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

Latest revision as of 14:09, 19 October 2019

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Resuspension of HiBiT-B2 and HiBiT-B5 DNA

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

HiBit-B5-Forward
HiBit-B5-Reverse
H3. HiBit-B2-Forward
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 :

20 μL of HiBit-B5-Forward + 20 μL of HiBit-B5-Reverse are mixed and incubated 10 min at 50°C
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 :

Reaction mix	Master mix (μL)
BbsI-HF	5
T4 Ligase	5
Buffer NEB CS	10
ATP 10 mM	10
Vf/tube	6

- The following tubes are prepared :

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

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

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

Cycle PCR	x3
Temperature (°C)	37	16	37	65	16
Time (min)	10	10	10	20	Indefinitely

3

4

5

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) :

Plasmid	p0-191 (HiBiT B2)				p0-192 (HiBiT B5)
Colonies	1	2	3	4	1	2	3	4
DNA concentration (ng/µL)	35.9	23.4	35.8	53.4	55	53.9	47.8	48.8

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7

8

9

Quality control of generated DNAs

- Prepare a master mix (for 9 reactions) :

Reaction mix	Master mix (μL)
Buffer NEB CS (10X)	9
BsaI-HF	2.7
Vf/tube	1.3

- Add 200 ng of DNA :

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
p0-191 (1)	35.9	5.6
p0-191 (2)	23.4	8.5
p0-191 (3)	35.8	5.6
p0-191 (4)	53.4	3.7
p0-192 (1)	55	3.6
p0-192 (2)	53.9	3.7
p0-192 (3)	47.8	4.8
p0-192 (4)	48.8	4.1

- 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

10

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 :

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

- The following constructs are prepared :

pCM1-1 (HiBiT-GFP)

pCM1-2 (GFP-HiBiT)

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

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

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

11

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

- Transformation samples :

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

DGAT
PLS1 (LPAAT)

Transformation of competent cells with DGAT and PLS1 to make stock

- Transformation samples:

9 µL of JM109 bacteria + 1 µL of reaction mix from DGAT
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 :

Reaction mix	Master mix (μL)
BsaI-HF	5
T4 Ligase	5
Buffer NEB CS	10
ATP 10 mM	10
Vf/tube	6

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

pCM1-7 (HiBiT-DGAT)

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

pCM1-9 (HiBiT-PLS1)

pCM1-10 (PLS1-HiBiT)
Negative control : pL1-4F (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

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13

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15

16

Determination of plasmid concentration with nanodrop

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

Plasmid	pCM1-1		pCM1-2		pCM1-3		PLS1		DGAT
Colonies	1	2	1	2	1	2	1	2	1	2
DNA concentration (ng/µL)	74.6	61.2	55.8	57.2	53.1	64.7	46.3	34.9	31.6	34.1

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

- Prepare a master mix for 8 reactions :

Reaction mix	Master mix (μL)
Buffer NEB CS (10X)	8
BbsI-HF	4
Vf/tube	1.5

- Add 100 ng of DNA :

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
pCM1-1 (1)	74.6	1.3
pCM1-1 (2)	61.2	1.6
pCM1-2 (1)	55.8	1.79
pCM1-2 (2)	57.2	1.72
pCM1-3 (1)	53.1	1.88
pCM1-3 (2)	64.7	1.55
pL1-2F	88	1.14

- 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) :

25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-7
25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-8
25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-9
25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-10
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) :

pCM1-4 (Nanoluc - GFP)

pCM1-5 (GFP - Nanoluc)
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

17

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) :

Plasmid	pCM1-8
DNA concentration (ng/µL)	440

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 :

Reaction mix	Master mix (μL)
BbsI-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) :

p0-DGAT (B3-B4)

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

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

Cycle PCR	x3
Temperature (°C)	37	16	37	65	16
Time (min)	10	10	10	20	Indefinitely

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 :

Reaction mix	Master mix (μL)
BbsI-HF	5
T4 Ligase	5
Buffer NEB CS	10
ATP 10 mM	10
Vf/tube	6

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

pCMM-1 (HiBiT-GFP)

pCMM-2 (GFP-HiBiT)

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

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

Cycle PCR	x3
Temperature (°C)	37	16	37	65	16
Time (min)	10	10	10	20	Indefinitely

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

- Transformation samples :

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

18

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

- Transformation samples :

25 µL of JM109 bacteria + 4 µL of reaction mix from p0-DGAT
25 µL of JM109 bacteria + 4 µL of reaction mix from p0-PLS1
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) :

Plasmid	pCM1-7				pCM1-8			pL1-3F
Colonies	1	2	3	4	1	2	07/17	4
DNA concentration (ng/µL)	148.4	114.2	87.9	172.9	115.3	136.4	440.8	29.4 (1/10 dilution)

Quality control of pCM1-7 and pCM1-8

- Prepare a master mix for 9 reactions :

Reaction mix	Master mix (μL)
Buffer NEB CS (10X)	9
BbsI-HF	4.5
Vf/tube	1.5

- Add 100 ng of DNA :

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
pCM1-7 (1)	148.4	0.6
pCM1-7 (2)	114.2	0.87
pCM1-7 (3)	87.9	1.13
pCM1-7 (4)	172.9	0.58
pCM1-8 (1)	115.3	0.87
pCM1-8 (2)	136.4	0.74
pCM1-8 (07/17)	440.8	0.23 (1/10 dilution)
pL1-3F	29.4 (1/10 dilution)	3.4

- 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.

19

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) :

Plasmid	pCM1-4		pCM1-5		pCMM-1		pCMM-2		pCMM-3
Colonies	1	2	1	2	1	2	1	2	1	2
DNA concentration (ng/µL)	168.6	100.9	171.8	152.2	192.5	232.8	253.1	247.7	226.1	226.4

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

- Prepare a master mix :

Reaction mix	pCMM Master mix (µL)	pCM1 Master mix (μL)
Number of reaction	5	7
Buffer NEB CS (10X)	5	7
Enzyme	1,5 (BsaI-HF)	2,1 (BbsI-HF)
Vf/tube	1.3	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)
pCM1-4 (1)	168.6	2.4
pCM1-4 (2)	100.9	4
pCM1-5 (1)	171.8	2.3
pCM1-5 (2)	152.2	2.6
pCMM-1 (1)	192.5	6.2
pCMM-1 (2)	232.8	5.2
pCMM-2 (1))	253.1	4.7
pCMM-2 (2)	247.7	4.8
pCMM-3 (1)	226.1	5.3
pCMM-3 (2)	226.4	5.3

- 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.

20

21

22

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.

23

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 :

Reaction mix	Master mix (μL) pCMM-4/5	Master mix (μL) pCM1-9/10	Master mix (μL) pCMM-6/7
Number of reaction	4	4	4
BsaI-HF	4	4 (BbsI-HF)	4
T4 Ligase	4	4	4
Buffer NEB CS	8	8	8
ATP 10 mM	8	8	8
Vf/tube	6	6	6

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

pCMM-4 (NanoLuc-GFP)

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

pCM1-9 (HiBiT-PLS1)

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

pCMM-6 (HiBiT-DGAT; HygroR)

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

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

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

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

For pCM1-9/10

Cycle PCR	x3
Temperature (°C)	37	16	37	65	16
Time (min)	10	10	10	20	Indefinitely

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

- Transformation samples :

25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-4
25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-5
25 µL of JM109 bacteria + 4 µL of reaction mix from pAGM8031
25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-9
25 µL of JM109 bacteria + 4 µL of reaction mix from pCM1-10
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.

24

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 :

25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-6
25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-7
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) :

Plasmid	pICH50900
DNA concentration (ng/µL)	70.2

25

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) :

Plasmid	pCMM-4		pCMM-5		pCM1-9		pCM1-10
Colonies	1	2	1	2	1	2	1	2
DNA concentration (ng/µL)	n/a (Miniprep didn't work)	296.3	313.6	278.7	183.1	180.9	183.4	141.7

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

- Prepare a master mix :

Reaction mix	pCMM Master mix (µL)	pCM1 Master mix (μL)
Number of reaction	2	2
Buffer NEB CS (10X)	2	2
Enzyme	1 (BsaI-HF)	1 (BbsI-HF)
Vf/tube	1.5	1.5

- Add 100 ng of DNA :

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
pCMM-4 (2)	296.3	0.67
p0-DGAT (2)	313.6	0.63
pCMM-5 (2)	278.7	0.72
pAGM8031	488	0.40
pCM1-9 (1)	183.1	0.54
pCM1-9 (2)	180,9	0,55
pCM1-10 (1)	183.4	0,54
pCM1-10 (2)	141.7	0,71
pL1-4F	29,4 (1/10 dilution from stock)	3.4

- 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 :

Reaction mix	Master mix (μL)
BbsI-HF	7
T4 Ligase	7
Buffer NEB CS	14
ATP 10 mM	14
Vf/tube	6

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

pCMM-8 (HiBiT-PLS1; HygroR)

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

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

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

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

Cycle PCR	x3
Temperature (°C)	37	16	37	75	16
Time (min)	10	10	10	20	Indefinitely

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

- Transformation samples :

25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-8
25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-9
25 µL of JM109 bacteria + 4 µL of reaction mix from pAGM8067
25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-14
25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-15
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.

26

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) :

Plasmid	pCMM-6		pCMM-7
Colonies	1	2	1	2
DNA concentration (ng/µL)	244.7	277	303	202.3

Quality control of pCMM-6 and pCMM-7

- Prepare a master mix for 6 reactions :

Reaction mix	Master mix (µL) pCMM
Buffer NEB CS (10X)	6
BsaI-HF	1,8
Vf/tube	1.3

- Add 200 ng of DNA :

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
pCMM-6 (1)	244.7	0.82
pCMM-6 (2)	277	0.72
pCMM-7 (1)	303	0.66
pCMM-7 (2)	202.3	0.99
pAGM8055	476	0.42

- 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.

27

28

29

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) :

Plasmid	pCMM-8		pCMM-9		pCMM-14		pCMM-15
Colonies	1	2	1	2	1	2	1	2
DNA concentration (ng/µL)	181.4	244.7	181.2	234.9	188.7	232.1	228.6	102.2

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

- Prepare a master mix for 11 reactions :

Reaction mix	Master mix (µL) pCMM
Buffer NEB CS (10X)	11
EcoRI	3.3
Vf/tube	1.3

- Add 100 ng of DNA :

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
pCMM-8 (1)	181.4	0.55
pCMM-8 (2)	244.7	0.41
pCMM-9 (1)	181.2	0.55
pCMM-9 (2)	234.9	0.43
pAGM8067	47,7 (1/10 dilution from stock)	2.1
pCMM-14 (1)	188.7	0.53
pCMM-14 (2)	232.1	0.43
pCMM-15 (1)	228.6	0.44
pCMM-15 (2)	102.2	0.98
pAGM8055	45,9 (1/10 dilution from stock)	2.18

- 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.
- 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.

30

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) :

Plasmid	pCMM-8
Colonies	3	4	5	6
DNA concentration (ng/µL)	869	1035	1094	958

Quality control of pCMM-15

- Prepare a master mix for 5 reactions :

Reaction mix	Master mix (µL) pCMM
Buffer NEB CS (10X)	5
EcoRI-HF	1.8
Vf/tube	1.3

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

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
pCMM-15 (3)	86.9	2.3
pCMM-15 (4)	103.5	1.9
pCMM-15 (5)	109.4	1.83
pCMM-15 (6)	95.8	2.08
pAGM8055	476	0.42

- 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

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.

31

Transformation of competent cells with pCMM-15

- Transformation samples :

25 µL of JM109 bacteria + 4 µL of reaction mix from pCMM-15
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) :

Plasmid	Conc. DNA (ng/μL)
pCMM-1	37.7
pCMM-2	32.7
pCMM-3	31.2
pCMM-4	19.9
pCMM-5	34
pCMM-6	24.2
pCMM-7	25.3
pCMM-8	21
pCMM-9	25
pCMM-14	26.3

1

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 :

Construct	Strain	Vol. DNA (μL)	Selection
pM5	+ctrl Paromomycin	1	TAP + Paromomycin
pL1-13	-ctrl Paromomycin	2.73
pCMM-1	M1	10.6
pCMM-2	M2	12.2
pCMM-3	M3	12.8
pCMM-4	M4	20.1
pCMM-5	M2	11.8
pL-13	+ ctrl Hygromycin	2.73	TAP + Hygromycin
pM5	- ctrl Hygromycin	1
pCMM-6	M6	16.5
pCMM-7	M7	15.8
pCMM-8	M8	19
pCMM-9	M9	16
pCMM-14	M14	15.2

- 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.

2

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) :

Plasmid	pCMM-15				pAGM8055
Colonies	1	2	3	4	1
DNA concentration (ng/µL)	1038	924	920	575	476

Quality Control digestion of pCMM-15

- Prepare a master mix for 5 reaction :

Reaction mix	Master mix (µL) pCMM
Buffer NEB CS (10X)	5
EcoRI-HF	1.5
Vf/tube	1.3

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

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
pCMM-15 (1)	103.8	1.92
pCMM-15 (2)	92.4	2.16
pCMM-15 (3)	92	2.17
pCMM-15 (4)	57.5	3.48
pAGM8055	47.6	8.28

- Add qsf 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.

3

4

5

Determination of plasmid concentration with nanodrop

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

Plasmid	pCMM-15
Clones	1	2	3	4
DNA concentration (ng/µL)	1038	924	920	575

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

- Prepare a master mix for 2 reaction :

Reaction mix	Master mix (µL) pCMM
Buffer NEB CS (10X)	2
EcoRI-HF	0.6
Vf/tube	1.3

- Add DNA :

Plasmid	Conc. DNA (ng/μL)	Vol. DNA (μL)
pCMM-15 (1)	1038	1.92 (1/10 dilution)
pCMM-15 (2)	924	2.16 (1/10 dilution)
pCMM-15 (3)	920	2.17 (1/10 dilution)
pCMM-15 (4)	575	3.48 (1/10 dilution)

- Add qsf 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.

6

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) :

Plasmid	pCMM-15
DNA concentration (ng/µL)	41.5

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 :

Construct	Strain	DNA (ng)	Vol. DNA (μL)	Selection
pl.22	+ ctrl Hygromycine	500	2,7	TAP + Hygromycine
iMl7	- Ctrl Hygromycine	100	3
pCMM-15	M1	100	2.4
pCMM-15	M2	200	4

- 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.

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Screening test on samples pCMM-2-2, pCMM-2-3, pCMM-3, pCMM-4-1 and pCMM-4-2 (replicat)

- The following samples are tested :

pCMM-2-2
pCMM-2-3
pCMM-3
pCMM-4-1
pCMM-4-2
WT

- 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

Standardized results compared to pCMM-3

pCMM2-2 pCMM2-3

NO₃ NH₄ NO₃ NH₄

250000 5637 787 2192 1019

125000 2332 309 1088 842

62500 1516 60 599 391

31250 1021 57 337 396

15625 544 27 364 102

Standardized results compared to WT

pCMM2-2 pCMM2-3

NO₃ NH₄ NO₃ NH₄

250000 5196.33333 435.666667 1751.33333 667.666667

125000 1385.66667 50.3333333 468.666667 427.333333

62500 1385.66667 -23.666667 468.666667 307.333333

31250 954.666667 7.33333333 261.666667 225.333333

15625 521.333333 14 341.333333 89

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Preparation of Chlamydomonas cells for fatty acids analysis

- Count cells (previously diluted) and put either in TAP+N or TAP-N medium at a final concentration of 5x10⁶ cells/mL and in a final volume of 2 mL : - For TAP-N cultures, take 10 million cells and centrifuge 3 min at 3000 g. - Wash the pellets twice using TAP-N medium. - Centrifuge again 3 min at 3000 g. - Resuspend the pellets in 2 mL of TAP-N. - For TAP+N cultures, take 10 million cells and centrifuge 3 min at 3000 g. - Resuspend the pellets in 2 mL of TAP+N. - Incubate under light and agitation.

Results

Only pCMM7-3 and pCMM15-3 NH₄ have grown.

- Weigh samples:

Sample	pCMM15-3	pCMM7-3	WT
Colonies	3	3	1
Weight (mg)	52	18	55

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

Latest revision as of 14:09, 19 October 2019

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+<center><div class="container"><img id="title-pagewiki2" src="https://2019.igem.org/wiki/images/9/95/T--Sorbonne_U_Paris--titrenotebook.png"></div></center>
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   <div id="juilday1"> <p class="bloc-date-calendrier">1</p> </div>
-<div id="juilday2"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday2Modal" style="cursor: pointer;" >2</p> </div>
+<div id="juilday2"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday2Modal" style="cursor: pointer;" >2</p> </div>
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 </div>
-<div id="juilday3"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday3Modal" style="cursor: pointer;" >3</p> </div>
+<div id="juilday3"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday3Modal" style="cursor: pointer;" >3</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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    <!--------------------END MODAL OR POP UP -------------------------->
-<div id="juilday4"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday4Modal" style="cursor: pointer;" >4</p> </div>
+<div id="juilday4"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday4Modal" style="cursor: pointer;" >4</p> </div>
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@@ Line 884: / Line 917: @@
 </p>
 <br>
-<h3>Results of the transformation with p0-191 and p0-192</h3>
+<h3>Transplanting colonies into liquid medium</h3>
 <p>- 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.</p>
@@ Line 895: / Line 928: @@
    <!--------------------END MODAL OR POP UP -------------------------->
-<div id="juilday5"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday5Modal" style="cursor: pointer;" >5</p> </div>
+<div id="juilday5"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday5Modal" style="cursor: pointer;" >5</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 976: / Line 1,009: @@
 <div id="juilday8"> <p class="bloc-date-calendrier" >8</p> </div>
-<div id="juilday9"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday9Modal" style="cursor: pointer;" >9</p> </div>
+<div id="juilday9"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday9Modal" style="cursor: pointer;" >9</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="juilday9Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 1,087: / Line 1,120: @@
 </div>
          </div>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C during 1h.</p>
@@ Line 1,093: / Line 1,126: @@
 <p>Prepare the agarose gel 1% in TAE buffer :<br>
-- 0,5 g of agarose powder is dissolve in 50 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
+- 0,5 g of agarose powder is dissolved in 50 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
 - 20 μL of Ethidium bromide (EtBr) solution is added. <br>
 - The solution is casted on an electrophoresis gel mould and let solidify at room temperature.<br>
@@ Line 1,207: / Line 1,240: @@
-<div id="juilday10"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday10Modal" style="cursor: pointer;" >10</p> </div>
+<div id="juilday10"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday10Modal" style="cursor: pointer;" >10</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 1,278: / Line 1,311: @@
          </div>
-<p>- The following construct are prepared :</p>
+<p>- The following constructs are prepared :</p>
 <ol>
@@ Line 1,367: / Line 1,400: @@
 </div>
-<div id="juilday11"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday11Modal" style="cursor: pointer;" >11</p> </div>
+<div id="juilday11"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday11Modal" style="cursor: pointer;" >11</p> </div>
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@@ Line 1,580: / Line 1,613: @@
-<div id="juilday12"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday12Modal" style="cursor: pointer;" >12</p> </div>
+<div id="juilday12"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday12Modal" style="cursor: pointer;" >12</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="juilday12Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 1,628: / Line 1,661: @@
 <div id="juilday14"> <p class="bloc-date-calendrier">14</p> </div>
-<div id="juilday15"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday15Modal" style="cursor: pointer;" >15</p> </div>
+<div id="juilday15"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday15Modal" style="cursor: pointer;" >15</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 1,687: / Line 1,720: @@
    <!--------------------END MODAL OR POP UP -------------------------->
-<div id="juilday16"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday16Modal" style="cursor: pointer;" >16</p> </div>
+<div id="juilday16"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday16Modal" style="cursor: pointer;" >16</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="juilday16Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 1,844: / Line 1,877: @@
          </div>
-<p>- Add qsp 10 μL of water. <br>
+<p>- Add qsf 10 μL of water. <br>
 - Incubate at 37°C during 1h.</p>
 <br>
@@ Line 2,026: / Line 2,059: @@
 </div>
-<div id="juilday17"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday17Modal" style="cursor: pointer;" >17</p> </div>
+<div id="juilday17"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday17Modal" style="cursor: pointer;" >17</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 2,355: / Line 2,388: @@
 </div>
-<div id="juilday18"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday18Modal" style="cursor: pointer;" >18</p> </div>
+<div id="juilday18"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday18Modal" style="cursor: pointer;" >18</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 2,531: / Line 2,564: @@
          </div>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C during 90 min.</p>
@@ Line 2,537: / Line 2,570: @@
 <p>Prepare the agarose gel 1% in TAE buffer :<br>
-- 0,5 g of agarose powder is dissolve in 50 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
+- 0,5 g of agarose powder is dissolved in 50 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
 - 20 μL of Ethidium bromide (EtBr) solution is added.<br>
 - The solution is casted on an electrophoresis gel mould and let solidify at room temperature.<br>
@@ Line 2,583: / Line 2,616: @@
-<div id="juilday19"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday19Modal" style="cursor: pointer;" >19</p> </div>
+<div id="juilday19"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday19Modal" style="cursor: pointer;" >19</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 2,766: / Line 2,799: @@
          </div>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C during 70 min.
 </p>
@@ Line 2,793: / Line 2,826: @@
 <div id="juilday21"> <p class="bloc-date-calendrier">21</p> </div>
-<div id="juilday22"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday22Modal" style="cursor: pointer;">22</p> </div>
+<div id="juilday22"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday22Modal" style="cursor: pointer;">22</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 2,820: / Line 2,853: @@
 <p>Prepare the agarose gel 1% in TAE buffer :<br>
-- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
+- 0,4 g of agarose powder is dissolved in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
 - 15 μL of Ethidium bromide (EtBr) solution is added.<br>
 - The solution is casted on an electrophoresis gel mould and let solidify at room temperature.<br>
@@ Line 2,947: / Line 2,980: @@
          </div>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C during 91 min.</p>
@@ Line 2,953: / Line 2,986: @@
 <p>Prepare the agarose gel 1% in TAE buffer :<br>
-- 0,2 g of agarose powder is dissolve in 20 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
+- 0,2 g of agarose powder is dissolved in 20 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
 - 7,5 μL of Ethidium bromide (EtBr) solution is added. <br>
 - The solution is casted on an electrophoresis gel mould and let solidify at room temperature.<br>
@@ Line 3,000: / Line 3,033: @@
-<div id="juilday23"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday23Modal" style="cursor: pointer;">23</p> </div>
+<div id="juilday23"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday23Modal" style="cursor: pointer;">23</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="juilday23Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 3,277: / Line 3,310: @@
-<div id="juilday24"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday24Modal" style="cursor: pointer;" >24</p> </div>
+<div id="juilday24"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday24Modal" style="cursor: pointer;" >24</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 3,375: / Line 3,408: @@
    <!--------------------END MODAL OR POP UP -------------------------->
-<div id="juilday25"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday25Modal" style="cursor: pointer;" >25</p> </div>
+<div id="juilday25"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday25Modal" style="cursor: pointer;" >25</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 3,567: / Line 3,600: @@
          </div>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C during 1h.</p>
 <br>
@@ Line 3,574: / Line 3,607: @@
 <p>Prepare the agarose gel 1% in TAE buffer :<br>
-- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
+- 0,4 g of agarose powder is dissolved in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
 - 20 μL of Ethidium bromide (EtBr) solution is added.<br>
 - The solution is casted on an electrophoresis gel mould and let solidify at room temperature.<br>
@@ Line 3,596: / Line 3,629: @@
 <p>Prepare the agarose gel 1% in TAE buffer :<br>
-- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
+- 0,4 g of agarose powder is dissolved in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
 - 20 μL of Ethidium bromide (EtBr) solution is added.<br>
 - The solution is casted on an electrophoresis gel mould and let solidify at room temperature.<br>
@@ Line 3,808: / Line 3,841: @@
-<div id="juilday26"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday26Modal" style="cursor: pointer;" >26</p> </div>
+<div id="juilday26"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday26Modal" style="cursor: pointer;" >26</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="juilday26Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 3,950: / Line 3,983: @@
 <br>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C during 1h.</p>
@@ Line 3,956: / Line 3,989: @@
 <p>Prepare the agarose gel 1% in TAE buffer :<br>
-- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
+- 0,4 g of agarose powder is dissolved in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
 - 20 μL of Ethidium bromide (EtBr) solution is added.<br>
 - The solution is casted on an electrophoresis gel mould and let solidify at room temperature.<br>
@@ Line 4,010: / Line 4,043: @@
 <div id="juilday28"> <p class="bloc-date-calendrier">28</p> </div>
-<div id="juilday29"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday29Modal" style="cursor: pointer;" >29</p> </div>
+<div id="juilday29"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday29Modal" style="cursor: pointer;" >29</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="juilday29Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 4,184: / Line 4,217: @@
          </div>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C during 1h.</p>
@@ Line 4,190: / Line 4,223: @@
 <p>Prepare the agarose gel 1% in TAE buffer :<br>
-- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
+- 0,4 g of agarose powder is dissolved in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
 - 15 μL of Ethidium bromide (EtBr) solution is added.<br>
 - The solution is casted on an electrophoresis gel mould and let solidify at room temperature.<br>
@@ Line 4,231: / Line 4,264: @@
 </div>
-<div id="juilday30"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday30Modal" style="cursor: pointer;" >30</p> </div>
+<div id="juilday30"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday30Modal" style="cursor: pointer;" >30</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="juilday30Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 4,372: / Line 4,405: @@
 <br>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C during 1h.</p>
@@ Line 4,378: / Line 4,411: @@
 <p>Prepare the agarose gel 1% in TAE buffer :<br>
-- 0,4 g of agarose powder is dissolve in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
+- 0,4 g of agarose powder is dissolved in 40 mL of TAE buffer 0,5X by heating the solution in the microwave.<br>
 - 20 μL of Ethidium bromide (EtBr) solution is added.<br>
 - The solution is casted on an electrophoresis gel mould and let solidify at room temperature.<br>
@@ Line 4,399: / Line 4,432: @@
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-<div id="juilday31"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#juilday31Modal" style="cursor: pointer;" >31</p> </div>
+<div id="juilday31"> <p class="bloc-date-calendrier active-juillet"  data-toggle="modal" data-target="#juilday31Modal" style="cursor: pointer;" >31</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 4,519: / Line 4,552: @@
 <div class="container" style="position: relative">
-      <div id="aoutday1"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday1Modal" style="cursor: pointer;" >1</p> </div>
+      <div id="aoutday1"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday1Modal" style="cursor: pointer;" >1</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday1Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 4,667: / Line 4,700: @@
    <!--------------------END MODAL OR POP UP -------------------------->
-<div id="aoutday2"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday2Modal" style="cursor: pointer;" >2</p> </div>
+<div id="aoutday2"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday2Modal" style="cursor: pointer;" >2</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 4,809: / Line 4,842: @@
 <br>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C during 110 min.</p>
@@ Line 4,850: / Line 4,883: @@
 <div id="aoutday4"> <p class="bloc-date-calendrier">4</p> </div>
-<div id="aoutday5"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday5Modal" style="cursor: pointer;" >5</p> </div>
+<div id="aoutday5"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday5Modal" style="cursor: pointer;" >5</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday5Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 4,979: / Line 5,012: @@
 </div>
-<p>- Add qsp 10 μL of water.<br>
+<p>- Add qsf 10 μL of water.<br>
 - Incubate at 37°C for 1h.</p>
 <br>
@@ Line 4,985: / Line 5,018: @@
 <h5><i>Chlamydomonas</I> culture dilution</h5>
-<p>The initial cell culture of Chlamydomonas (UVM7) is saturated with a concentration of 15.10^6 cells/mL : <br>
+<p>The initial cell culture of Chlamydomonas (UVM7) is saturated with a concentration of 15.10<sup>6</sup> cells/mL : <br>
-Prepare cell culture of 1.10^6 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.</p>
+Prepare cell culture of 1.10<sup>6</sup> 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.</p>
 </div>
@@ Line 4,996: / Line 5,029: @@
    <!--------------------END MODAL OR POP UP -------------------------->
-<div id="aoutday6"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday6Modal" style="cursor: pointer;" >6</p> </div>
+<div id="aoutday6"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday6Modal" style="cursor: pointer;" >6</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday6Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,069: / Line 5,102: @@
 <h5>Transformation of <I>Chlamydomonas reinhardtii</I></h5>
-<p>- Verify into the countness of chlamy swing, and the concentration culture (it must be 1-5.106 cells/mL).<br>
+<p>- Verify into the countness of chlamy swing, and the concentration culture (it must be 1-5.10<sup>6</sup> cells/mL).<br>
 - Aliquote 50 mL of culture in 50 mL falcon tubes 2 times (1 tube = 2 transformations).<br>
 - Centrifuge 5 min at 1500 g, RT.<br>
@@ Line 5,136: / Line 5,169: @@
    <!--------------------END MODAL OR POP UP -------------------------->
-<div id="aoutday7"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday7Modal" style="cursor: pointer;" >7</p> </div>
+<div id="aoutday7"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday7Modal" style="cursor: pointer;" >7</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday7Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,179: / Line 5,212: @@
 <div id="aoutday12"> <p class="bloc-date-calendrier">12</p> </div>
-<div id="aoutday13"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday13Modal" style="cursor: pointer;" >13</p> </div>
+<div id="aoutday13"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday13Modal" style="cursor: pointer;" >13</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday13Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,202: / Line 5,235: @@
 <h5>Preparation of TAP-N medium</h5>
-<p>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.
+<p>In order to activate the genes that are under the control of the Nia1 promoter, chlamy has to be cultivated in ammonium starvation (-NH<sub>4</sub>). To allow cell growth, we prepare a TAP-N medium containing nitrate (NO<sub>3</sub>) as a nitrogen source.
 <br><br>
-- Prepare intermediate solution of NO3 : dissolve 0,85 g of solid NaNO3 in 100 mL of H2O.<br>
+- Prepare intermediate solution of NO<sub>3</sub> : dissolve 0,85 g of solid NaNO<sub>3</sub> in 100 mL of H2O.<br>
 - Filtrate the intermediate solution with a vacuum pump.<br>
-- Add 2 mL of NaNO3 (100 mM) to 50 mL of TAP to a final concentration of 400 mM.</p>
+- Add 2 mL of NaNO<sub>3</sub> (100 mM) to 50 mL of TAP to a final concentration of 400 mM.</p>
 <br>
@@ Line 5,306: / Line 5,339: @@
 </div>
-<div id="aoutday14"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday14Modal" style="cursor: pointer;" >14</p> </div>
+<div id="aoutday14"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday14Modal" style="cursor: pointer;" >14</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 5,468: / Line 5,501: @@
 <div id="aoutday18"> <p class="bloc-date-calendrier">18</p> </div>
-<div id="aoutday19"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday19Modal" style="cursor: pointer;" >19</p> </div>
+<div id="aoutday19"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday19Modal" style="cursor: pointer;" >19</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday19Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,484: / Line 5,517: @@
 <div class="modal-body color-div" style="width: 40%; height: 500px;overflow: auto; background-color: #f5e3a0;">
-<p class="aoutday19" > <span class="title-calendrier">MONDAY 19<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Marta De Almeida</p>
+<p class="aoutday19" > <span class="title-calendrier">MONDAY 19<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Selma Bennacer<br>Marta De Almeida</p>
 </div>
@@ Line 5,506: / Line 5,539: @@
       </div>
          </div>
    <!--------------------END MODAL OR POP UP -------------------------->
+<div class="modal liste1NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+       <img style="width: 100%;" src="https://2019.igem.org/wiki/images/a/ab/T--Sorbonne_U_Paris--liste1NB.png" >
+    </div>
+  </div>
+</div>
-<div id="aoutday20"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday20Modal" style="cursor: pointer;" >20</p> </div>
+<div id="aoutday20"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday20Modal" style="cursor: pointer;" >20</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
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@@ Line 5,524: / Line 5,566: @@
 <div class="modal-body color-div" style="width: 40%; height: 500px;overflow: auto; background-color: #f5e3a0;">
-<p class="aoutday20" > <span class="title-calendrier">TUESDAY 20<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Marta De Almeida</p>
+<p class="aoutday20" > <span class="title-calendrier">TUESDAY 20<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Selma Bennacer<br>Marta De Almeida</p>
 </div>
 <div class="modal-body" style="width: 100%; height: 500px;overflow: auto;">
-<p>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.</p>
- <p>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.</p>
+<h5>Cell counting on samples pCMM-1, pCMM-2 and pCMM-5</h5>
-<p>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.</p>
-<p>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.</p>
+<p>The cultures from the constructs pCMM1 to pCMM5 have not grown with the same speed, we selected the ones that were saturated : </p>
+<br>
+<ol>
+<li> pCMM-1-2 </li>
+<li > pCMM-1-4 </li>
+<li> pCMM-1-5 </li>
+<li> pCMM-2-1 </li>
+<li> pCMM-2-4 </li>
+<li> pCMM-2-5 </li>
+<li> pCMM-5-1 </li>
+<li> pCMM-5-3 </li>
+<li> WT UVM</li>
+</ol>
+<br>
+<p>- Prepare 2 mL tubes for cell counting.<br>
+- Add 32 µL of TAP medium, 4 µL of cell culture and 4 µL I2(1/10 dilution).<br>
+- Deposit 15 µL between the cell counting slide and the cover glass, 2 measures per culture.<br>
+- Count cells on automatic cell counter (final concentration needed : 1.10<sup>6</sup> cells/mL, don't forget the dilution factor x10).<br>
+- Harvest the volume needed to have 1.10<sup>6</sup> cells.<br>
+- Centrifuge cells at 3000g 3 min and discard the supernatant.<br>
+- Resuspend in 1 mL of TAP or TAP-N medium (both conditions for each culture).<br>
+- Incubated 4h under agitation.<br>
+</p>
+<br>
+<h5>Screening test on pCMM-1, pCMM-2 and pCMM-5</h5>
+<p>- Transfer 25µL of liquid culture on opaque 96-well plate :</p>
+<center><img src="https://2019.igem.org/wiki/images/0/03/T--Sorbonne_U_Paris--tableau38NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau38NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/5/5e/T--Sorbonne_U_Paris--tableau39NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau39NB"></center>
+<br>
+<p>- For HiBiT screen, add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well. <br>
+- Incubate 10 min. <br>
+- Add  25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well. <br>
+- Measure the luminescence after 2h, 23h and 24h.<br>
+- For Nanoluc screen (pCMM5-1 and pCMM5-3), transfer 25 µL of liquid culture on opaque 96-well plate. <br>
+- Add 25 µL (1:1) Nano-Glo®  Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well. <br>
+- Read luminescence and absorbance at 448 nm.
+</p>
+<h4>Results</h4>
+<center><img src="https://2019.igem.org/wiki/images/c/c2/T--Sorbonne_U_Paris--tableau40NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau40NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/5/58/T--Sorbonne_U_Paris--tableau41NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau41NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/3/3c/T--Sorbonne_U_Paris--tableau42NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau42NB"></center>
+<br>
+<p>Due to a high signal in some of the TAP conditions, we had difficulties to use these results.<br> We decided to use a new TAP medium containing either NH4 or NO3, based on the concentrations used in <I>Lopes et al, 1997. </I></p>
 </div>
 </div>
@@ Line 5,539: / Line 5,636: @@
       </div>
          </div>
    <!--------------------END MODAL OR POP UP -------------------------->
+<div class="modal tableau38NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+       <img style="width: 100%;" src="https://2019.igem.org/wiki/images/0/03/T--Sorbonne_U_Paris--tableau38NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau39NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/5/5e/T--Sorbonne_U_Paris--tableau39NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau40NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/c/c2/T--Sorbonne_U_Paris--tableau40NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau41NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/5/58/T--Sorbonne_U_Paris--tableau41NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau42NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/3/3c/T--Sorbonne_U_Paris--tableau42NB.png" >
+    </div>
+  </div>
+</div>
-<div id="aoutday21"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday21Modal" style="cursor: pointer;" >21</p> </div>
+<div id="aoutday21"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday21Modal" style="cursor: pointer;" >21</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday21Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,557: / Line 5,699: @@
 <div class="modal-body color-div" style="width: 40%; height: 500px;overflow: auto; background-color: #f5e3a0;">
-<p class="aoutday21" > <span class="title-calendrier">WEDNESDAY 21<sup>st</sup> OF AUGUST 2019</span></FONT><br><br><br>Marta De Almeida</p>
+<p class="aoutday21" > <span class="title-calendrier">WEDNESDAY 21<sup>st</sup> OF AUGUST 2019</span></FONT><br><br><br>Selma Bennacer<br>Marta De Almeida</p>
 </div>
 <div class="modal-body" style="width: 100%; height: 500px;overflow: auto;">
-<p>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.</p>
- <p>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.</p>
+<h5>Screening test on the rest of the clones pCMM1 to pCMM5</h5>
-<p>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.</p>
-<p>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.</p>
+<p>- The following samples are tested :</p>
+<br>
+<ol>
+<li> pCMM-1-1 </li>
+<li > pCMM-1-3 </li>
+<li> pCMM-2-2 </li>
+<li> pCMM-2-3 </li>
+<li > pCMM-3 </li>
+<li> pCMM-4-1</li>
+<li > pCMM-4-2 </li>
+<li> pCMM-5-3 </li>
+</ol>
+<br>
+<p>- Transfer 25 µL of liquid culture on opaque 96-well plate<br>
+- For HiBiT screen, add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well. <br>
+- Incubate 10 min. <br>
+- Add  25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well. <br>
+- Measure the luminescence after 2h, 23h and 24h.<br>
+- For Nanoluc screen (pCMM5-3), transfer 25 µL of liquid culture on opaque 96-well plate.<br>
+- Add 25 µL (1:1) Nano-Glo®  Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well. <br>
+- Read luminescence and absorbance at 448 nm.  </p>
 </div>
 </div>
@@ Line 5,573: / Line 5,738: @@
    <!--------------------END MODAL OR POP UP -------------------------->
-<div id="aoutday22"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday22Modal" style="cursor: pointer;" >22</p> </div>
+<div id="aoutday22"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday22Modal" style="cursor: pointer;" >22</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday22Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,589: / Line 5,754: @@
 <div class="modal-body color-div" style="width: 40%; height: 500px;overflow: auto; background-color: #f5e3a0;">
-<p class="aoutday22" > <span class="title-calendrier">THURSDAY 22<sup>nd</sup> OF AUGUST 2019</span></FONT><br><br><br>Marta De Almeida</p>
+<p class="aoutday22" > <span class="title-calendrier">THURSDAY 22<sup>nd</sup> OF AUGUST 2019</span></FONT><br><br><br>Selma Bennacer<br>Marta De Almeida</p>
 </div>
 <div class="modal-body" style="width: 100%; height: 500px;overflow: auto;">
-<p>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.</p>
- <p>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.</p>
+<h5>Preparation of TAP-N+NO<sub>3</sub> and TAP-N+NH<sub>4</sub> medium</h5>
-<p>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.</p>
-<p>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.</p>
+<p>In order to activate the genes that are under the control of the Nia1 promoter, chlamy has to be cultivated in ammonium starvation (-NH<sub>4</sub>). 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.</p>
+<h4>TAP-N+NH<sub>4</sub></h4>
+<p>- Prepare a stock solution of 187,5 mM: add 1,002 g of solid NH<sub>4</sub>Cl in 100 mL H<sub>2</sub>0.<br>
+- Prepare a final solution with concentration of 7,5 mM (R. Lopes, 1999): 2 mL of stock solution in 50 mL of TAP-N.
+<br><br>
+<i>Detailed calculation:
+<br><br>
+NH<sub>4</sub>Cl: solid         Final concentration used for chlamy culture: 7,5 mM
+<br><br>
+Vf = 200 mL                Cf = 7,5 mmol/L = 7,5.10-3 mol/L<br>
+Vi = 4% . 200 mL        Ci = (7,5.10-3.0,2)/ (0,04.0,2) = 0,1875 mol/L
+<br><br>
+Mm (NH<sub>4</sub>Cl) = 53,49 g/mol        Vf = 100 mL<br>
+m = C.M.V = 0,1875.53,49.0,1 = 1,002 g
+<br><br>
+Stock solution with concentration of 187,5 mM: 1,002g NH<sub>4</sub>Cl in 100 mL H20<br>
+Final solution with concentration of 7,5 mM: 2 mL of stock solution in 50 mL of TAP-N
+</i></p>
+<h4>TAP-N+NO<sub>3</sub></h4>
+<p>- Prepare a stock solution of 100 mM: add 0,85 g of solid NaNO<sub>3</sub> in 100 mL H<sub>2</sub>O.<br>
+- Prepare a final final solution with concentration of 400 mM: 2 mL of stock solution in 50 mL TAP-N.
+<br><br>
+<I>Detailed calculation :
+<br><br>
+Intermediate solution of NO<sub>3</sub>: Mr (NaNO<sub>3</sub>) = 85,01 g/mol             Vi=0,04.20 mL<br>
+                                                  Vf = 200 mL                                      Cf=0,1 mol/L
+,85 g of solid NaNO<sub>3</sub> in 100 mL H2O
+<br><br>
+Dilution of intermediate solution into TAP-N medium: 2 mL NaNO<sub>3</sub> (100 mM) in 50 mL TAP<br>
+Final concentration of medium: 400 mM
+</i></p>
+<h5>Transplantation of positive colonies into liquid medium (1st batch)</h5>
+<p>- Prepare Falcon tubes with 5 mL TAP.<br>
+- With "Hockey stick", pick a small quantity of colony from culture dish : </p>
+<br>
+<ol>
+<li> pCMM-1-2 </li>
+<li > pCMM-1-4 </li>
+<li> pCMM-1-5 </li>
+<li> pCMM-2-1 </li>
+<li> pCMM-2-4 </li>
+<li> pCMM-2-5 </li>
+<li> pCMM-5-1 </li>
+<li> pCMM-5-3 </li>
+<li> WT UVM</li>
+</ol>
+<br>
+<p>- Lay out colony on falcon wall and slowly resuspend with TAP.</p>
+<h5>Transfer of colonies in TAP-N+NO<sub>3</sub> and TAP-N+NH<sub>4</sub> medium (2nd batch)</h5>
+<p>- The following samples are used : </p>
+<br>
+<ol>
+<li> pCMM-1-1 </li>
+<li > pCMM-1-3 </li>
+<li> pCMM-2-2 </li>
+<li> pCMM-2-3 </li>
+<li> pCMM-3 </li>
+<li> pCMM-4-1 </li>
+<li> pCMM-4-2 </li>
+<li> pCMM-5-2 </li>
+<li> pCMM-5-3 </li>
+<li> WT UVM</li>
+</ol>
+<br>
+<p>- Count and collect 106 cells.<br>
+- Centrifugation 5 min at 3000 g.<br>
+- Resuspend each colony in 1 mL of TAP-N+NO<sub>3</sub> or TAP-N+NH<sub>4</sub>.
+</p>
 </div>
 </div>
@@ Line 5,606: / Line 5,850: @@
    <!--------------------END MODAL OR POP UP -------------------------->
-<div id="aoutday23"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday23Modal" style="cursor: pointer;" >23</p> </div>
+<div id="aoutday23"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday23Modal" style="cursor: pointer;" >23</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday23Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,622: / Line 5,866: @@
 <div class="modal-body color-div" style="width: 40%; height: 500px;overflow: auto; background-color: #f5e3a0;">
-<p class="aoutday23" > <span class="title-calendrier">FRIDAY 23<sup>RD</sup> OF AUGUST 2019</span></FONT><br><br><br>Marta De Almeida</p>
+<p class="aoutday23" > <span class="title-calendrier">FRIDAY 23<sup>RD</sup> OF AUGUST 2019</span></FONT><br><br><br>Selma Bennacer<br>Marta De Almeida</p>
 </div>
 <div class="modal-body" style="width: 100%; height: 500px;overflow: auto;">
-<p>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.</p>
- <p>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.</p>
+<h5>Screening test on 1st batch samples</h5>
-<p>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.</p>
-<p>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.</p>
+<p>- The following samples are tested : </p>
+<br>
+<ol>
+<li> pCMM-1-1 </li>
+<li > pCMM-1-3 </li>
+<li> pCMM-2-2 </li>
+<li> pCMM-2-3 </li>
+<li> pCMM-3 </li>
+<li> pCMM-4-1 </li>
+<li> pCMM-4-2 </li>
+<li> pCMM-5-2 </li>
+<li> pCMM-5-3 </li>
+<li> WT UVM</li>
+</ol>
+<br>
+<p>- Transfer 25 µL of liquid culture on opaque 96-well plate :</p>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/6/61/T--Sorbonne_U_Paris--tableau43NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau43NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/e/e4/T--Sorbonne_U_Paris--tableau44NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau44NB"></center>
+<br>
+<p>- For HiBiT screen, add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well.<br>
+-  Incubate 10 min. <br>
+- Before reading the luminescence, add  25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well. <br>
+- Read luminescence after 4 hours and 19 hours.<br>
+- For Nanoluc screen (pCMM-5-3), transfer 25 µL of liquid culture on opaque 96-well plate. <br>
+- Add 25 µL (1:1) Nano-Glo®  Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well. <br>
+- Read luminescence and absorbance at 448 nm. </p>
+<h4>Results</h4>
+<center><img src="https://2019.igem.org/wiki/images/4/4f/T--Sorbonne_U_Paris--tableau45NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau45NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/8/8d/T--Sorbonne_U_Paris--tableau46NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau46NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/6/61/T--Sorbonne_U_Paris--tableau43NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau43NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/1/13/T--Sorbonne_U_Paris--graph1NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".graph1NB"></center>
+<br>
+<h5>Transplantation of positive colonies into liquid medium</h5>
+<p>- Resuspension of validated colonies : </p>
+<br>
+<ol>
+<li> pCMM-2-2 </li>
+<li > pCMM-2-3 </li>
+<li> pCMM-3 </li>
+<li> pCMM-4-1 </li>
+<li> pCMM-4-2 </li>
+<li> WT</li>
+</ol>
+<br>
+<p>- Prepare Falcon tubes with 5 mL of TAP.<br>
+- With "Hockey stick", pick a small quantity of colony from culture dish.<br>
+- Lay out colony on falcon wall and slowly resuspend with TAP.
+</p>
 </div>
 </div>
@@ Line 5,637: / Line 5,944: @@
       </div>
          </div>
    <!--------------------END MODAL OR POP UP -------------------------->
+<div class="modal tableau43NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+       <img style="width: 100%;" src="https://2019.igem.org/wiki/images/6/61/T--Sorbonne_U_Paris--tableau43NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau44NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/e/e4/T--Sorbonne_U_Paris--tableau44NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau45NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/4/4f/T--Sorbonne_U_Paris--tableau45NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau46NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/8/8d/T--Sorbonne_U_Paris--tableau46NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau47NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/9/98/T--Sorbonne_U_Paris--tableau47NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal graph1NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/1/13/T--Sorbonne_U_Paris--graph1NB.png" >
+    </div>
+  </div>
+</div>
 <div id="aoutday24"> <p class="bloc-date-calendrier">24</p> </div>
@@ Line 5,643: / Line 6,003: @@
 <div id="aoutday26"> <p class="bloc-date-calendrier">26</p> </div>
-<div id="aoutday27"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday27Modal" style="cursor: pointer;" >27</p> </div>
+<div id="aoutday27"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday27Modal" style="cursor: pointer;" >27</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday27Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,659: / Line 6,019: @@
 <div class="modal-body color-div" style="width: 40%; height: 500px;overflow: auto; background-color: #f5e3a0;">
-<p class="aoutday27" > <span class="title-calendrier">TUESDAY 27<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Marta De Almeida</p>
+<p class="aoutday27" > <span class="title-calendrier">TUESDAY 27<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Selma Bennacer<br>Marta De Almeida</p>
 </div>
 <div class="modal-body" style="width: 100%; height: 500px;overflow: auto;">
-<p>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.</p>
- <p>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.</p>
+<h5>Cell counting on samples pCMM-2-2, pCMM-2-3, pCMM-3, pCMM-4-1 and pCMM-4-2</h5>
-<p>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.</p>
-<p>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.</p>
+<p>- The following samples are used :</p>
+<br>
+<ol>
+<li> pCMM-2-2 </li>
+<li> pCMM-2-3 </li>
+<li> pCMM-3 </li>
+<li> pCMM-4-1 </li>
+<li> pCMM-4-2 </li>
+<li> WT </li>
+</ol>
+<br>
+<p>- Prepare 2 mL tubes for cell counting.<br>
+- Add 32 µL of TAP medium, 4 µL of cell culture and 4 µL I2 (dilution 1/10).<br>
+- Deposit 15 µL between the cell counting slide and the cover glass, 2 measures per culture.<br>
+- Count cells on automatic cell counter (don't forget the dilution factor x10, final concentration needed : 6.10<sup>6</sup> cells/mL).<br>
+- Harvest the volume needed to have 1.10<sup>6</sup> cells.<br>
+- Centrifuge cells at 3000 g for 3 min, discard the supernatant. <br>
+- Resuspend in 1 mL of TAP-N+NH<sub>4</sub> or TAP-N+NO<sub>3</sub> medium (both conditions for each culture).<br>
+- Incubate 4h and 26h at room temperature.<br>
+- Recount cells.</p>
+<br>
+<h5>Screening test on pCMM-2-2, pCMM-2-3, pCMM-3, pCMM-4-1 and pCMM-4-2</h5>
+<p>- Transfer 100 µL of culture on 96 well plate :</p>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/c/c9/T--Sorbonne_U_Paris--tableau48NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau48NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/6/63/T--Sorbonne_U_Paris--tableau49NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau49NB"></center>
+<br>
+<p>- Distribute 50 µL of TAP in neighbour wells for cascade dilutions.<br>
+- Harvest 50 µL of first well and transfer in neighbour well, mix and repeat.<br>
+- In the last well, harvest 50 µL and throw out.<br>
+- Add 50 µL of Nano-Glo® HiBiT Lytic Buffer and 2 µL of LgBiT Protein in each well, incubate 10 min.<br>
+- 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).<br>
+- Read luminescence.
+</p>
+<h4>Results</h4>
+<center><img src="https://2019.igem.org/wiki/images/6/60/T--Sorbonne_U_Paris--tableau50NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau50NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/1/12/T--Sorbonne_U_Paris--tableau51NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau51NB"></center>
+<br>
+<h5>Screening test on 1st batch samples</h5>
+<p>- The following samples are tested :</p>
+ <br>
+<ol>
+<li> pCMM7-1 </li>
+<li > pCMM7-2 </li>
+<li> pCMM7-5 </li>
+<li> pCMM9-1 </li>
+<li> pCMM9-3 </li>
+<li> pCMM9-5 </li>
+<li> pCMM9-6 </li>
+<li> WT UVM</li>
+</ol>
+<br>
+<p>- Count cells in order to harvest 10<sup>6</sup> cells.
+- Centrifuge and throw out supernatant.
+- Resuspend in 1 mL of TAP-N+NH<sub>4</sub> or TAP-N+NO<sub>3</sub> medium (both conditions for each culture).
+- Transfer 25 µL of liquid culture on opaque 96-well plate:
+</p>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/c/cb/T--Sorbonne_U_Paris--tableau52NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau52NB"></center>
+<br>
+<p>- For HiBiT screen, add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well.<br>
+- Incubate 10 min. <br>
+- Add  25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well. <br>
+- Measure the luminescence after 2h, 23h and 24h.<br>
+- For Nanoluc screen, transfer 25 µL of liquid culture on opaque 96-well plate. <br>
+- Add 25 µL (1:1) Nano-Glo®  Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well. <br>
+- Read luminescence and absorbance at 448 nm.
+</p>
+<h4>Results</h4>
+<center><img src="https://2019.igem.org/wiki/images/7/7b/T--Sorbonne_U_Paris--tableau53NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau53NB"></center>
+<br>
+<h5>Transplantation colonies that have not grown into liquid medium (2nd batch)</h5>
+<p>- The following samples are used : </p>
+<br>
+<ol>
+<li> pCMM7-3 </li>
+<li > pCMM7-4 </li>
+<li> pCMM8-1 </li>
+<li> pCMM9-2 </li>
+<li> pCMM9-4 </li>
+<li> pCMM15-1 </li>
+<li> pCMM15-2 </li>
+<li> pCMM15-3 </li>
+<li> pCMM15-4 </li>
+<li> pCMM15-5 </li>
+<li> pCMM15-6 </li>
+<li> pCMM15-7 </li>
+</ol>
+<br>
+<p>- Harvest clones from culture dish and resuspension in 5 mL of TAP medium.</p>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/6/68/T--Sorbonne_U_Paris--graph2NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".graph2NB"></center>
+<br>
 </div>
 </div>
@@ Line 5,674: / Line 6,150: @@
       </div>
          </div>
    <!--------------------END MODAL OR POP UP -------------------------->
+<div class="modal tableau48NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+       <img style="width: 100%;" src="https://2019.igem.org/wiki/images/c/c9/T--Sorbonne_U_Paris--tableau48NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau49NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/6/63/T--Sorbonne_U_Paris--tableau49NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau50NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/6/60/T--Sorbonne_U_Paris--tableau50NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau51NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/1/12/T--Sorbonne_U_Paris--tableau51NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau52NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/c/cb/T--Sorbonne_U_Paris--tableau52NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau53NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/7/7b/T--Sorbonne_U_Paris--tableau53NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal graph2NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/6/68/T--Sorbonne_U_Paris--graph2NB.png" >
+    </div>
+  </div>
+</div>
-<div id="aoutday28"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday28Modal" style="cursor: pointer;" >28</p> </div>
+<div id="aoutday28"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday28Modal" style="cursor: pointer;" >28</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday28Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,692: / Line 6,230: @@
 <div class="modal-body color-div" style="width: 40%; height: 500px;overflow: auto; background-color: #f5e3a0;">
-<p class="aoutday28" > <span class="title-calendrier">WEDNESDAY 28<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Marta De Almeida</p>
+<p class="aoutday28" > <span class="title-calendrier">WEDNESDAY 28<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Selma Bennacer<br>Marta De Almeida</p>
 </div>
 <div class="modal-body" style="width: 100%; height: 500px;overflow: auto;">
-<p>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.</p>
- <p>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.</p>
+<h5>Screening test on 2nd batch samples</h5>
-<p>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.</p>
-<p>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.</p>
+<p>- The following samples are tested :</p>
+<br>
+<ol>
+<li> pCMM7-3 </li>
+<li > pCMM7-4 </li>
+<li> pCMM8-1 </li>
+<li> pCMM9-2 </li>
+<li> pCMM9-4 </li>
+<li> pCMM15-1 </li>
+<li> pCMM15-2 </li>
+<li> pCMM15-3 </li>
+<li> pCMM15-4 </li>
+<li> pCMM15-5 </li>
+<li> pCMM15-6 </li>
+<li> pCMM15-7 </li>
+</ol>
+<br>
+<p>- Count cells in order to harvest 10<sup>6</sup> cells.<br>
+- Centrifuge and resuspend in 1 mL.<br>
+- Transfer 25 µL of liquid culture on opaque 96-well plate:</p>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/d/d4/T--Sorbonne_U_Paris--tableau54NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau54NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/1/1d/T--Sorbonne_U_Paris--tableau55NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau55NB"></center>
+<br>
+<p>- Add 50 µL (1:1)  Nano-Glo® HiBiT Lytic Buffer and 2 µL 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.
+- Read luminescence.</p>
+<h4>Results</h4>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/2/20/T--Sorbonne_U_Paris--tableau56NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau56NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/b/be/T--Sorbonne_U_Paris--tableau57NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau57NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/9/98/T--Sorbonne_U_Paris--graph3.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".graph3NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/0/07/T--Sorbonne_U_Paris--graph4.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".graph4NB"></center>
+<br>
 </div>
 </div>
@@ Line 5,707: / Line 6,290: @@
       </div>
          </div>
    <!--------------------END MODAL OR POP UP -------------------------->
+<div class="modal tableau54NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+       <img style="width: 100%;" src="https://2019.igem.org/wiki/images/d/d4/T--Sorbonne_U_Paris--tableau54NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau55NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/1/1d/T--Sorbonne_U_Paris--tableau55NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau56NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/2/20/T--Sorbonne_U_Paris--tableau56NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau57NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/b/be/T--Sorbonne_U_Paris--tableau57NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal graph3NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/9/98/T--Sorbonne_U_Paris--graph3.png" >
+    </div>
+  </div>
+</div>
+<div class="modal graph4NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/0/07/T--Sorbonne_U_Paris--graph4.png" >
+    </div>
+  </div>
+</div>
 <div id="aoutday29"> <p class="bloc-date-calendrier">29</p> </div>
-<div id="aoutday30"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#aoutday30Modal" style="cursor: pointer;" >30</p> </div>
+<div id="aoutday30"> <p class="bloc-date-calendrier active-aout"  data-toggle="modal" data-target="#aoutday30Modal" style="cursor: pointer;" >30</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="aoutday30Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,727: / Line 6,363: @@
 <div class="modal-body color-div" style="width: 40%; height: 500px;overflow: auto; background-color: #f5e3a0;">
-<p class="aoutday30" > <span class="title-calendrier">FRIDAY 30<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Marta De Almeida</p>
+<p class="aoutday30" > <span class="title-calendrier">FRIDAY 30<sup>th</sup> OF AUGUST 2019</span></FONT><br><br><br>Selma Bennacer<br>Marta De Almeida</p>
 </div>
 <div class="modal-body" style="width: 100%; height: 500px;overflow: auto;">
-<p>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.</p>
- <p>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.</p>
+<h5>Screening test on samples pCMM-2-2, pCMM-2-3, pCMM-3, pCMM-4-1 and pCMM-4-2 (replicat)</h5>
-<p>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.</p>
-<p>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.</p>
+<p>- The following samples are tested :</p>
+<br>
+<ol>
+<li> pCMM-2-2 </li>
+<li> pCMM-2-3 </li>
+<li> pCMM-3 </li>
+<li> pCMM-4-1 </li>
+<li> pCMM-4-2 </li>
+<li> WT </li>
+</ol>
+<br>
+<p>- Transfer 25 µL of liquid culture on opaque 96-well plate :</p>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/b/b2/T--Sorbonne_U_Paris--tableau58NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau58NB"></center>
+<br>
+<p>- Add 25 µL (1:1) Nano-Glo® HiBiT Lytic Buffer and 1 µL LgBiT Protein in each well.<br>
+- Incubate 10 min. <br>
+- Before reading the luminescence, add 25 µL Nano-Glo® HiBiT Lytic Buffer and 1 µL Nano-Glo® HiBiT Lytic Substrate in each well.<br>
+- Read luminescence.
+</p>
+<h4>Results</p>
+<center><img src="https://2019.igem.org/wiki/images/6/6a/T--Sorbonne_U_Paris--tableau59NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau59NB"></center>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/6/68/T--Sorbonne_U_Paris--tableau60NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".tableau60NB"></center>
+<br>
+<div class="d-flex justify-content-center">
+<div class="table-responsive-sm">
+    <table class="tablenotebook text-center">
+    <thead>
+  <tr>
+   <th class="th-notebook-aout" colspan="5">Standardized results compared to pCMM-3 </th>
+  </tr>
+</thead>
+  <tbody>
+  <tr>
+   <td class="td-notebook"></td>
+   <td class="td-notebook" colspan="2">pCMM2-2</td>
+<td class="td-notebook" colspan="2">pCMM2-3</td>
+  </tr>
+  <tr >
+   <td class="td-notebook"></td>
+   <td class="td-notebook">NO<sub>3</sub></td>
+         <td class="td-notebook">NH<sub>4</sub></td>
+      <td class="td-notebook">NO<sub>3</sub></td>
+         <td class="td-notebook">NH<sub>4</sub></td>
+  </tr>
+        <tr >
+   <td class="td-notebook">250000</td>
+   <td class="td-notebook">5637</td>
+            <td class="td-notebook">787</td>
+             <td class="td-notebook">2192</td>
+             <td class="td-notebook">1019</td>
+  </tr>
+          <tr >
+   <td class="td-notebook">125000</td>
+   <td class="td-notebook">2332</td>
+            <td class="td-notebook">309</td>
+            <td class="td-notebook">1088</td>
+            <td class="td-notebook">842</td>
+  </tr>
+      <tr >
+   <td class="td-notebook">62500</td>
+   <td class="td-notebook">1516</td>
+            <td class="td-notebook">60</td>
+        <td class="td-notebook">599</td>
+            <td class="td-notebook">391</td>
+  </tr>
+      <tr >
+   <td class="td-notebook">31250</td>
+   <td class="td-notebook">1021</td>
+            <td class="td-notebook">57</td>
+        <td class="td-notebook">337</td>
+            <td class="td-notebook">396</td>
+  </tr>
+      <tr >
+   <td class="td-notebook">15625</td>
+   <td class="td-notebook">544</td>
+            <td class="td-notebook">27</td>
+          <td class="td-notebook">364</td>
+            <td class="td-notebook">102</td>
+  </tr>
+  </tbody>
+</table>
+</div>
+        </div>
+<br>
+<div class="d-flex justify-content-center">
+<div class="table-responsive-sm">
+    <table class="tablenotebook text-center">
+    <thead>
+  <tr>
+   <th class="th-notebook-aout" colspan="5">Standardized results compared to WT  </th>
+  </tr>
+</thead>
+  <tbody>
+  <tr>
+   <td class="td-notebook"></td>
+   <td class="td-notebook" colspan="2">pCMM2-2</td>
+<td class="td-notebook" colspan="2">pCMM2-3</td>
+  </tr>
+  <tr >
+   <td class="td-notebook"></td>
+   <td class="td-notebook">NO<sub>3</sub></td>
+         <td class="td-notebook">NH<sub>4</sub></td>
+      <td class="td-notebook">NO<sub>3</sub></td>
+         <td class="td-notebook">NH<sub>4</sub></td>
+  </tr>
+        <tr >
+   <td class="td-notebook">250000</td>
+   <td class="td-notebook">5196.33333</td>
+            <td class="td-notebook">435.666667</td>
+             <td class="td-notebook">1751.33333</td>
+             <td class="td-notebook">667.666667</td>
+  </tr>
+          <tr >
+   <td class="td-notebook">125000</td>
+   <td class="td-notebook">1385.66667</td>
+            <td class="td-notebook">50.3333333</td>
+            <td class="td-notebook">468.666667</td>
+            <td class="td-notebook">427.333333</td>
+  </tr>
+      <tr >
+   <td class="td-notebook">62500</td>
+   <td class="td-notebook">1385.66667</td>
+            <td class="td-notebook">-23.666667</td>
+        <td class="td-notebook">468.666667</td>
+            <td class="td-notebook">307.333333</td>
+  </tr>
+      <tr >
+   <td class="td-notebook">31250</td>
+   <td class="td-notebook">954.666667</td>
+            <td class="td-notebook">7.33333333</td>
+        <td class="td-notebook">261.666667</td>
+            <td class="td-notebook">225.333333</td>
+  </tr>
+      <tr >
+   <td class="td-notebook">15625</td>
+   <td class="td-notebook">521.333333</td>
+            <td class="td-notebook">14</td>
+          <td class="td-notebook">341.333333</td>
+            <td class="td-notebook">89</td>
+  </tr>
+  </tbody>
+</table>
+</div>
+        </div>
+<br>
+<center><img src="https://2019.igem.org/wiki/images/6/6d/T--Sorbonne_U_Paris--graph5NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".graph5NB"></center>
+<br>
 </div>
 </div>
@@ Line 5,742: / Line 6,538: @@
       </div>
          </div>
    <!--------------------END MODAL OR POP UP -------------------------->
+<div class="modal tableau58NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+       <img style="width: 100%;" src="https://2019.igem.org/wiki/images/b/b2/T--Sorbonne_U_Paris--tableau58NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau59NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/6/6a/T--Sorbonne_U_Paris--tableau59NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal tableau60NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/6/68/T--Sorbonne_U_Paris--tableau60NB.png" >
+    </div>
+  </div>
+</div>
+<div class="modal graph5NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/6/6d/T--Sorbonne_U_Paris--graph5NB.png" >
+    </div>
+  </div>
+</div>
 <div id="aoutday31"> <p class="bloc-date-calendrier">31</p> </div>
@@ Line 5,763: / Line 6,595: @@
 <div id="septday5"> <p class="bloc-date-calendrier">5</p> </div>
-<div id="septday6"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#septday6Modal" style="cursor: pointer;" >6</p> </div>
+<div id="septday6"> <p class="bloc-date-calendrier active-septembre"  data-toggle="modal" data-target="#septday6Modal" style="cursor: pointer;" >6</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="septday6Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,784: / Line 6,616: @@
 <div class="modal-body" style="width: 100%; height: 500px;overflow: auto;">
-<p>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.</p>
- <p>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.</p>
+<h5>Preparation of <I>Chlamydomonas</I> cells for fatty acids analysis </h5>
-<p>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.</p>
-<p>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.</p>
+<p>- Count cells (previously diluted) and put either in TAP+N or TAP-N medium at a final concentration of 5x10<sup>6</sup> cells/mL and in a final volume of 2 mL :
+- For TAP-N cultures, take 10 million cells and centrifuge 3 min at 3000 g.
+- Wash the pellets twice using TAP-N medium.
+- Centrifuge again 3 min at 3000 g.
+- Resuspend the pellets in 2 mL of TAP-N.
+- For TAP+N cultures, take 10 million cells and centrifuge 3 min at 3000 g.
+- Resuspend the pellets in 2 mL of TAP+N.
+- Incubate under light and agitation.
+</p>
+<h4>Results</h4>
+<p>Only pCMM7-3 and pCMM15-3 NH<sub>4</sub> have grown.
+<br><br>
+- Weigh samples:
+</p>
+<br>
+<div class="d-flex justify-content-center">
+<div class="table-responsive">
+    <table class="tablenotebook text-center">
+    <thead>
+  <tr>
+   <th class="th-notebook-septembre">Sample</th>
+   <th class="td-notebook">pCMM15-3</th>
+ <th class="td-notebook">pCMM7-3</th>
+ <th class="td-notebook">WT</th>
+  </tr>
+</thead>
+  <tbody>
+  <tr>
+   <td class="th-notebook-septembre">Colonies</td>
+   <td class="td-notebook">3</td>
+      <td class="td-notebook">3</td>
+      <td class="td-notebook" >1</td>
+  </tr>
+  <tr >
+   <td class="th-notebook-septembre">Weight (mg)</td>
+   <td class="td-notebook">52</td>
+      <td class="td-notebook" >18</td>
+      <td class="td-notebook" >55</td>
+  </tr>
+  </tbody>
+</table>
+</div>
+</div>
 </div>
 </div>
@@ Line 5,801: / Line 6,679: @@
 <div id="septday10"> <p class="bloc-date-calendrier">10</p> </div>
-<div id="septday11"> <p class="bloc-date-calendrier"  data-toggle="modal" data-target="#septday11Modal" style="cursor: pointer;" >11</p> </div>
+<div id="septday11"> <p class="bloc-date-calendrier active-septembre"  data-toggle="modal" data-target="#septday11Modal" style="cursor: pointer;" >11</p> </div>
   <!-------------- START MODAL OR POP UP ------------------------------>
          <div id="septday11Modal" class="modal fade bs-example-modal-lg" tabindex="-1" role="dialog" aria-labelledby="myModalLabel" aria-hidden="true" >
@@ Line 5,821: / Line 6,699: @@
 <div class="modal-body" style="width: 100%; height: 500px;overflow: auto;">
-<p>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.</p>
- <p>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.</p>
+<h5>Preparation of the samples pCMM15-3 NH4, pCMM7-3 and WT for fatty acids analysis</h5>
-<p>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.</p>
-<p>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.</p>
+<p>- Add to the pellets 2,5 mL of methanol, chloroform and water in this order.<br>
+- Centrifuge 15 min at 5.000 rpm and 4°C.<br>
+- Transfer the chloroform phase to another tube and complete it up with 3 mL of chloroform.<br>
+- Keep in ice.
+</p>
+<h4>Palm oil solution</h4>
+<p>- With a spatula, place the equivalent of one drop of palm oil at the bottom of a tube without touching the walls.<br>
+- Add 10 mL hexane.<br>
+- Keep the tube in ice.
+</p>
+<br>
+<h5>Separation of lipid reserves of pCMM15-3 NH4, pCMM7-3 and WT </h5>
+<p>- Prepare a solution of petroleum ether/ ethyl ether/ acetic acid (70/30/0,4, V/V/V), put it in the chromatograph tank and seal it.<br>
+- Prepare a silica gel plate: trace a line at 2 cm of the bottom edge of the plate for the deposit of the sample and a line at 1 cm of the top edge for the end of migration.<br>
+- Put 1 mL lipid solution in 5 mL SVL tube.<br>
+- Evaporate until dry under a nitrogen gas stream.<br>
+- Resuspend in 100 µL chloroform.<br>
+- With a Hamilton syringe and a hairdryer, put 30 µL of the samples on the plate, drop by drop.<br>
+- Place the silica gel plate inside the chromatograph tank and seal it.<br>
+- While the solvent reach the top line, take out the plate and let it dry.<br>
+- Spray primuline (1 g/L in acetone 80%) all over the plate.<br>
+- Reveal under UV light (340 nm).
+</p>
+<h4>Results</h4>
+<center><img src="https://2019.igem.org/wiki/images/6/64/T--Sorbonne_U_Paris--gel11NB.png" style="width:100%; cursor:zoom-in" data-toggle="modal" data-target=".graph11NB"></center>
 </div>
 </div>
@@ Line 5,832: / Line 6,741: @@
          </div>
    <!--------------------END MODAL OR POP UP -------------------------->
+<div class="modal graph11NB" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
+  <div class="modal-dialog modal-lg">
+    <div class="modal-content">
+ <div class="container" ><button type="button" class="close" data-dismiss="modal" aria-label="Close" ><span aria-hidden="true">&times;</span> </button></div>
+      <img style="width: 100%;" src="https://2019.igem.org/wiki/images/6/64/T--Sorbonne_U_Paris--gel11NB.png" >
+    </div>
+  </div>
+</div>
 <div id="septday12"> <p class="bloc-date-calendrier">12</p> </div>

Standardized results compared to pCMM-3
	pCMM2-2		pCMM2-3
	NO₃	NH₄	NO₃	NH₄
250000	5637	787	2192	1019
125000	2332	309	1088	842
62500	1516	60	599	391
31250	1021	57	337	396
15625	544	27	364	102