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.

Transplanting colonies into liquid medium

- 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 qsf 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 dissolved 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 constructs 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 qsf 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 qsf 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 dissolved 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 qsf 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 dissolved 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 qsf 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 dissolved 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 qsf 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 dissolved 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 dissolved 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 qsf 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 dissolved 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.