iGEM 2019 — Team Sorbonne_U_Paris

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# Team:Sorbonne U Paris/Notebook

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![](https://static.igem.org/mediawiki/2019/9/95/T--Sorbonne_U_Paris--titrenotebook.png)

1

2

×

TUESDAY 2nd OF JULY 2019

Octave Joliot
Pauline Morin

### Resuspension of HiBiT-B2 and HiBiT-B5 DNA

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

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

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

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

### Digestion/Ligation reaction to assemble HiBiT-B2 and HiBiT-B5 into level 0 plasmid

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

- A master mix is prepared for 5 reactions :

| 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 :

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

![](https://static.igem.org/mediawiki/2019/8/8c/T--Sorbonne_U_Paris--tableau1NB.png)

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

![](https://static.igem.org/mediawiki/2019/4/4c/T--Sorbonne_U_Paris--tableau2NB.png)

- 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 |

×

![](https://static.igem.org/mediawiki/2019/8/8c/T--Sorbonne_U_Paris--tableau1NB.png)

×

![](https://static.igem.org/mediawiki/2019/4/4c/T--Sorbonne_U_Paris--tableau2NB.png)

3

×

WEDNESDAY 3rd OF JULY 2019

Octave Joliot

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

-The following tubes are prepared :

1. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with HiBiT-B2 and pAGM1276
2. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with HiBiT-B5 and pAGM1301
3. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pAGM1276 alone (negative control)
4. 25 µL of JM109 bacteria + 4 µL of reaction mix from digestion/ligation reaction with pAGM1301 alone (negative control)
5. 25 µL of JM109 bacteria alone

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µLof LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000 g, discard surnageant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/ Spectinomycin (50 µg/mL) Petri dish and incubate at 37°C overnight.

4

×

THURSDAY 4th OF JULY 2019

Octave Joliot

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

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

### Transplanting colonies into liquid medium

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

5

×

FRIDAY 5th OF JULY 2019

Octave Joliot

### Plasmid extraction

- Extract plasmid DNA p0-191 and p0-192 using Ozyme [ZymoPURE™ Plasmid Miniprep Kit](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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 |

6

7

8

9

×

TUESDAY 9th OF JULY 2019

Pauline Morin
Manon Moussay

### 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.

![](https://static.igem.org/mediawiki/2019/9/92/T--Sorbonne_U_Paris--gel1NB.png)

### 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 |

×

![](https://static.igem.org/mediawiki/2019/9/92/T--Sorbonne_U_Paris--gel1NB.png)

10

×

WEDNESDAY 10th OF JULY 2019

Octave Joliot

### 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 :

1. pCM1-1 (HiBiT-GFP)

![](https://static.igem.org/mediawiki/2019/4/40/T--Sorbonne_U_Paris--tableau3NB.png)

2. pCM1-2 (GFP-HiBiT)

![](https://static.igem.org/mediawiki/2019/b/b0/T--Sorbonne_U_Paris--tableau4NB.png)

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

![](https://static.igem.org/mediawiki/2019/2/2f/T--Sorbonne_U_Paris--tableau5NB.png)

- 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 |

×

![](https://static.igem.org/mediawiki/2019/4/40/T--Sorbonne_U_Paris--tableau3NB.png)

×

![](https://static.igem.org/mediawiki/2019/b/b0/T--Sorbonne_U_Paris--tableau4NB.png)

×

![](https://static.igem.org/mediawiki/2019/2/2f/T--Sorbonne_U_Paris--tableau5NB.png)

11

×

THURSDAY 11th OF JULY 2019

Octave Joliot
Pauline Morin
Manon Moussay

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

- Transformation samples :

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

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000 g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Ampicillin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

### Resuspension of DGAT and PLS1 DNA

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

1. DGAT
2. PLS1 (LPAAT)

### Transformation of competent cells with DGAT and PLS1 to make stock

- Transformation samples:

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

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Kanamycin (50 µg/mL) Petri dish and incubate at 37°C overnight.

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

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

- A master mix is prepared for 5 reactions :

| 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) :

1. pCM1-7 (HiBiT-DGAT)

![](https://static.igem.org/mediawiki/2019/2/23/T--Sorbonne_U_Paris--tableau6NB.png)

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

![](https://static.igem.org/mediawiki/2019/6/68/T--Sorbonne_U_Paris--tableau7NB.png)

4. pCM1-9 (HiBiT-PLS1)

![](https://static.igem.org/mediawiki/2019/2/20/T--Sorbonne_U_Paris--tableau8NB.png)

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

![](https://static.igem.org/mediawiki/2019/f/f8/T--Sorbonne_U_Paris--tableau9NB.png)

- 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 |

×

![](https://static.igem.org/mediawiki/2019/2/23/T--Sorbonne_U_Paris--tableau6NB.png)

×

![](https://static.igem.org/mediawiki/2019/6/68/T--Sorbonne_U_Paris--tableau7NB.png)

×

![](https://static.igem.org/mediawiki/2019/2/20/T--Sorbonne_U_Paris--tableau8NB.png)

×

![](https://static.igem.org/mediawiki/2019/f/f8/T--Sorbonne_U_Paris--tableau9NB.png)

12

×

FRIDAY 12th OF JULY 2019

Octave Joliot

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

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

### Transplanting colonies into liquid medium

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

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

13

14

15

×

MONDAY 15th OF JULY 2019

Pauline Morin
Manon Moussay

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

- Transformation samples :

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

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

### Plasmid extraction

- Extract plasmid DNA DGAT and PLS1 using Ozyme [ZymoPURE™ Plasmid Miniprep Kit.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf) In default of Ozyme kit buffer, Macherey Nagel kit buffer was used.

- Extract plasmid DNA pCM1-1, pCM1-2, pCM1-3 and pL1-2F using Ozyme [ZymoPURE™ Plasmid Miniprep Kit.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf) In default of Ozyme kit buffer, Macherey Nagel kit buffer was used.

16

×

TUESDAY16th OF JULY 2019

Octave Joliot

### 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

![](https://static.igem.org/mediawiki/2019/8/87/T--Sorbonne_U_Paris--gel2NB.png)

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

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

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

### Transplanting the pCM1-8 colony into liquid medium

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

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

- Transformation samples (see 07/11 samples) :

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

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Kanamycin (50 µg/mL) Petri dish and incubate at 37°C overnight.

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

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

- A master mix is prepared for 4 reactions :

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

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

1. pCM1-4 (Nanoluc - GFP)

![](https://static.igem.org/mediawiki/2019/6/61/T--Sorbonne_U_Paris--tableau10NB.png)

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

![](https://static.igem.org/mediawiki/2019/b/b3/T--Sorbonne_U_Paris--tableau11NB.png)

- 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 |

×

![](https://static.igem.org/mediawiki/2019/8/87/T--Sorbonne_U_Paris--gel2NB.png)

×

![](https://static.igem.org/mediawiki/2019/6/61/T--Sorbonne_U_Paris--tableau10NB.png)

×

![](https://static.igem.org/mediawiki/2019/b/b3/T--Sorbonne_U_Paris--tableau11NB.png)

17

×

WEDNESDAY 17th OF JULY 2019

Octave Joliot

### 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.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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) :

1. p0-DGAT (B3-B4)

![](https://static.igem.org/mediawiki/2019/a/a2/T--Sorbonne_U_Paris--tableau12NB.png)

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

![](https://static.igem.org/mediawiki/2019/6/68/T--Sorbonne_U_Paris--tableau13NB.png)

- 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) :

1. pCMM-1 (HiBiT-GFP)

![](https://static.igem.org/mediawiki/2019/2/25/T--Sorbonne_U_Paris--tableau14NB.png)

2. pCMM-2 (GFP-HiBiT)

![](https://static.igem.org/mediawiki/2019/7/7c/T--Sorbonne_U_Paris--tableau15NB.png)

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

![](https://static.igem.org/mediawiki/2019/5/54/T--Sorbonne_U_Paris--tableau16NB.png)

- 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 :

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

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria with pCMM1, 2 and 3 on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.
- Spread bacteria with pCM1-4 and 5 on a LB/Ampicillin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

×

![](https://static.igem.org/mediawiki/2019/a/a2/T--Sorbonne_U_Paris--tableau12NB.png)

×

![](https://static.igem.org/mediawiki/2019/6/68/T--Sorbonne_U_Paris--tableau13NB.png)

×

![](https://static.igem.org/mediawiki/2019/2/25/T--Sorbonne_U_Paris--tableau14NB.png)

×

![](https://static.igem.org/mediawiki/2019/7/7c/T--Sorbonne_U_Paris--tableau15NB.png)

×

![](https://static.igem.org/mediawiki/2019/5/54/T--Sorbonne_U_Paris--tableau16NB.png)

18

×

THURSDAY 18th OF JULY 2019

Octave Joliot

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

- Transformation samples :

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

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

### Plasmid extraction

- Extract plasmid DNA pCM1-7, pCM1-8 and pL1-3F using Ozyme [ZymoPURE™ Plasmid Miniprep Kit.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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

![](https://static.igem.org/mediawiki/2019/1/16/T--Sorbonne_U_Paris--gel3NB.png)

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.

×

![](https://static.igem.org/mediawiki/2019/1/16/T--Sorbonne_U_Paris--gel3NB.png)

19

×

FRIDAY 19th OF JULY 2019

Octave Joliot
Pauline Morin
Manon Moussay

### Plasmid extraction

- Extract plasmid DNA pCM1-4, pCM1-5, pCMM-1, pCMM-2 and pCMM-3 using Ozyme [ZymoPURE™ Plasmid Miniprep Kit.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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

×

MONDAY 22nd OF JULY 2019

Pauline Morin
Manon Moussay

### 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

![](https://static.igem.org/mediawiki/2019/4/48/T--Sorbonne_U_Paris--gel4NB.png)

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.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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

![](https://static.igem.org/mediawiki/2019/a/ab/T--Sorbonne_U_Paris--gel5NB.png)

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.

×

![](https://static.igem.org/mediawiki/2019/4/48/T--Sorbonne_U_Paris--gel4NB.png)

×

![](https://static.igem.org/mediawiki/2019/a/ab/T--Sorbonne_U_Paris--gel5NB.png)

23

×

TUESDAY 23rd OF JULY 2019

Octave Joliot

### 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) :

1. pCMM-4 (NanoLuc-GFP)

![](https://static.igem.org/mediawiki/2019/4/49/T--Sorbonne_U_Paris--tableau17NB.png)

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

![](https://static.igem.org/mediawiki/2019/d/df/T--Sorbonne_U_Paris--tableau18NB.png)

4. pCM1-9 (HiBiT-PLS1)

![](https://static.igem.org/mediawiki/2019/8/86/T--Sorbonne_U_Paris--tableau19NB.png)

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

![](https://static.igem.org/mediawiki/2019/9/9b/T--Sorbonne_U_Paris--tableau20NB.png)

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

![](https://static.igem.org/mediawiki/2019/7/7c/T--Sorbonne_U_Paris--tableau21NB.png)

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

![](https://static.igem.org/mediawiki/2019/a/a7/T--Sorbonne_U_Paris--tableau22NB.png)

- 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 :

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

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria with pCMM on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.
- Spread bacteria with pCM1 on a LB/Ampicillin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

×

![](https://static.igem.org/mediawiki/2019/4/49/T--Sorbonne_U_Paris--tableau17NB.png)

×

![](https://static.igem.org/mediawiki/2019/d/df/T--Sorbonne_U_Paris--tableau18NB.png)

×

![](https://static.igem.org/mediawiki/2019/8/86/T--Sorbonne_U_Paris--tableau19NB.png)

×

![](https://static.igem.org/mediawiki/2019/9/9b/T--Sorbonne_U_Paris--tableau20NB.png)

×

![](https://static.igem.org/mediawiki/2019/7/7c/T--Sorbonne_U_Paris--tableau21NB.png)

×

![](https://static.igem.org/mediawiki/2019/a/a7/T--Sorbonne_U_Paris--tableau22NB.png)

24

×

WEDNESDAY 24th OF JULY 2019

Octave Joliot

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

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

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

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

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

- Transformation samples :

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

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

### Plasmid extraction

- Extract plasmid DNA pICH50900 (end linker) using Ozyme [ZymoPURE™ Plasmid Miniprep Kit.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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

×

THURSDAY 25th OF JULY 2019

Octave Joliot

### 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.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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

![](https://static.igem.org/mediawiki/2019/2/2e/T--Sorbonne_U_Paris--gel6NB.png )

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

![](https://static.igem.org/mediawiki/2019/0/08/T--Sorbonne_U_Paris--gel7NB.png)

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) :

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

![](https://static.igem.org/mediawiki/2019/5/5e/T--Sorbonne_U_Paris--tableau23NB.png)

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

![](https://static.igem.org/mediawiki/2019/d/d5/T--Sorbonne_U_Paris--tableau24NB.png)

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

![](https://static.igem.org/mediawiki/2019/8/86/T--Sorbonne_U_Paris--tableau25NB.png)

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

![](https://static.igem.org/mediawiki/2019/a/ac/T--Sorbonne_U_Paris--tableau26NB.png)

- 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 :

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

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
- Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

×

![](https://static.igem.org/mediawiki/2019/2/2e/T--Sorbonne_U_Paris--gel6NB.png)

×

![](https://static.igem.org/mediawiki/2019/0/08/T--Sorbonne_U_Paris--gel7NB.png)

×

![](https://static.igem.org/mediawiki/2019/5/5e/T--Sorbonne_U_Paris--tableau23NB.png)

×

![](https://static.igem.org/mediawiki/2019/d/d5/T--Sorbonne_U_Paris--tableau24NB.png)

×

![](https://static.igem.org/mediawiki/2019/8/86/T--Sorbonne_U_Paris--tableau25NB.png)

×

![](https://static.igem.org/mediawiki/2019/a/ac/T--Sorbonne_U_Paris--tableau26NB.png)

26

×

FRIDAY 26th OF JULY 2019

Octave Joliot

### Plasmid extraction

- Extract plasmid DNA pCMM-6 and pCMM-7 using Ozyme [ZymoPURE™ Plasmid Miniprep Kit.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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

![](https://static.igem.org/mediawiki/2019/e/e6/T--Sorbonne_U_Paris--gel8NB.png)

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.

×

![](https://static.igem.org/mediawiki/2019/e/e6/T--Sorbonne_U_Paris--gel8NB.png)

27

28

29

×

MONDAY 29th OF JULY 2019

Octave Joliot

### Plasmid extraction

- Extract plasmid DNA pCMM-8, pCMM-9, pCMM-14 and pCMM-15 using Ozyme [ZymoPURE™ Plasmid Miniprep Kit.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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

![](https://static.igem.org/mediawiki/2019/0/04/T--Sorbonne_U_Paris--gel9NB.png )

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.

×

![](https://static.igem.org/mediawiki/2019/0/04/T--Sorbonne_U_Paris--gel9NB.png)

30

×

TUESDAY 30th OF JULY 2019

Octave Joliot

### Plasmid extraction

- Extract plasmid DNA pCMM-15 using Ozyme [ZymoPURE™ Plasmid Miniprep Kit.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

### 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

×

WEDNESDAY 31th OF JULY 2019

Octave Joliot

### Transformation of competent cells with pCMM-15

- Transformation samples :

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

- Add DNA to bacteria and mix gently by pipetting up and down.
- Incubate 10 min on ice, then 55 sec at 42°C, then back on ice for 2 min.
- Add 700 µL of LB and incubate 1h at 37°C.
-Centrifuge 2 min at 4,000g, discard supernatant (keep only 100 µL), resuspend the bacteria.
- Spread bacteria on a LB/Spectinomycin (50 µg/mL)/X-gal (50 µg/mL) Petri dish and incubate at 37°C overnight.

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

-Purification of plasmids pCMM-1 to pCMM-9 and pCMM-14 using [NucleoSpinⓇ Gel and PCR Clean-up kit](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf) 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 |

![](https://static.igem.org/mediawiki/2019/d/dd/T--Sorbonne_U_Paris--calendarjuly.png)

1

×

THURSDAY 1st OF AUGUST 2019

Laurent Chen

##### 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

×

FRIDAY 2nd OF AUGUST 2019

Laurent Chen

##### 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.](https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf)

##### 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

![](https://static.igem.org/mediawiki/2019/3/34/T--Sorbonne_U_Paris--gel10NB.png)

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.

×

![](https://static.igem.org/mediawiki/2019/3/34/T--Sorbonne_U_Paris--gel10NB.png)

3

4

5

×

MONDAY 5th OF AUGUST 2019

Marta De Almeida

##### 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

×

TUESDAY 6th OF AUGUST 2019

Marta De Almeida

##### 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.](https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf)

##### 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.

7

×

WEDNESDAY 7th OF AUGUST 2019

Marta De Almeida

##### Transplantation of *Chlamydomonas* colonies onto solid medium

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

8

9

10

11

12

13

×

TUESDAY 13th OF AUGUST 2019

Selma Bennacer
Marta De Almeida

##### Preparation of TAP-N medium

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

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

##### Screening test on pCMM-1 to pCMM-5

- The following samples are tested :

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

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

![](https://static.igem.org/mediawiki/2019/d/dc/T--Sorbonne_U_Paris--tableau27NB.png)

![](https://static.igem.org/mediawiki/2019/3/30/T--Sorbonne_U_Paris--tableau28NB.png)

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

![](https://static.igem.org/mediawiki/2019/b/b6/T--Sorbonne_U_Paris--tableau29NB.png)

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

![](https://static.igem.org/mediawiki/2019/6/60/T--Sorbonne_U_Paris--tableau30NB.png)

![](https://static.igem.org/mediawiki/2019/0/06/T--Sorbonne_U_Paris--tableau31NB.png)

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

×

![](https://static.igem.org/mediawiki/2019/d/dc/T--Sorbonne_U_Paris--tableau27NB.png )

×

![](https://static.igem.org/mediawiki/2019/3/30/T--Sorbonne_U_Paris--tableau28NB.png )

×

![](https://static.igem.org/mediawiki/2019/b/b6/T--Sorbonne_U_Paris--tableau29NB.png)

×

![](https://static.igem.org/mediawiki/2019/6/60/T--Sorbonne_U_Paris--tableau30NB.png )

×

![](https://static.igem.org/mediawiki/2019/0/06/T--Sorbonne_U_Paris--tableau31NB.png)

14

×

WEDNESDAY 14th OF AUGUST 2019

Selma Bennacer
Marta De Almeida

##### Transplantation of positive colonies into liquid medium

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

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

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

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

- The following samples are tested :

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

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

![](https://static.igem.org/mediawiki/2019/f/f1/T--Sorbonne_U_Paris--tableau32NB.png)

![](https://static.igem.org/mediawiki/2019/2/2d/T--Sorbonne_U_Paris--tableau33NB.png)

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

![](https://static.igem.org/mediawiki/2019/3/31/T--Sorbonne_U_Paris--tableau34NB.png)

![](https://static.igem.org/mediawiki/2019/f/fb/T--Sorbonne_U_Paris--tableau35NB.png)

- 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

![](https://static.igem.org/mediawiki/2019/b/bd/T--Sorbonne_U_Paris--tableau36NB.png)

![](https://static.igem.org/mediawiki/2019/6/6e/T--Sorbonne_U_Paris--tableau37NB.png)

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

×

![](https://static.igem.org/mediawiki/2019/f/f1/T--Sorbonne_U_Paris--tableau32NB.png)

×

![](https://static.igem.org/mediawiki/2019/2/2d/T--Sorbonne_U_Paris--tableau33NB.png)

×

![](https://static.igem.org/mediawiki/2019/3/31/T--Sorbonne_U_Paris--tableau34NB.png)

×

![](https://static.igem.org/mediawiki/2019/f/fb/T--Sorbonne_U_Paris--tableau35NB.png)

×

![](https://static.igem.org/mediawiki/2019/b/bd/T--Sorbonne_U_Paris--tableau36NB.png)

×

![](https://static.igem.org/mediawiki/2019/6/6e/T--Sorbonne_U_Paris--tableau37NB.png)

15

16

17

18

19

×

MONDAY 19th OF AUGUST 2019

Selma Bennacer
Marta De Almeida

##### Transplantation of positive colonies into liquid medium

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

![](https://static.igem.org/mediawiki/2019/a/ab/T--Sorbonne_U_Paris--liste1NB.png)

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

×

![](https://static.igem.org/mediawiki/2019/a/ab/T--Sorbonne_U_Paris--liste1NB.png)

20

×

TUESDAY 20th OF AUGUST 2019

Selma Bennacer
Marta De Almeida

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

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

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

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

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

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

![](https://static.igem.org/mediawiki/2019/0/03/T--Sorbonne_U_Paris--tableau38NB.png)

![](https://static.igem.org/mediawiki/2019/5/5e/T--Sorbonne_U_Paris--tableau39NB.png)

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

#### Results

![](https://static.igem.org/mediawiki/2019/c/c2/T--Sorbonne_U_Paris--tableau40NB.png)

![](https://static.igem.org/mediawiki/2019/5/58/T--Sorbonne_U_Paris--tableau41NB.png)

![](https://static.igem.org/mediawiki/2019/3/3c/T--Sorbonne_U_Paris--tableau42NB.png)

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

×

![](https://static.igem.org/mediawiki/2019/0/03/T--Sorbonne_U_Paris--tableau38NB.png)

×

![](https://static.igem.org/mediawiki/2019/5/5e/T--Sorbonne_U_Paris--tableau39NB.png)

×

![](https://static.igem.org/mediawiki/2019/c/c2/T--Sorbonne_U_Paris--tableau40NB.png)

×

![](https://static.igem.org/mediawiki/2019/5/58/T--Sorbonne_U_Paris--tableau41NB.png)

×

![](https://static.igem.org/mediawiki/2019/3/3c/T--Sorbonne_U_Paris--tableau42NB.png)

21

×

WEDNESDAY 21st OF AUGUST 2019

Selma Bennacer
Marta De Almeida

##### Screening test on the rest of the clones pCMM1 to pCMM5

- The following samples are tested :

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

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

22

×

THURSDAY 22nd OF AUGUST 2019

Selma Bennacer
Marta De Almeida

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

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

#### TAP-N+NH4

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

*Detailed calculation:

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

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

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

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

#### TAP-N+NO3

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

*Detailed calculation :

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

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

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

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

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

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

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

- The following samples are used :

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

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

23

×

FRIDAY 23RD OF AUGUST 2019

Selma Bennacer
Marta De Almeida

##### Screening test on 1st batch samples

- The following samples are tested :

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

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

![](https://static.igem.org/mediawiki/2019/6/61/T--Sorbonne_U_Paris--tableau43NB.png)

![](https://static.igem.org/mediawiki/2019/e/e4/T--Sorbonne_U_Paris--tableau44NB.png)

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

#### Results

![](https://static.igem.org/mediawiki/2019/4/4f/T--Sorbonne_U_Paris--tableau45NB.png)

![](https://static.igem.org/mediawiki/2019/8/8d/T--Sorbonne_U_Paris--tableau46NB.png)

![](https://static.igem.org/mediawiki/2019/6/61/T--Sorbonne_U_Paris--tableau43NB.png)

![](https://static.igem.org/mediawiki/2019/1/13/T--Sorbonne_U_Paris--graph1NB.png)

##### Transplantation of positive colonies into liquid medium

- Resuspension of validated colonies :

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

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

×

![](https://static.igem.org/mediawiki/2019/6/61/T--Sorbonne_U_Paris--tableau43NB.png)

×

![](https://static.igem.org/mediawiki/2019/e/e4/T--Sorbonne_U_Paris--tableau44NB.png)

×

![](https://static.igem.org/mediawiki/2019/4/4f/T--Sorbonne_U_Paris--tableau45NB.png)

×

![](https://static.igem.org/mediawiki/2019/8/8d/T--Sorbonne_U_Paris--tableau46NB.png)

×

![](https://static.igem.org/mediawiki/2019/9/98/T--Sorbonne_U_Paris--tableau47NB.png)

×

![](https://static.igem.org/mediawiki/2019/1/13/T--Sorbonne_U_Paris--graph1NB.png)

24

25

26

27

×

TUESDAY 27th OF AUGUST 2019

Selma Bennacer
Marta De Almeida

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

- The following samples are used :

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

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

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

- Transfer 100 µL of culture on 96 well plate :

![](https://static.igem.org/mediawiki/2019/c/c9/T--Sorbonne_U_Paris--tableau48NB.png)

![](https://static.igem.org/mediawiki/2019/6/63/T--Sorbonne_U_Paris--tableau49NB.png)

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

#### Results

![](https://static.igem.org/mediawiki/2019/6/60/T--Sorbonne_U_Paris--tableau50NB.png)

![](https://static.igem.org/mediawiki/2019/1/12/T--Sorbonne_U_Paris--tableau51NB.png)

##### Screening test on 1st batch samples

- The following samples are tested :

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

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

![](https://static.igem.org/mediawiki/2019/c/cb/T--Sorbonne_U_Paris--tableau52NB.png)

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

#### Results

![](https://static.igem.org/mediawiki/2019/7/7b/T--Sorbonne_U_Paris--tableau53NB.png)

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

- The following samples are used :

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

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

![](https://static.igem.org/mediawiki/2019/6/68/T--Sorbonne_U_Paris--graph2NB.png)

×

![](https://static.igem.org/mediawiki/2019/c/c9/T--Sorbonne_U_Paris--tableau48NB.png)

×

![](https://static.igem.org/mediawiki/2019/6/63/T--Sorbonne_U_Paris--tableau49NB.png)

×

![](https://static.igem.org/mediawiki/2019/6/60/T--Sorbonne_U_Paris--tableau50NB.png)

×

![](https://static.igem.org/mediawiki/2019/1/12/T--Sorbonne_U_Paris--tableau51NB.png)

×

![](https://static.igem.org/mediawiki/2019/c/cb/T--Sorbonne_U_Paris--tableau52NB.png)

×

![](https://static.igem.org/mediawiki/2019/7/7b/T--Sorbonne_U_Paris--tableau53NB.png)

×

![](https://static.igem.org/mediawiki/2019/6/68/T--Sorbonne_U_Paris--graph2NB.png)

28

×

WEDNESDAY 28th OF AUGUST 2019

Selma Bennacer
Marta De Almeida

##### Screening test on 2nd batch samples

- The following samples are tested :

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

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

![](https://static.igem.org/mediawiki/2019/d/d4/T--Sorbonne_U_Paris--tableau54NB.png)

![](https://static.igem.org/mediawiki/2019/1/1d/T--Sorbonne_U_Paris--tableau55NB.png)

- 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.

#### Results

![](https://static.igem.org/mediawiki/2019/2/20/T--Sorbonne_U_Paris--tableau56NB.png)

![](https://static.igem.org/mediawiki/2019/b/be/T--Sorbonne_U_Paris--tableau57NB.png)

![](https://static.igem.org/mediawiki/2019/9/98/T--Sorbonne_U_Paris--graph3.png)

![](https://static.igem.org/mediawiki/2019/0/07/T--Sorbonne_U_Paris--graph4.png)

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![](https://static.igem.org/mediawiki/2019/d/d4/T--Sorbonne_U_Paris--tableau54NB.png)

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![](https://static.igem.org/mediawiki/2019/1/1d/T--Sorbonne_U_Paris--tableau55NB.png)

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![](https://static.igem.org/mediawiki/2019/b/be/T--Sorbonne_U_Paris--tableau57NB.png)

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![](https://static.igem.org/mediawiki/2019/9/98/T--Sorbonne_U_Paris--graph3.png)

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![](https://static.igem.org/mediawiki/2019/0/07/T--Sorbonne_U_Paris--graph4.png)

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FRIDAY 30th OF AUGUST 2019

Selma Bennacer
Marta De Almeida

##### 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 :

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

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

![](https://static.igem.org/mediawiki/2019/b/b2/T--Sorbonne_U_Paris--tableau58NB.png)

- 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

![](https://static.igem.org/mediawiki/2019/6/6a/T--Sorbonne_U_Paris--tableau59NB.png)

![](https://static.igem.org/mediawiki/2019/6/68/T--Sorbonne_U_Paris--tableau60NB.png)

| 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 |

![](https://static.igem.org/mediawiki/2019/6/6d/T--Sorbonne_U_Paris--graph5NB.png)

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![](https://static.igem.org/mediawiki/2019/7/70/T--Sorbonne_U_Paris--calendaraout.png)

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FRIDAY 6th OF SEPTEMBER 2019

Selma Bennacer

##### 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 |

7

8

9

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WEDNESDAY 11th OF SEPTEMBER 2019

Laurent Chen
Pauline Morin
Manon Moussay

##### Preparation of the samples pCMM15-3 NH4, pCMM7-3 and WT for fatty acids analysis

- Add to the pellets 2,5 mL of methanol, chloroform and water in this order.
- Centrifuge 15 min at 5.000 rpm and 4°C.
- Transfer the chloroform phase to another tube and complete it up with 3 mL of chloroform.
- Keep in ice.

#### Palm oil solution

- With a spatula, place the equivalent of one drop of palm oil at the bottom of a tube without touching the walls.
- Add 10 mL hexane.
- Keep the tube in ice.

##### Separation of lipid reserves of pCMM15-3 NH4, pCMM7-3 and WT

- 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.
- 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.
- Put 1 mL lipid solution in 5 mL SVL tube.
- Evaporate until dry under a nitrogen gas stream.
- Resuspend in 100 µL chloroform.
- With a Hamilton syringe and a hairdryer, put 30 µL of the samples on the plate, drop by drop.
- Place the silica gel plate inside the chromatograph tank and seal it.
- While the solvent reach the top line, take out the plate and let it dry.
- Spray primuline (1 g/L in acetone 80%) all over the plate.
- Reveal under UV light (340 nm).

#### Results

![](https://static.igem.org/mediawiki/2019/6/64/T--Sorbonne_U_Paris--gel11NB.png)

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![](https://static.igem.org/mediawiki/2019/2/2f/T--Sorbonne_U_Paris--calendarseptember.png)


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# Team:Sorbonne U Paris/Experiments

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![](https://static.igem.org/mediawiki/2019/1/12/T--Sorbonne_U_Paris--dechirureseule1.png)

### MoClo Assembly

The Modular Cloning allows the assembly of parts easily and rapidly with the Golden Gate cloning strategy. The principle is to assemble basic gene parts from donor plasmids to complex multigenic DNA in an acceptor vector in two steps using type IIS restriction enzymes.
Basic parts are cloned into level 0 via BpiI. The level 0 vector is based on pUC19 and contains a spectinomycin resistance gene. It also contains BsaI and BpiI restriction sites and lacZ cassette which is removed during the cloning process, allowing blue/white selection after transformation in *E. Coli*. With BsaI restriction-ligation, level 0 modules are removed from level 0 vector to be assembled in a level 1 vector and form a transcription unit. This level 1 vector also contains a lacZ cassette and an ampicillin resistance gene. Parts are assembled in the correct order in one step thanks to the element-specific fusion sites located at the edges. As for level 1, transcriptional units can be removed from the vector with BpiI to be assembled in a level 2 or M vector and form multigene constructs. Level 2 vector contains a lacZ cassette and a kanamycin resistance gene. These multigene constructs are then used to transform *Chlamydomonas* cells and obtain your new strains.

![](https://static.igem.org/mediawiki/2019/2/2c/T--Sorbonne_U_Paris--expmoclo.png)

### Transformation of bacteria

Bacteria are transformed with DNA constructs. Bacteria undergo a thermal choc to enable DNA penetration and are incubated with the constructs.

![](https://static.igem.org/mediawiki/2019/d/da/T--Sorbonne_U_Paris--exptransfobact.png)

### Transplanting a colony into a liquid medium

Microorganisms are transferred from agar plate to liquid medium for optimal growth.

![](https://static.igem.org/mediawiki/2019/b/b5/T--Sorbonne_U_Paris--exprepiq.png)

### Plasmid extraction

The plasmids are extracted from the bacteria using a plasmid extraction kit such as the Ozyme ZymoPURE Plasmid Miniprep or the Promega PureYield Miniprep System. The plasmid DNA is extracted, based on the alkaline lysis technique of the bacterial cells. The DNA is purified by means of a silica membrane which selectively retains DNA and selectively ionic strength.

![](https://static.igem.org/mediawiki/2019/7/78/T--Sorbonne_U_Paris--expextractionplasmide.png)

### DNA concentration measurement

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

![](https://static.igem.org/mediawiki/2019/2/25/T--Sorbonne_U_Paris--expnanodrop.png)

### Digestion for quality control of generated DNA

Generated DNA are digested with the restriction enzymes to check its quality by electrophoresis.

![](https://static.igem.org/mediawiki/2019/f/f5/T--Sorbonne_U_Paris--expdigestion.png)

### DNA gel electrophoresis

To check the quality of the DNA after transformation, DNA is extracted, digested and the fragments are separated according to their size by electrophoresis on agarose gel. The size of the DNA fragment is evaluated in comparison with a size marker. The migration profile should be similar to a predicted gel that can be done using a cloning software such as SerialCloner.

![](https://static.igem.org/mediawiki/2019/1/11/T--Sorbonne_U_Paris--expgelelectropho.png)

### Transformation of *Chlamydomonas reinhardtii*

Our constructs are integrated in the genome of wild-type *C. reinhardtii* using the electroporation technology. For that, an electrical field is applied to wild-type *C. reinhardtii* in order to increase the permeability of its membrane, allowing the plasmid to be introduced into the microalga.

![](https://static.igem.org/mediawiki/2019/2/2b/T--Sorbonne_U_Paris--exptransfochlamy.png)

### Measurement of our constructs expression with the HiBiT protein tagging system

In order to measure the expression of our enzymes, we implemented the HiBiT technology developed by Promega into the *C. reinhardtii* MoClo kit. The HiBiT tag is a 11 amino acid tag that is essentially one part of the NanoBiT® enzyme. When the HiBiT-tagged protein is incubated with the LgBiT, which is the other part of the NanoBiT, and its substrate, a functional NanoBiT is assembled and emit a measurable and quantifiable luminescence signal.

![](https://static.igem.org/mediawiki/2019/4/4b/T--Sorbonne_U_Paris--exphibit.png)

### Lipid extraction

To determine the impact of our strategy on the fatty acid content of the strains, lipids must be extracted and analysed. The content in neutral lipids is analysed qualitatively by thin-layer chromatography with the technique of Mangold and quantitatively by gas chromatography.

![](https://static.igem.org/mediawiki/2019/f/fb/T--Sorbonne_U_Paris--explipidextr.png)

# *For more details, go on our Protocols document :*

[FULL PROTOCOLS](https://static.igem.org/mediawiki/2019/7/77/T--Sorbonne_U_Paris--protocols.pdf)

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# Team:Sorbonne U Paris/Results

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![](https://static.igem.org/mediawiki/2019/0/03/T--Sorbonne_U_Paris--plasmide7.png)

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![](https://static.igem.org/mediawiki/2019/f/f9/T--Sorbonne_U_Paris--plasmide8.png)

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![](https://static.igem.org/mediawiki/2019/5/5e/T--Sorbonne_U_Paris--plasmide15.png)

![](https://static.igem.org/mediawiki/2019/3/37/T--Sorbonne_U_Paris--titreresults.png)

### Building plasmids

All plasmids were constructed using the Modular Cloning (MoClo) method 1 adapted to *Chlamydomonas Reinhardtii* 2. This method, based on Golden-Gate assembly 3, allows first the assembly of parts such as promoters, coding sequences, signal peptides, etc. into a transcriptional unit in only one reaction and then, the assembly of full transcriptional units into multi-gene constructs again in a single reaction. Using this method, we were able to construct without difficulty all our plasmids.

## HiBiT characterization

We aimed to produce plasmids that would be used to transform *C. Reinhardtii* in order to characterize the HiBiT tag in this organism, as well as adding a new part to the MoClo kit for this alga.

To construct plasmids expressing the HiBiT tag, we ordered two oligonucleotides for the two positions we wanted to use our tag, in N-terminal or C-terminal. We let them hybridize, and then start the reaction to build level 0 plasmids. To be sure that the initial oligonucleotide sequence was correct, we’ve sequenced all the plasmids obtained. Thus, we obtained level 0 plasmids with the HiBiT tag in N-terminal or C-terminal position.
Using parts of the MoClo kit provided by Pierre Crozet, we successfully constructed the level 1 plasmids build to express either GFP tagged with the HiBiT sequence in N-terminal position (pCM1-1) or C-terminal position (pCM1-2), GFP tagged with NanoLuc in N-terminal position (pCM1-4) or C-terminal position (pCM1-5) or GFP without any tag (pCM1-3). To confirm the presence of the transcriptional units in the plasmid, we digested the plasmids with Bbs-I, and performed an electrophorese on agarose gel. Doing so we were able to identify clones containing the transcriptional units.

We did not sequence our plasmids to ensure that they all contain the full transcriptional unit with right sequence because first, the Golden Gate assembly does not allow the circularization of a plasmid not containing all parts; and second, we did not use PCR to amplify DNA or any other process involving the synthesis of DNA, other than the replication of plasmids by the bacteria machinery, thus avoiding replication error leading to a modification of the DNA sequence.

In order to select algae cells transformed with our DNA, we needed to add to our constructions a resistance gene to an antibiotic. To build multigenic constructions we used again the MoClo assembly method to make level M plasmids, with two transcriptional units: one enabling the expression of the tagged GFP and the other allowing the cells containing the plasmids to resist to paromomycin.
Using again the digestion/ligation reaction, we digested with Bbs-I the level 1 construct we obtained before as well as a level 1 construct enabling the expression of the paromomycin resistance gene.
Thus, we obtained constructs enabling the expression of GFP tagged with the HiBiT sequence in N-terminal position (pCMM-1) or C-terminal position (pCMM-2), GFP tagged with NanoLuc in N-terminal position (pCMM-4) or C-terminal position (pCMM-5) or GFP without any tag (pCMM-3) in addition to the paromomycin resistance gene.
We verified our construction as before, but using Bsa-I instead of Bbs-I

![](https://static.igem.org/mediawiki/2019/3/3f/T--Sorbonne_U_Paris--results1.png)

## Lipids metabolism modification

The first step was to insert the coding sequence of the enzymes in level 0 plasmids. We ordered synthetic DNA corresponding to the coding of FAT-A, FAT-B2, DGAT and LPAAT of *Elaeis guineensis* (African oil palm) already verified by sequencing, but unfortunately on the four sequences we’ve order, we received only two, the sequences coding for DGAT and for LPAAT. The other two, coding for FAT-A and FAT-B2, were too rich in cytosine and guanine to be synthesized.
To speed up the process, we chose to try to build level 1 containing the enzyme straight with the synthetic DNA we received, skipping the level 0 step. After the first digestion of the plasmids we received containing DGAT or LPAAT with Bbs-I, we add the reaction mix to the reaction with others level 0 parts and started the digestion reaction with Bsa-I to make level 1 constructs. In parallel we transformed bacteria with the DNA we received and then made level 0 for both enzymes. Although we did not follow the classical way of the Modular Cloning, we still obtained clones for bacteria transformed with plasmids enabling the expressing of DGAT tagged with HiBiT in N-terminal or C-terminal proving the power of MoClo assembly. As the reactions to make level 1 plasmids with LPAAT did not work we had to do it again but in the proper way, starting with the LPAAT coding sequence in a level 0 plasmid. We made the digestion ligation reaction with Bsa-I using level 0 plasmids containing the different parts of the level 1 plasmids we wanted to build.
Again, we confirmed the presence of our insert by digesting with Bbs-I and by separating the digested DNA by electrophoresis on an agarose gel.
Thus, we obtained plasmids enabling the expression of DGAT tagged with HiBiT in N-terminal (pCM1-7) or in C-terminal (pCM1-8), or LPAAT tagged as well with HiBiT in N-terminal (pCM1-9) or C-terminal (pCM1-10).

![](https://static.igem.org/mediawiki/2019/6/6f/T--Sorbonne_U_Paris--results2.png)

Following the same path than before, we inserted transcriptional units with our enzymes into level M construct. This time, we used a Hygromycin resistance gene to select cells. As well, we made level M constructs with both enzymes and the Hygromycin resistance gene.
We made the digestion/ligation reaction using Bsb-I with level 1 plasmids obtained before and with level 1 plasmids enabling the expression of the hygromycin resistance gene.
We obtained plasmids enabling the expression of DGAT tagged with HiBiT in N-terminal (pCMM-6) or in C-terminal (pCMM-7), or LPAAT tagged as well with HiBiT in N-terminal (pCMM-8) or C-terminal (pCMM-9), or both DGAT and LPAAT tagged with HiBiT in N-terminal (pCMM-14) or C-terminal (pCMM-15), and for all plasmids the hygromycin resistance gene.

### Part Characterization

## HiBiT tag

Our goal here was to characterize the expression of the HiBiT tag in *C. Reinhardtii*, and show that it could be a reliable method to detect and quantify the expression of protein in this organism.

#### Transformation of *C. Reinhardtii*

Before adding the DNA to the cells, we had to digest first the plasmids containing the insert of interest to enable the insertion in the alga genome of our transcriptional units. We digested plasmids with Bsa-I and purified the DNA. The insert and the backbone were too close in size to be separated by electrophoresis on agarose gel and we did not have access to relevant restriction enzymes that would cut only in the backbone. Thus, we chose to transform cells without separating the two fragments.

![](https://static.igem.org/mediawiki/2019/f/f9/T--Sorbonne_U_Paris--results3.png)

Transformation was done by electroporation, and after one day of recovery, cells were cultured on media with paromomycin, selecting cells expressing the resistance gene. We observed several colonies, indicating that some algae express at least the paromomycin resistance gene.

![](https://static.igem.org/mediawiki/2019/b/b8/T--Sorbonne_U_Paris--results4.png)

As the DNA can be cut during the transformation and/or inserted in non-expressed region of the genome, we had to check as well the presence of the tagged GFP in cells. To do so, we screened colonies of cells using the HiBiT tag, culturing cells onto media with Nitrate as nitrogen source to activate the pNIT promoter.
Thereby, we identified colonies expressing the construct. After adding the LgBiT and the substrate we measured the bioluminescence produced by colonies, identifying colonies expressing the tagged protein, as well as pointing out colonies highly expressing the protein.

![](https://static.igem.org/mediawiki/2019/c/c7/T--Sorbonne_U_Paris--results5.png)

#### Characterization of the HiBiT tag

Now being sure that the HiBiT tag was expressed in cells, we further investigated the characteristic of the quantification by the HiBiT system. As well, we tested the activation or repression of the pNIT promoter depending on the nitrogen source.

#### Validation of the pNIT promoter

To be sure that the regulation of the expression of the tagged GFP was efficient, we cultured cells in media with nitrate or ammonium as nitrogen source to respectively activate or repress the expression of the tagged protein. For most of our clones, we observed, as expected, expression of the tagged protein only in media with nitrate. Interestingly, we observed a clone for which the expression was constitutive, not depending on the nitrogen source. Our hypothesis was that, in this clone, the insert was inserted in region under the control of a constitutive promoter, abolishing the regulatory properties of the promoter in the insert. Cells transformed with pCMM-4 and pCMM-5, thus expected to express a GFP tagged with NanoLuc showed very low level of expression.

![](https://static.igem.org/mediawiki/2019/4/42/T--Sorbonne_U_Paris--results6.png)

#### Linearity of the signal

To test whether the bioluminescence signal was linear in a wide range, we cultured cells from a unique clone selected after the screening, either in media with nitrate or ammonium and diluted the cells to have a wide range of expression level. After addition of the LgBiT and substrate, we observed a linear increase of the NanoLuc activity with the number of cells.

![](https://static.igem.org/mediawiki/2019/8/85/T--Sorbonne_U_Paris--resultats7.png)

## DGAT and LPAAT

Using again the HiBiT tag, our goal was to determine whether the enzymes were expressed or not.

#### Transformation of *C. Reinhardtii*

We first digested the level M construct with Bsa-I and, after purifying the DNA, we let the DNA migrate by electrophoresis on an agarose gel. For the insert with two genes (the enzyme and the resistance gene) we could not distinguish the band corresponding to the insert and the one corresponding to the backbone so we transformed cells without separating the two fragments. For constructs with 3 genes (both enzymes and the resistance gene) we decided to stay in the same condition for the transformation and thus did not separate the DNA from insert and backbone as we have done before.
Transformation was done by electrophoresis, and after one day of recovery cells were cultured in media Hygromycin to select cells expressing the resistance gene. Some colonies grew demonstrating that at least the hygromycin resistance gene is expressed.

![](https://static.igem.org/mediawiki/2019/4/44/T--Sorbonne_U_Paris--results8.png)

Cells were then cultured in media with nitrate as nitrogen source to activate the pNIT promoter. The bioluminescence activity of each colony, after addition of LgBit and substrate, was measured highlighting colonies expressing the enzymes. For cells transformed with constructs having two enzymes, we could only measure the addition of both signals without being able to distinguish the contribution of each enzymes and thus, we could not be sure that both enzymes were expressed.

![](https://static.igem.org/mediawiki/2019/b/b8/T--Sorbonne_U_Paris--results9.png)

#### Expression of the enzymes

Again, we tested the expression of the enzymes depending on the nitrogen source. For two clones, the enzymes were expressed independently of the nitrogen source.

![](https://static.igem.org/mediawiki/2019/a/ae/T--Sorbonne_U_Paris--results10.png)

### Modification of lipid metabolism

We showed here the modifications of the lipid profile in presence of the enzymes, DGAT and LPAT.

## Cell culture condition

Cells were first cultured in a medium with nitrate to activate the pNIT promoter and then, after multiple wash, they were cultured in medium without any source of nitrogen, in order to trigger the accumulation of triglycerides by cells, as described in the literature 4.
After 6 days of culture in nitrogen-free media, most of the cells died. Thus, we decided to continue to work on cells cultured in media with ammonium to try to investigate the effect of DGAT and LPAAT on the lipid profile in normal condition. As mentioned before, after the transformation, we obtained clones expressing the enzymes independently of the nitrogen source, so we used these clones, one expressing the DGAT, and the other expressing both LPAAT and DGAT, to explore the potential modification of the lipid profile.

## Expression of the enzymes

To be sure that the enzymes were well expressed, we tested the presence of the enzymes thanks to the HiBiT system. We confirmed that the first clone was expressing the DGAT, and that the other clone was expressing at least one of the two enzymes, DGAT or LPAAT.

![](https://static.igem.org/mediawiki/2019/6/6e/T--Sorbonne_U_Paris--results11.png)

## Modification of lipid profile

We investigated the modification of the lipid profile by doing a Thin Layer Chromatography (TLC) using identified lipids as standards to be compared with the profile of our clones. For the clone expressing the DGAT, we observed that one spot corresponding to the diglyceride was missing in comparison with a wild type strain making the lipid profile for this clone similar to the palm oil profile. This result is coherent with the mechanism of the DGAT. The presence of the enzyme results in an increased consumption of diglyceride, shifting the balance of lipids in the cells.

![](https://static.igem.org/mediawiki/2019/9/9d/T--Sorbonne_U_Paris--resultats12.png)

For the clone potentially expressing both DGAT and LPAAT, there was no difference in the lipid profile with wild type strain. We know, thanks to the HiBiT quantification, that this clone expresses at least one enzyme, and as well, the lipid profile of this clone was different from the profile of the DGAT-expressing clone. Thus, we thought about several hypotheses :

* This clone does not express the DGAT, or at such a low level that it does not have any effect on the lipid profile. And so, it is expressing the LPAAT, which is coherent with the profile we observed. Indeed, if the LPAAT is expressed, we were expecting an increase of the diglyceride quantity, that we cannot detect with the TLC as it is not a quantitative method.
* This clone expresses both DGAT and LPAAT, meaning that the decrease of the diglyceride induced by the exogenous DGAT is offset by the increase of diglyceride production induced by the exogenous LPAAT, resulting in a lipid profile similar to a wild type profile.

1. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6(2): e16765. [doi:10.1371/journal.pone.0016765](https://www.ncbi.nlm.nih.gov/pubmed/21364738)
2. Crozet, Pierre, et al. "Birth of a photosynthetic chassis: A MoClo toolkit enabling synthetic biology in the microalga Chlamydomonas reinhardtii." ACS synthetic biology 7.9 (2018): 2074-2086.
3. Engler C, Kandzia R, Marillonnet S (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3(11): e3647. [doi:10.1371/journal.pone.0003647.](https://www.ncbi.nlm.nih.gov/pubmed/?term=10.1371%2Fjournal.pone.0003647)
4. Deng, Xiaodong, Xiaowen Fei, and Yajun Li. "The effects of nutritional restriction on neutral lipid accumulation in Chlamydomonas and Chlorella." African Journal of Microbiology Research 5, no. 3 (2011): 260-270.

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