selection is relevant.
For the plate transformed with sample 5, there was no colonies.
→ the Spectinomycin is efficient.

Results of the transformation with p0-191 and p0-192

- Pick 4 white colonies of each transformation plate (p0-191 and p0-192) and incubate them in LB/ Spectinomycin (50 µg/mL) medium at 30°C overnight.

Plasmid extraction

- Extract plasmid DNA p0-191 and p0-192 using Ozyme ZymoPURE™ Plasmid Miniprep Kit

Determination of plasmid concentration with nanodrop

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

Quality control of generated DNAs

- Prepare a master mix (for 9 reactions) :

- Add 200 ng of DNA :

- Add qsp 10 μL of water.
- Incubate at 37°C during 1h.

DNA gel electrophoresis

Prepare the agarose gel 1% in TAE buffer :
- 0,5 g of agarose powder is dissolve in 50 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 20 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 8 μL of DNA + 2 μL of PromegaTM Blue/Orange Loading Dye 6X
- Ladder : 1 μL of PromegaTM Blue/Orange Loading Dye 6X + 1 μL of PromegaTM 200 bp DNA Step Ladder
- Run the gel for 30 min at 100V.

Results

For the restriction analysis, we expect two fragments generated (2235 bp and 52 bp).
→ On the gel, we can see a single strip in all the samples as expected (we can't see the 52 bp fragments), so we can conclude that HiBiT-B2 and HiBiT-B5 have been inserted in the corresponding level 0 plasmid.

Sequencing of the extracted plasmids p0-191 and p0-192

-Add 2 µL of primer at 10 µM.
-Add DNA and water :

Plasmid	Conc. DNA (ng/μL)	Cf wished in 15 μL	μL of DNA	μL of H2O
p0-191 (1)	35.9	30	12.5	2.5
p0-191 (2)	23.4	15	9.6	5.4
p0-191 (3)	35.8	30	12.6	2.4
p0-191 (4)	53.4	30	8.4	6.6
p0-192 (1)	55	30	8.2	6.8
p0-192 (2)	53.9	30	8.3	6.7
p0-192 (3)	47.8	30	9.4	5.6
p0-192 (4)	48.8	30	9.2	5.8

Results of sequencing of p0-191 and p0-192

p0-191: One error in the replication origin in all plasmids, no error in the HiBiT sequence.
→ use the clone p0-191 (4) (the sequencing results were stronger).

p0-192: Again one error in the replication origin in all plasmids, one error in the HiBiT sequence for the clone n°4, and some dirty signal for clones n°2 and 3.
→ use the clone p0-192 (1).

Digestion/Ligation reaction to assemble HiBiT-B2 (p0-191) and HiBiT-B5 (p0-192) into a level 1 plasmid with GFP

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 4 reactions :

- The following construct are prepared :

1. pCM1-1 (HiBiT-GFP)



2. pCM1-2 (GFP-HiBiT)



3. pCM1-3 (GFP)
4. Negative control : pL1-2F (empty vector)

- 6 μL/tube of master mix are added.
- Reaction parameters :

Transformation of competent cells with pCM1-1, pCM1-2 and pCM1-3

- Transformation samples :

1. 25 µL of JM109 bacteria + 4µL of reaction mix from digestion/ligation reaction with pCM1-1 (HiBiT-GFP)
2. 25 µL of JM109 bacteria + 4µL of reaction mix from digestion/ligation reaction with pCM1-2 (GFP-HiBiT)
3. 25 µL of JM109 bacteria + 4µL of reaction mix from digestion/ligation reaction with pCM1-3 (GFP alone)
4. 25 µL of JM109 bacteria + 4µL of reaction mix from digestion/ligation reaction with pL-2F (empty vector)

- 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 µL of 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)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

Resuspension of DGAT and PLS1 DNA

- Oligos are resuspended in water to get a final concentration of 20 ng/μL :

1. DGAT
2. PLS1 (LPAAT)

Transformation of competent cells with DGAT and PLS1 to make stock

- Transformation samples:

1. 9 µL of JM109 bacteria + 1 µL of reaction mix from DGAT
2. 9 µL of JM109 bacteria + 1 µL of reaction mix from PLS1

- 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 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Kanamycin (50 µg/mL) Petri dish and incubate at 37°C overnight.

Digestion/Ligation reaction to assemble DGAT and PLS1 into level 1 plasmid with HiBiT-tag

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 5 reactions :

- The following tubes are prepared (for more details see the excel table provided) :

1. pCM1-7 (HiBiT-DGAT)



2. pCM1-8 (DGAT-HiBiT)
3. Negative control : pL1-3F (empty vector)



4. pCM1-9 (HiBiT-PLS1)



5. pCM1-10 (PLS1-HiBiT)
6. Negative control : pL1-4F (empty vector)

- 6 μL/tube of master mix are added.
- PCR thermocycler parameters :

Results of the transformation with pCM1-1, pCM1-2, pCM1-3, DGAT and PLS1

For plate pL1-2F, there are white colonies.
→ the white/blue selection is not very relevant.
For plates pCM1-1, pCM1-2 and pCM1-3, few colonies grew.
→ the transformation was not very efficient.
For plates with DGAT and PLS1, bacteria grew.
→ the transformation was efficient.

Transplanting colonies into liquid medium

- Pick 2 colonies of pCM1-1, pCM1-2 and pCM1-3 transformation plates and incubate them in LB/ Ampicillin (50 µg/mL)/X-gal (50 µg/mL) liquid medium at room temperature during the weekend.

- Pick 2 colonies of DGAT and PLS1 transformation plates and incubate them in LB/ Kanamycin (50 µg/mL)/X-gal (50 µg/mL) liquid medium at room temperature during the weekend.

Transformation of competent cells with pCM1-7, pCM1-8, pCM1-9 and pCM1-10

- Transformation samples :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pCM1-7
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pCM1-8
3. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pCM1-9
4. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pCM1-10
5. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pL1-3F
6. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pL1-4F
7. 25 µL of JM109 bacteria alone

- Add DNA to bacteria and mix gently by stirring with the cone (do not pipette up and down).
- Incubate 10 min on ice, then 20 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/ Ampicillin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

Plasmid extraction

- Extract plasmid DNA DGAT and PLS1 using Ozyme ZymoPURE™ Plasmid Miniprep Kit. In default of Ozyme kit buffer, Macherey Nagel kit buffer was used.

- Extract plasmid DNA pCM1-1, pCM1-2, pCM1-3 and pL1-2F using Ozyme ZymoPURE™ Plasmid Miniprep Kit. In default of Ozyme kit buffer, Macherey Nagel kit buffer was used.

Determination of plasmid concentration with nanodrop

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

Quality control of pCM1-1, pCM1-2 and pCM1-3

- Prepare a master mix for 8 reactions :

- Add 100 ng of DNA :

- Add qsp 10 μL of water.
- Incubate at 37°C during 1h.

DNA gel electrophoresis

Prepare the agarose gel 1% in TAE buffer :
- 0,5 g of agarose powder is dissolve in 50 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 20 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 8 μL of DNA + 2 μL of PromegaTM Blue/Orange Loading Dye 6X
- Ladder : 1 μL of PromegaTM Blue/Orange Loading Dye 6X + 1 μL of PromegaTM 200 bp DNA Step Ladder
- Run the gel for 25 min at 100V.

Results

For the restriction analysis of pCM1-1, pCM1-2 and pCM1-3, we expect two fragments generated (4352 bp and about 2450 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that the HiBiT-GFP fragments, the GFP-HiBiT fragments and the GFP fragment have been inserted in the corresponding level 1 plasmid.

Results of the transformation with pCM1-7, pCM1-8, pCM1-9 and pCM1-10

For plate pCM1-8, there is 1 colony.
→ the digestion/ligation reaction or the transformation was not very efficient.
For the other plates, bacteria did not grow.
→ the digestion/ligation reaction or the transformation did not work.

Transplanting the pCM1-8 colony into liquid medium

- Pick the colony of pCM1-8 transformation plate and incubate in 2 mL LB/Ampicillin (50 µg/mL) /X-gal (50 µg/mL) liquid medium at 30°C overnight.

Transformation of competent cells with pCM1-7, pCM1-8, pCM1-9 and pCM1-10 again

- Transformation samples (see 07/11 samples) :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-7
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-8
3. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-9
4. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-10
5. 25 µL of JM109 bacteria + 4 µL of reaction mix from pL1-4F

- 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 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Kanamycin (50 µg/mL) Petri dish and incubate at 37°C overnight.

Digestion/Ligation reaction to assemble Nanoluc into level 1 plasmid with GFP

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 4 reactions :

Reaction mix	Master mix (μL)
BsaI-HF	4
T4 Ligase	4
Buffer NEB CS	8
ATP 10 mM	8
Vf/tube	6

- The following tubes are prepared (for more details see the excel table provided) :

1. pCM1-4 (Nanoluc - GFP)



2. pCM1-5 (GFP - Nanoluc)
3. Negative control : pL1-2F (empty vector)

- 6 μL/tube of master mix are added.
- PCR thermocycler parameters :

Cycle PCR	x3
Temperature (°C)	40	16	50	80	16
Time (min)	10	10	10	20	Indefinitely

Results of the transformation with pCM1-7, pCM1-8, pCM1-9 and pCM1-10

For plates pL1-4F, pCM1-9 and pCM1-10, bacteria didn’t grow.
→ the digestion/ligation reaction did not work.
For plate pL1-3F there are many blue colonies (100%).
→ the alpha complementation is working.
→ the white/blue selection is relevant.
For plates pCM1-7 and pCM1-8, there are many white colonies (about 70% of white colonies).
→ the digestion/ligation reaction was very efficient.

Transplanting the pCM1-7 and pCM1-8 colonies into liquid medium

- Pick 2 white colonies of pCM1-7 and pCM1-8 transformation plates and incubate them in 2 mL LB/Ampicillin (50 µg/mL) /X-gal (50 µg/mL) liquid medium at 30°C overnight.

Plasmid extraction

- Extract plasmid DNA from pCM1-8 colony using ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Digestion/Ligation reaction to assemble DGAT-B3-B4 and PLS1-B3-B4 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 4 reactions :

- The following tubes are prepared (for more details see the excel table provided) :

1. p0-DGAT (B3-B4)



2. p0-PLS1 (B3-B4)
3. Negative control : pAGM1287 (empty vector)

- 6 μL/tube of master mix are added.
- PCR thermocycler parameters :

Digestion/Ligation reaction to assemble HiBiT-B2 and HiBiT-B5 into a level M plasmid with GFP

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 5 reactions :

- The following tubes are prepared (for more details see the excel table provided) :

1. pCMM-1 (HiBiT-GFP)



2. pCMM-2 (GFP-HiBiT)



3. pCMM-3 (GFP)
4. Negative control : pAGM8031 (empty vector)

- 6 μL/tube of master mix are added.
- PCR thermocycler parameters :

Transformation of competent cells with pCMM-1, pCMM-2, pCMM-3, pCM1-4 and pCM1-5

- Transformation samples :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-1
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-2
3. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-3
4. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-4
5. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-5
6. 25 µL of JM109 bacteria + 4 µL of reaction mix from pL1-2F

- 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 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria with pCMM1, 2 and 3 on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.
- Spread bacteria with pCM1-4 and 5 on a LB/Ampicillin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

Transformation of competent cells with p0-DGAT and p0-PLS1

- Transformation samples :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from p0-DGAT
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from p0-PLS1
3. 25 µL of JM109 bacteria + 4 µL of reaction mix from pAGM1287

- 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 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

Plasmid extraction

- Extract plasmid DNA pCM1-7, pCM1-8 and pL1-3F using Ozyme ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Quality control of pCM1-7 and pCM1-8

- Prepare a master mix for 9 reactions :

- Add 100 ng of DNA :

- Add qsp 10 μL of water.
- Incubate at 37°C during 90 min.

DNA gel electrophoresis

Prepare the agarose gel 1% in TAE buffer :
- 0,5 g of agarose powder is dissolve in 50 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 20 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 8 μL of DNA + 2 μL of PromegaTM Blue/Orange Loading Dye 6X
- Ladder : 1 μL of PromegaTM Blue/Orange Loading Dye 6X + 1 μL of PromegaTM 200 bp DNA Step Ladder
- Run the gel for 30 min at 100V.

Results

For the restriction analysis of pCM1-7 and pCM1-8, we expect two fragments generated (4352 bp and about 3100 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that the HiBiT-DGAT fragments and the DGAT-HiBiT fragments have been inserted in the corresponding level 1 plasmid.

Results of the transformation with pCM1-4, pCM1-5, pCMM-1, pCMM-2 and pCMM-3

For each plate, there are many colonies (almost all white).
→ the digestion/ligation reaction was very efficient.

Transplanting the pCM1-4, pCM1-5, pCMM-1, pCMM-2 and pCMM-3 colonies into liquid medium

- Pick 2 white colonies of pCM1-4 and pCM1-5 transformation plates and incubate them in 2 mL LB/Ampicillin (50 µg/mL) /X-gal (50 µg/mL) liquid medium at 30°C overnight.
- Pick 2 white colonies of pCMM-1, pCMM-2 and pCMM-3 transformation plates and incubate them in 2 mL LB/Spectinomycin (50 µg/mL) /X-gal (50 µg/mL) liquid medium at 30°C overnight.

Plasmid extraction

- Extract plasmid DNA pCM1-4, pCM1-5, pCMM-1, pCMM-2 and pCMM-3 using Ozyme ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Quality control of pCM1-4, pCM1-5, pCMM-1, pCMM-2 and pCMM-3 (1st part)

- Prepare a master mix :

- Add 200 ng of DNA (pCMM samples are diluted 6 times and pCM1 samples 2 times) :

- Add qsp 10 μL of water.
- Incubate at 37°C during 70 min.

Results of the transformation with p0-DGAT and p0-PLS1

For plate pAGM1287 there are many colonie, almost all blue.
→ the alpha complementation is working.
→ the white/blue selection is relevant.
For plates p0-DGAT and p0-PLS1 there are many colonies, almost all white.
→ the digestion/ligation reaction was very efficient.

Transplanting the p0-DGAT and p0-PLS1 colonies into liquid medium

- Pick 2 white colonies of p0-DGAT and p0-PLS1 transformation plates and incubate them in LB/Spectinomycine (50 µg/µL)/X-gal (50 µg/µL) liquid medium at room temperature for the week-end.

Quality control of pCM1-4, pCM1-5, pCMM-1, pCMM-2 and pCMM-3 (2nd part)

DNA gel electrophoresis

Prepare the agarose gel 1% in TAE buffer :
- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 15 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 8 μL of DNA + 2 μL of PromegaTM Blue/Orange Loading Dye 6X
- Ladder : 1 μL of PromegaTM Blue/Orange Loading Dye 6X + 1 μL of PromegaTM 200 bp DNA Step Ladder
- Run the gel for 25 min at 100V.

Results

For the restriction analysis of pCM1-4 and pCM1-5, we expect two fragments generated (4352 bp and about 2900 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that the NanoLuc-GFP fragments and the GFP-NanoLuc fragments have been inserted in the corresponding level 1 plasmid.

For the restriction analysis of pCMM-1, pCMM-2 and pCMM-3, we expect two fragments generated (4623 bp and 4002 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that the ParoR + GFP-HiBiT fragments, ParoR + HiBiT-GFP fragments and the ParoR + GFP-NanoLuc fragments have been inserted in the corresponding level M plasmid.

Plasmid extraction

- Extract plasmid DNA p0-DGAT and p0-PLS1 using Ozyme ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Plasmid	p0-DGAT		p0-PLS1
Colonies	1	2	1	2
DNA concentration (ng/µL)	91.1	90.8	72.4	85.4

Quality control of p0-DGAT and p0-PLS1

- Prepare a master mix for 5 reaction :

Reaction mix	Master mix (µL)
Buffer NEB CS (10X)	5
BsaI-HF	1,5
Vf/tube	1.3

- Add 200 ng of DNA (pCMM samples are diluted 6 times and pCM1 samples 2 times) :

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
p0-DGAT (1)	91.1	2.2
p0-DGAT (2)	90.8	2.2
p0-PLS1 (1)	72.4	2.8
p0-PLS1 (2)	85.4	2.3

- Add qsp 10 μL of water.
- Incubate at 37°C during 91 min.

DNA gel electrophoresis

Prepare the agarose gel 1% in TAE buffer :
- 0,2 g of agarose powder is dissolve in 20 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 7,5 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 8 μL of DNA + 2 μL of PromegaTM Blue/Orange Loading Dye 6X
- Ladder : 1 μL of PromegaTM Blue/Orange Loading Dye 6X + 1 μL of PromegaTM 200 bp DNA Step Ladder
- Run the gel for 25 min at 100V.

Results

For the restriction analysis of p0-DGAT, we expect two fragments generated (about 2300 bp and about 1700 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that DGAT fragment has been inserted in the corresponding level 0 plasmid.

For the restriction analysis of p0-PLS1, we expect two fragments generated (about 2300 bp and about 1200 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that PLS1 fragment has been inserted in the corresponding level 0 plasmid.

Digestion/Ligation reaction to assemble NanoLuc into a level M plasmid with GFP, HiBiT and DGAT into a level M plasmid with HygroR and PLS1 into level 1 with HiBiT.

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 :

- The following tubes are prepared (for more details see the excel table provided) :

1. pCMM-4 (NanoLuc-GFP)



2. pCMM-5 (GFP-NanoLuc)
3. Negative control : pAGM8031 (empty vector)



4. pCM1-9 (HiBiT-PLS1)



5. pCM1-10 (PLS1-HiBiT)
6. Negative control : pL1-4F (empty vector)



7. pCMM-6 (HiBiT-DGAT; HygroR)



8. pCMM-7 (DGAT-HiBiT; HygroR)
9. Negative control : pAGM8055 (empty vector)

- 6 μL/tube of master mix are added.
- PCR thermocycler parameters :

For pCMM-4/5 and pCMM-6/7 :

For pCM1-9/10

Transformation of competent cells with pCM1-9, pCM1-10, pCMM-4 and pCMM-5

- Transformation samples :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-4
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-5
3. 25 µL of JM109 bacteria + 4 µL of reaction mix from pAGM8031
4. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-9
5. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-10
6. 25 µL of JM109 bacteria + 4 µL of reaction mix from pL1-4F

- 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 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria with pCMM on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.
- Spread bacteria with pCM1 on a LB/Ampicillin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

Results of the transformation with pCMM-4, pCMM-5, pCM1-9 and pCM1-10

For plates pAGM8031, there are some white colonies (false positive).
→ the alpha complementation is not working.
→ the white/blue selection is not relevant.
For plates pCMM-4 and pCMM-5, there are lots of white colonies (few blue colonies).
→ the digestion/ligation reaction was very efficient.
For plates pL1-4F, there are some white colonies (false positive).
→ the alpha complementation is not working.
→ the white/blue selection is not relevant.
For plates pCM1-9 and pCM1-10, there are lots of white colonies (few blue colonies).
→ the digestion/ligation reaction was very efficient.

Transplanting the pCMM-4, pCMM-5, pCM1-9 and pCM1-10 colonies into liquid medium

- Pick 2 white colonies of pCMM-4 and pCMM-5 transformation plates and incubate them in LB/Spectinomycine (50 µg/µL)/X-gal (50 µg/µL) liquid medium at 30°C overnight.
- Pick 2 white colonies of pCM1-9 and pCM1-10 transformation plates and incubate them in LB/Amplicillin (50 µg/µL)/X-gal (50 µg/µL) liquid medium at 30°C overnight.

Transformation of competent cells with pCMM-6 and pCMM-7

- Transformation samples :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-6
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-7
3. 25 µL of JM109 bacteria + 4 µL of reaction mix from pAGM8055

- 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 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

Plasmid extraction

- Extract plasmid DNA pICH50900 (end linker) using Ozyme ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Results of the transformation with pCMM-6 and pCMM-7

For plate pAGM8055, there are some white colonies (false positive).
→ the alpha complementation is not working.
→ the white/blue selection is not relevant.
For plates pCMM-6 and pCMM-7, there are lots of white colonies (few blue colonies).
→ the digestion/ligation reaction was very efficient.

Transplanting the pCMM-6 and pCMM-7 colonies into liquid medium

- Pick 2 white colonies on the plates pCMM-6 and pCMM-7 and incubate them in LB/Spectinomycine (50 µg/µL)/X-gal (50 µg/µL) liquid medium at 30°C overnight.

Plasmid extraction

- Extract plasmid DNA pCMM-4, pCMM-5, pCM1-9 and pCM1-10 using Ozyme ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Quality control of pCMM-4, pCMM-5, pCM1-9 and pCM1-10

- Prepare a master mix :

- Add 100 ng of DNA :

- Add qsp 10 μL of water.
- Incubate at 37°C during 1h.

DNA gel electrophoresis (pCMM)

Prepare the agarose gel 1% in TAE buffer :
- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 20 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 10 μL of DNA + 2 μL of NEBTM Gel Loading Dye, Purple (6X)
- Ladder : 1,5 μL of NEB 1kb plus DNA ladder
- Run the gel for 25 min at 100V.

Results

For the restriction analysis of pCMM-4, we expect two fragments generated (4635 and 4492 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that both transcriptional units were inserted in the corresponding level M plasmid.

For the restriction analysis of pCMM-5, we expect two fragments generated (4635 and 4492 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that both transcriptional units were inserted in the corresponding level M plasmid.

DNA gel electrophoresis (pCM1)

Prepare the agarose gel 1% in TAE buffer :
- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 20 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 10 μL of DNA + 2 μL of NEBTM Gel Loading Dye, Purple (6X)
- Ladder : 1,5 μL of NEB 1kb plus DNA ladder
- Run the gel for 25 min at 100V.

Results

For the restriction analysis of pCM1-9, we expect two fragments generated (4352 bp and 2514 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that all fragments were inserted in the corresponding level 1 plasmid.

For the restriction analysis of pCM1-10, we expect two fragments generated (4352 bp and 2489 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that all fragments were inserted in the corresponding level 1 plasmid.

Digestion/Ligation reaction to assemble HiBiT and PLS1 into a level M plasmid with HygroR and HiBiT, DGAT and PLS1 into a level M plasmid with HygroR

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 7 reactions :

- The following tubes are prepared (for more details see the excel table provided) :

1. pCMM-8 (HiBiT-PLS1; HygroR)



2. pCMM-9 (PLS1-HiBiT; HygroR)
3. Negative control : pAGM8067 (empty vector)



4. pCMM-14 (HiBiT-DGAT; HiBiT-PLS1; HygroR)



5. pCMM-15 (DGAT-HiBiT; PLS1-HiBiT; HygroR)
6. Negative control : pAGM8055 (empty vector)

- 6 μL/tube of master mix are added.
- PCR thermocycler parameters :

Transformation of competent cells with pCMM-8, pCMM-9, pCMM-14 and pCMM-15

- Transformation samples :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-8
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-9
3. 25 µL of JM109 bacteria + 4 µL of reaction mix from pAGM8067
4. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-14
5. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-15
6. 25 µL of JM109 bacteria + 4 µL of reaction mix from pAGM8055

- 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 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

Plasmid extraction

- Extract plasmid DNA pCMM-6 and pCMM-7 using Ozyme ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Quality control of pCMM-6 and pCMM-7

- Prepare a master mix for 6 reactions :

- Add 200 ng of DNA :

- Add qsp 10 μL of water.
- Incubate at 37°C during 1h.

DNA gel electrophoresis

Prepare the agarose gel 1% in TAE buffer :
- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 20 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 10 μL of DNA + 2 μL of PromegaTM Gel Loading Dye (6X)
- Ladder : 1µL PromegaTM 200bp DNA step ladder + 1µL PromegaTM Gel Loading Dye (6X)
- Run the gel for 25 min at 100V.

Results

For the restriction analysis of pCMM-6, we expect two fragments generated against one in the empty vector (4600 pb and 4891 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that both transcriptional units were inserted in the corresponding level M plasmid.

For the restriction analysis of pCMM-7, we expect two fragments generated against one in the empty vector (4600 pb and 4866 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that both transcriptional units were inserted in the corresponding level M plasmid.

Results of the transformation with pCMM-8, pCMM-9, pCMM-14 and pCMM-15

For plates pCMM-8, pCMM-9, pCMM-14 and pCMM-15, there are some white colonies.
→ the transformation was efficient.

For plates pAGM8067 and pAGM8055, there are blue colonies.
→ the alpha complementation is working.
→ the white/blue selection is relevant.

Transplanting the pCMM-8, pCMM-9, pCMM-14 and pCMM-15 colonies into liquid medium

- Pick 2 white colonies on the plates pCMM-8, pCMM-9, pCMM-14 and pCMM-15 and incubate them in LB/Spectinomycine (50 µg/µL)/X-gal (50 µg/µL) liquid medium at 30°C overnight.

Plasmid extraction

- Extract plasmid DNA pCMM-8, pCMM-9, pCMM-14 and pCMM-15 using Ozyme ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Quality control of pCMM-8, pCMM-9, pCMM-14 and pCMM-15

- Prepare a master mix for 11 reactions :

- Add 100 ng of DNA :

- Add qsp 10 μL of water.
- Incubate at 37°C during 1h.

DNA gel electrophoresis

Prepare the agarose gel 1% in TAE buffer :
- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 15 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 10 μL of DNA + 2 μL of PromegaTM Gel Loading Dye (6X)
- Ladder : 1µL PromegaTM 200bp DNA step ladder + 1µL PromegaTM Gel Loading Dye (6X)
- Run the gel for 25 min at 100V.

Results

For the restriction analysis of pCMM-8, we expect two fragments generated against only one in an empty vector (6392 bp and 2514 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that both transcriptional units were inserted in the corresponding level M plasmid.

For the restriction analysis of pCMM-9, we expect two fragments generated against only one in an empty vector (6392 bp and 2489 bp).
→ On the gel, we can see two strips in all the samples as expected, so we can conclude that both transcriptional units were inserted in the corresponding level M plasmid.

For the restriction analysis of pCMM-14, we expect three fragments generated against only one in an empty vector (6392 bp, 3099 bp and 2514 bp).
→ On the gel, we can see three strips in all the samples as expected, so we can conclude that the three transcriptional units were inserted in the corresponding level M plasmid.

For the restriction analysis of pCMM-15, we expect three fragments generated against only one in an empty vector (6392 bp, 3074 bp and 2489 bp).
→ On the gel, we can see only two strip for both clone (strips observed are different between clones), therefore, these plasmids doesn't have the three transcriptional units inserted.

Plasmid extraction

- Extract plasmid DNA pCMM-15 using Ozyme ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Quality control of pCMM-15

- Prepare a master mix for 5 reactions :

- Add 200 ng of DNA (all DNA from samples were diluted 1/10) :

- Add qsp 10 μL of water.
- Incubate at 37°C during 1h.

DNA gel electrophoresis

Prepare the agarose gel 1% in TAE buffer :
- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 20 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 10 μL of DNA + 2 μL of PromegaTM Gel Loading Dye (6X)
- Ladder : 1µL PromegaTM 200bp DNA step ladder + 1µL PromegaTM Gel Loading Dye (6X)
- Run the gel for 25 min at 100V.

Results

We expect three fragments generated against one in the empty vector (see predicted gel from 29/07).
→ On the gel, we can see only two strip for both clone (strips observed are different between clones), therefore, these plasmids doesn't have the three transcriptional units inserted.

Transformation of competent cells with pCMM-15

- Transformation samples :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-15
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from pAGM8055

- 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 µL of LB and incubate 1h at 37°C.
-Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

Purification of pCMM-1 to pCMM-9 and pCMM-14 for the transformation of Chlamydomonas reinhardtii

-Purification of plasmids pCMM-1 to pCMM-9 and pCMM-14 using NucleoSpinⓇ Gel and PCR Clean-up kit from Macherey-Nagel

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

Transformation of Chlamydomonas reinhardtii

- Aliquote 50 mL of culture in 50 mL falcon tubes 7 times (1 tube = 2 transformations).
- Centrifuge 5 min at 1500 g, RT.
- Discard the supernatant.
- Resuspend each pellet with 500 µL of TAP + 60 mM Sucrose (i.e. concentrating it 100X).
- Aliquote 250 µL of cells into a 0,4 cm gapped cuvette.
- Add 400 ng of DNA :

- Incubate 15 min on ice.
- Place each cuvette in the electroporation machine and use 800V (2000V/cm) and 25 uF with NO shunt resistance.
- Transfer the cells in 10 mL of TAP + Sucrose (60 mM).
- Let the cells recover overnight (at least 16h) while being rotated.

Results of the transformation with pCMM-15

For plate pAGM8055, transformation control plate had the same results but with less overall colonies. For plate pCMM-15, there are many white colonies with a few blue ones. → the digestion/ligation reaction was very efficient.

Transplanting pCMM-15 colonies into liquid medium

- Pick 4 white colonies of the pCMM-15 transformation plate and incubated them in LB/Spectinomycin (50 µg/mL) liquid medium at 30°C overnight.

Miniprep of the 4 pCMM-15 clones from 01.08.19

- Extract plasmid DNA pCMM-15 of 4 clones using Ozyme ZymoPURE™ Plasmid Miniprep Kit.

Determination of plasmid concentration with nanodrop

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

Quality Control digestion of pCMM-15

- Prepare a master mix for 5 reaction :

- Add 200 ng of DNA (all DNA from samples were diluted 1/10) :

- Add qsp 10 μL of water.
- Incubate at 37°C during 110 min.

DNA gel electrophoresis

Prepare the agarose gel 1% in TAE buffer :
- 0,2 g of agarose powder is dissolved in 20 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 7 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let it solidify at room temperature.
Prepare the samples :
- DNA samples : 10 μL of DNA + 2 μL of NEBTM Gel Loading Dye, Purple (6X)
- Ladder : 3 μL of NEB 1kb plus DNA ladder
- Run the gel for 40 min at 100V.

Results

For the restriction analysis of pCMM-15, we expect three fragments generated against one in the empty vector (see predicted gel from 29/07).
→ On the gel, we can see three strips in the samples 1,2 and 3 as expected but we can see only two strip for clone 4, so we can conclude that both transcriptional units were inserted in the corresponding level M plasmid only for clones 1 to 3.

Determination of plasmid concentration with nanodrop

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

Digestion of pCMM-15 to obtain the linear insert for Chlamydomonas transformation (1st part)

- Prepare a master mix for 2 reaction :

- Add DNA :

- Add qsp 10 μL of water.
- Incubate at 37°C for 1h.

Chlamydomonas culture dilution

The initial cell culture of Chlamydomonas (UVM7) is saturated with a concentration of 15.10⁶ cells/mL :
Prepare cell culture of 1.10⁶ cells/mL for optimal growth. For that, add 13,3 mL of initial cell culture in a 250 mL flask containing 200 mL of TAP medium.

Quality Control digestion of pCMM-15 (2nd part)

DNA gel electrophoresis

Prepare the agarose gel 0,8% in TAE buffer :
- 0,16 g of agarose powder is dissolved in 20 mL of TAE buffer 0,5X by heating the solution in the microwave.
- 7 μL of Ethidium bromide (EtBr) solution is added.
- The solution is casted on an electrophoresis gel mould and let solidify at room temperature.
Prepare the samples :
- DNA samples : 10 μL of DNA + 2 μL of PromegaTM Gel Loading Dye (6X)
- Ladder : 1µL PromegaTM 200bp DNA step ladder + 1µL PromegaTM Gel Loading Dye (6X)
- Run the gel for 25 min at 100V.

Results

We expect three fragments generated against one in the empty vector (see predicted gel from 29/07).
→ On the gel, we can see three strips in pCMM15 digestion as expected, so we can conclude that the insert (high size) is separated from the plasmid backbone (low size).

Extraction of the insert of pCMM-15

- To isolate the insert, cut the agarose gel under blue light in. - Extract plasmid DNA pCMM-15 of 4 clones using Macherey-Nagel NucleoSpinTM Gel and PCR Clean-up kit.

Determination of insert concentration with nanodrop

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

Transformation of Chlamydomonas reinhardtii

- Verify into the countness of chlamy swing, and the concentration culture (it must be 1-5.10⁶ cells/mL).
- Aliquote 50 mL of culture in 50 mL falcon tubes 2 times (1 tube = 2 transformations).
- Centrifuge 5 min at 1500 g, RT.
- Discard the supernatant.
- Resuspend each pellet with 500 µL of TAP + 60 mM Sucrose (i.e. concentrating it 100X).
- Aliquote 250 µL of cells into a 0,4 cm gapped cuvette.
- Add 10 ng to 1 g of DNA :

- Incubate 15 min on ice.
- Place each cuvette in the electroporation machine and use 800V (2000V/cm) and 25 uF with NO shunt resistance.
- Transfer the cells in 10 mL of TAP + Sucrose (60 mM).
- Let the cells recover overnight (at least 16h) while being rotated.

Transplantation of Chlamydomonas colonies onto solid medium

- Centrifuge 3 min at 2000 g.
- Resuspend in 500 µL TAP.
- Plate onto selective TAP agar/ Hygromycine (15 µg/mL) plates using "hockey stick spreader" under the hood, dim light.
- Transfer the plates in the light.
- Wait about 5 days for the colonies to grow.

Preparation of TAP-N medium

In order to activate the genes that are under the control of the Nia1 promoter, chlamy has to be cultivated in ammonium starvation (-NH4). To allow cell growth, we prepare a TAP-N medium containing nitrate (NO3) as a nitrogen source.

- Prepare intermediate solution of NO3 : dissolve 0,85 g of solid NaNO3 in 100 mL of H2O.
- Filtrate the intermediate solution with a vacuum pump.
- Add 2 mL of NaNO3 (100 mM) to 50 mL of TAP to a final concentration of 400 mM.

Screening test on pCMM-1 to pCMM-5

- The following samples are tested :

1. pCMM-1 : ParoR; pNIT1-HiBiT-GFP-mSTOP-tPSAD
2. pCMM-2 : ParoR; pNIT1-GFP-HiBiT-tPSAD
3. pCMM-3 : ParoR; pNIT1-GFP-mSTOP-tPSAD
4. pCMM-4 : ParoR; pNIT1-NanoLuc-GFP-mSTOP-tPASD
5. pCMM-5 : ParoR; pNIT1-GFP-NanoLuc-tPSAD

- Resuspend Chlamydomonas strains in 50 µL of TAP-N liquid medium on a 96-well plate :

- Incubate 4h at room temperature under medium light conditions.
- Transfer 25 µL of liquid culture on opaque 96-well plate :

- For pCMM-1,2 and 3 (HiBiT screen), add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well.
- Incubate 10 min.
- Add 25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- For pCMM-4 and 5 (NanoLuc screen), add 25 µL (1:1) Nano-Glo® Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Read luminescence and absorbance at 448 nm.

The results of the HiBiT screening show different positive clones for Nanoluciferase luminescence.
The results of the GFP measure can't be used because of the positive signal in the negative control.

Transplantation of positive colonies into liquid medium

- Add 25 µL of screening test positive colonies to 5 mL of TAP liquid medium and to TAP/ paromomycine (15 μg/mL) plate:

1. pCMM-1-1 : B1
2. pCMM-1-2 : D1
3. pCMM-1-3 : E2
4. pCMM-1-4 : F2
5. pCMM-1-5 : G2
6. pCMM-2-1 : A3
7. pCMM-2-2 : C3
8. pCMM-2-3 : H3
9. pCMM-2-4 : A4
10. pCMM-2-5 : E4
11. pCMM-4-1 : G8
12. pCMM-4-2 : A9
13. pCMM-5-1 : C10
14. pCMM-5-2 : D10
15. pCMM-5-3 : B11

- Incubate 4h at room temperature under medium light conditions.

Screening test on pCMM-6 to pCMM-9, pCMM-14 and pCMM-15

- The following samples are tested :

1. pCMM-6 : pNIT1-HiBiT-DGAT-mSTOP-tPSAD; HygroR
2. pCMM-7 : pNIT1-DGAT-HiBiT-tPSAD; HygroR
3. pCMM-8 : pNIT1-HiBiT-PLS1-mSTOP-tPSAD; HygroR
4. pCMM-9 : pNIT1-PLS1-HiBiT-tPSAD; HygroR
5. pCMM-14 : pNIT1-HiBiT-DGAT-tPSAD; pNIT1-HiBiT-PLS1-tPSAD; HygroR
6. pCMM-15 : pNIT1-DGAT-HiBiT-tPSAD; pNIT1-PLS1-HiBiT-tPSAD; HygroR

- Resuspend Chlamydomonas strains in 50 µL of TAP-N liquid medium on a 96-well plate :

- Incubate 4h at room temperature under medium light conditions.
- Transfer 25 µL of liquid culture on opaque 96-well plate :

- Add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well.
- Incubate 10 min.
- Before reading the luminescence, add 25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Read luminescence.

Results

The results of the HiBiT screening show different positive clones for Nanoluciferase luminescence. We can see that C-ter position of the HiBit is higher expressed than N-ter position.

Transplantation of positive colonies into liquid medium

- Add 25 µL of screening test positive colonies to 5 mL of TAP liquid medium and to TAP/ hygromycine (15 μg/mL) plate (for position, refer to plates 1 and 2) :

- Incubate 4h at room temperature under medium light conditions.

Cell counting on samples pCMM-1, pCMM-2 and pCMM-5

The cultures from the constructs pCMM1 to pCMM5 have not grown with the same speed, we selected the ones that were saturated :

1. pCMM-1-2
2. pCMM-1-4
3. pCMM-1-5
4. pCMM-2-1
5. pCMM-2-4
6. pCMM-2-5
7. pCMM-5-1
8. pCMM-5-3
9. WT UVM

- Prepare 2 mL tubes for cell counting.
- Add 32 µL of TAP medium, 4 µL of cell culture and 4 µL I2(1/10 dilution).
- Deposit 15 µL between the cell counting slide and the cover glass, 2 measures per culture.
- Count cells on automatic cell counter (final concentration needed : 1.10⁶ cells/mL, don't forget the dilution factor x10).
- Harvest the volume needed to have 1.10⁶ cells.
- Centrifuge cells at 3000g 3 min and discard the supernatant.
- Resuspend in 1 mL of TAP or TAP-N medium (both conditions for each culture).
- Incubated 4h under agitation.

Screening test on pCMM-1, pCMM-2 and pCMM-5

- Transfer 25µL of liquid culture on opaque 96-well plate :

- For HiBiT screen, add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well.
- Incubate 10 min.
- Add 25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Measure the luminescence after 2h, 23h and 24h.
- For Nanoluc screen (pCMM5-1 and pCMM5-3), transfer 25 µL of liquid culture on opaque 96-well plate.
- Add 25 µL (1:1) Nano-Glo® Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Read luminescence and absorbance at 448 nm.

Results

Due to a high signal in some of the TAP conditions, we had difficulties to use these results.
We decided to use a new TAP medium containing either NH4 or NO3, based on the concentrations used in Lopes et al, 1997.

Screening test on the rest of the clones pCMM1 to pCMM5

- The following samples are tested :

1. pCMM-1-1
2. pCMM-1-3
3. pCMM-2-2
4. pCMM-2-3
5. pCMM-3
6. pCMM-4-1
7. pCMM-4-2
8. pCMM-5-3

- Transfer 25 µL of liquid culture on opaque 96-well plate
- For HiBiT screen, add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well.
- Incubate 10 min.
- Add 25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Measure the luminescence after 2h, 23h and 24h.
- For Nanoluc screen (pCMM5-3), transfer 25 µL of liquid culture on opaque 96-well plate.
- Add 25 µL (1:1) Nano-Glo® Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Read luminescence and absorbance at 448 nm.

Preparation of TAP-N+NO3 and TAP-N+NH4 medium

In order to activate the genes that are under the control of the Nia1 promoter, chlamy has to be cultivated in ammonium starvation (-NH4). We decided to mix our own TAP+N solution in the same conditions as our TAP-N for a better comparison between our control and activated results. For sterile solutions, filtrate the intermediate solutions with a vacuum pump.

TAP-N+NH4

- Prepare a stock solution of 187,5 mM: add 1,002 g of solid NH4Cl in 100 mL H20.
- Prepare a final solution with concentration of 7,5 mM (R. Lopes, 1999): 2 mL of stock solution in 50 mL of TAP-N.

Detailed calculation:

NH4Cl: solid Final concentration used for chlamy culture: 7,5 mM

Vf = 200 mL Cf = 7,5 mmol/L = 7,5.10-3 mol/L
Vi = 4% . 200 mL Ci = (7,5.10-3.0,2)/ (0,04.0,2) = 0,1875 mol/L

Mm (NH4Cl) = 53,49 g/mol Vf = 100 mL
m = C.M.V = 0,1875.53,49.0,1 = 1,002 g

Stock solution with concentration of 187,5 mM: 1,002g NH4Cl in 100 mL H20
Final solution with concentration of 7,5 mM: 2 mL of stock solution in 50 mL of TAP-N

TAP-N+NO3

- Prepare a stock solution of 100 mM: add 0,85 g of solid NaNO3 in 100 mL H2O.
- Prepare a final final solution with concentration of 400 mM: 2 mL of stock solution in 50 mL TAP-N.

Detailed calculation :

Intermediate solution of NO3: Mr (NaNO3) = 85,01 g/mol Vi=0,04.20 mL
Vf = 200 mL Cf=0,1 mol/L 0,85 g of solid NaNO3 in 100 mL H2O

Dilution of intermediate solution into TAP-N medium: 2 mL NaNO3 (100 mM) in 50 mL TAP
Final concentration of medium: 400 mM

Transplantation of positive colonies into liquid medium (1st batch)

- Prepare Falcon tubes with 5 mL TAP.
- With "Hockey stick", pick a small quantity of colony from culture dish :

1. pCMM-1-2
2. pCMM-1-4
3. pCMM-1-5
4. pCMM-2-1
5. pCMM-2-4
6. pCMM-2-5
7. pCMM-5-1
8. pCMM-5-3
9. WT UVM

- Lay out colony on falcon wall and slowly resuspend with TAP.

Transfer of colonies in TAP-N+NO3 and TAP-N+NH4 medium (2nd batch)

- The following samples are used :

1. pCMM-1-1
2. pCMM-1-3
3. pCMM-2-2
4. pCMM-2-3
5. pCMM-3
6. pCMM-4-1
7. pCMM-4-2
8. pCMM-5-2
9. pCMM-5-3
10. WT UVM

- Count and collect 106 cells.
- Centrifugation 5 min at 3000 g.
- Resuspend each colony in 1 mL of TAP-N+NO3 or TAP-N+NH4.

Screening test on 1st batch samples

- The following samples are tested :

1. pCMM-1-1
2. pCMM-1-3
3. pCMM-2-2
4. pCMM-2-3
5. pCMM-3
6. pCMM-4-1
7. pCMM-4-2
8. pCMM-5-2
9. pCMM-5-3
10. WT UVM

- Transfer 25 µL of liquid culture on opaque 96-well plate :

- For HiBiT screen, add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well.
- Incubate 10 min.
- Before reading the luminescence, add 25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Read luminescence after 4 hours and 19 hours.
- For Nanoluc screen (pCMM-5-3), transfer 25 µL of liquid culture on opaque 96-well plate.
- Add 25 µL (1:1) Nano-Glo® Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Read luminescence and absorbance at 448 nm.

Results

Transplantation of positive colonies into liquid medium

- Resuspension of validated colonies :

1. pCMM-2-2
2. pCMM-2-3
3. pCMM-3
4. pCMM-4-1
5. pCMM-4-2
6. WT

- Prepare Falcon tubes with 5 mL of TAP.
- With "Hockey stick", pick a small quantity of colony from culture dish.
- Lay out colony on falcon wall and slowly resuspend with TAP.

Cell counting on samples pCMM-2-2, pCMM-2-3, pCMM-3, pCMM-4-1 and pCMM-4-2

- The following samples are used :

1. pCMM-2-2
2. pCMM-2-3
3. pCMM-3
4. pCMM-4-1
5. pCMM-4-2
6. WT

- Prepare 2 mL tubes for cell counting.
- Add 32 µL of TAP medium, 4 µL of cell culture and 4 µL I2 (dilution 1/10).
- Deposit 15 µL between the cell counting slide and the cover glass, 2 measures per culture.
- Count cells on automatic cell counter (don't forget the dilution factor x10, final concentration needed : 6.10⁶ cells/mL).
- Harvest the volume needed to have 1.10⁶ cells.
- Centrifuge cells at 3000 g for 3 min, discard the supernatant.
- Resuspend in 1 mL of TAP-N+NH4 or TAP-N+NO3 medium (both conditions for each culture).
- Incubate 4h and 26h at room temperature.
- Recount cells.

Screening test on pCMM-2-2, pCMM-2-3, pCMM-3, pCMM-4-1 and pCMM-4-2

- Transfer 100 µL of culture on 96 well plate :

- Distribute 50 µL of TAP in neighbour wells for cascade dilutions.
- Harvest 50 µL of first well and transfer in neighbour well, mix and repeat.
- In the last well, harvest 50 µL and throw out.
- Add 50 µL of Nano-Glo® HiBiT Lytic Buffer and 2 µL of LgBiT Protein in each well, incubate 10 min.
- Before reading the luminescence, add 50 µL Nano-Glo® HiBiT Lytic Buffer and 2 µL Nano-Glo® HiBiT Lytic Substrate in each well (for Nanoluc conditions: Buffer + Substrate premix).
- Read luminescence.

Results

Screening test on 1st batch samples

- The following samples are tested :

1. pCMM7-1
2. pCMM7-2
3. pCMM7-5
4. pCMM9-1
5. pCMM9-3
6. pCMM9-5
7. pCMM9-6
8. WT UVM

- Count cells in order to harvest 10⁶ cells. - Centrifuge and throw out supernatant. - Resuspend in 1 mL of TAP-N+NH4 or TAP-N+NO3 medium (both conditions for each culture). - Transfer 25 µL of liquid culture on opaque 96-well plate:

- For HiBiT screen, add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well.
- Incubate 10 min.
- Add 25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Measure the luminescence after 2h, 23h and 24h.
- For Nanoluc screen, transfer 25 µL of liquid culture on opaque 96-well plate.
- Add 25 µL (1:1) Nano-Glo® Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.
- Read luminescence and absorbance at 448 nm.

Results

Transplantation colonies that have not grown into liquid medium (2nd batch)

- The following samples are used :

1. pCMM7-3
2. pCMM7-4
3. pCMM8-1
4. pCMM9-2
5. pCMM9-4
6. pCMM15-1
7. pCMM15-2
8. pCMM15-3
9. pCMM15-4
10. pCMM15-5
11. pCMM15-6
12. pCMM15-7

- Harvest clones from culture dish and resuspension in 5 mL of TAP medium.

Screening test on 2nd batch samples

- The following samples are tested :

1. pCMM7-3
2. pCMM7-4
3. pCMM8-1
4. pCMM9-2
5. pCMM9-4
6. pCMM15-1
7. pCMM15-2
8. pCMM15-3
9. pCMM15-4
10. pCMM15-5
11. pCMM15-6
12. pCMM15-7

- Count cells in order to harvest 106 cells.
- Centrifuge and resuspend in 1 mL.
- Transfer 25 µL of liquid culture on opaque 96-well plate:

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.

Inter has ruinarum varietates a Nisibi quam tuebatur accitus Vrsicinus, cui nos obsecuturos iunxerat imperiale praeceptum, dispicere litis exitialis certamina cogebatur abnuens et reclamans, adulatorum oblatrantibus turmis, bellicosus sane milesque semper et militum ductor sed forensibus iurgiis longe discretus, qui metu sui discriminis anxius cum accusatores quaesitoresque subditivos sibi consociatos ex isdem foveis cerneret emergentes, quae clam palamve agitabantur, occultis Constantium litteris edocebat inplorans subsidia, quorum metu tumor notissimus Caesaris exhalaret.