Team:Sorbonne U Paris/Experiments

Experiments

Experiments



Design in silico :

All in silico sequence designs and analyses were performed with Serial Cloner and Geneious prime.

MoClo Assembly Conditions:

The Modular Cloning (MoClo, Weber et al, 2009) provides a quick and reliable method to assemble basic parts such as promoters, CDSs or terminators. The MoClo relies on the Golden Gate cloning strategy (Engler et al, 2008).

Materials :

To make our MoClo Assembly protocols, we used an excel file 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". For each reaction this table enabled us to calculate the appropriate volumes of DNA based on the DNA concentrations of the used solutions, as well as as the amount of water to reach the final volume (20 µL).

Master mix (for 2 ligations):

*The choice of the restriction enzyme depends on the level of the construction wanted. For level 0 and level M constructions BbsI-HF must be used, while for level 1 construction BsaI-HF must be used.

The reaction is performed in a thermocycler to allow several cycles of digestion (at37°C) and ligation (at16°C). The program used includes a final digestion to remove background (37°C) and the denaturation of the enzymes (with a step at 65°C for 20 min). The samples are then cooled to 16°C until they are taken out of the thermocycler.

Transformation of bacteria:

Chemically competent E. coli DH10β was used for heat shock transformation.

Materials :

- NEB 10-beta Competent E. coli
- Digestion/Ligation products (assay or negative control)
- LB medium (Sigma)
- Ice bucket
- LB + X-Gal + antibiotics* solid medium
- Shaking incubator with modular temperature

*The antibiotic depends on the construction wanted, for level 0 and level M the media contains spectinomycin at 50μg/mL, for level 1 the media contains ampicillin at 50μg/mL.

Methods:

1. Take competent bacteria out of -80°C and thaw on ice.
2. Mix 4 μL of Digestion/Ligation products with 25 μL of E. coli.
3. Incubate the competent cell/DNA mixture on ice (4°C) for 10 min
4. Heat shock each transformation tube by placing the tube at 42°C for 45 seconds.
5. Put the tubes back on ice (4°C) for 2 min.
6. Add 700 μL of LB medium (without antibiotic) and incubate 1h at 37°C with shaking (400rpm).
7. Spin for 2 min at 4.000 rcf to pellet the cells.
8. Discard the supernatant, leaving only about 100 μL of media.
9. Resuspend cells in the remaining medium.
10. Spread 100 μL on a culture plate containing LB + X-gal (50 μg/mL) + antibiotic* (see above) (50 μg/mL).
11. Incubate plates at 37°C overnight.

Transplanting a colony into a liquid medium:

Transfer micro-organisms from agar to liquid medium for growth.

Materials :

- LB/Antibiotic (50 μg/mL)/X-gal (50 μg/mL) liquid medium
- Culture plates with transformed bacteria

Methods:

1. Collect a white clone of interest using a tip (white clones correspond to recombinant clones whereas blue clones correspond to clones with empty vectors).
2. Drop the tip in 2 mL of liquid medium in a 15mL Falcon tube.
3. Incubate overnight at 37°C or at 30°C if the plasmid is bigger than 10kb.
4. Harvest bacterial culture and pellet by centrifuging at 4.000 rcf for 2 min.
5. Discard supernatant.

Minipreps were carried out according to the manufacturer's instructions.

Plasmid extraction:

The plasmids were extracted from the recombinant clones using the MACHEREY‑NAGEL NucleoSpin® Plasmid kit. The plasmid DNA is extracted based on the alkaline lysis of the bacterial cells. The DNA is purified by means of a silica membrane which selectively retains DNA.

Materials :

- Ozyme ZymoPURE™ Plasmid Miniprep Kit
- Liquid culture of bacteria

Methods:

1. Harvest bacterial culture and pellet by centrifuging at 4.000 g for 2 min.
2. Discard supernatant.
3. Minipreps were carried out according to the manufacturer's instructions.

DNA concentration measurement:

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

Materials:

- NanoDrop 2000 spectrophotometer (ThermoFisher Scientific)
- Lint - free laboratory wipe
- Highly pure MiliQ water
- Elution buffer from the Plasmid Kit
- Samples

Methods:

DNA concentration measurement was performed according to the NanoDrop 2000 spectrophotometer protocol.

1. Make the blank with the buffer used to extract the DNA
2. Pipet 1 to 2 μL of sample directly onto the measurement pedestal*.

*Between measurements: wipe the sample off of both the upper and lower pedestals with a clean, dry lint.

When the measurement is complete, raise the sampling arm and wipe the sample off of both the upper and lower pedestals using a dry, lint-free laboratory wipe, pipet 2μL of water to clean any DNA remaining on the pedestals, and dry it with lint-free wipe.

Quality Control of generated DNAs:

All plasmids were controlled by differential restriction to check the size of the insert.

Materials :

- BsaI-HF, BbsI-HF, EcoRI (NEB)
- Purified DNACut
- Smart buffer 10X(NEB)
- Sterile Water

Methods:

1. Add 100-200ng of DNA.
2. Add water to a final volume of 8.7μL.
3. Prepare the mix for all reactions

For one reaction : 

4. Dispatch 1.3 μL per tube.
5. Incubate 1 hour at 37°C

Preparation of DNA samples for sequencing:

Preparation of DNA samples for sequencing were carried out according to the manufacturer’s instructions (Eurofins) : add 2µL of primer at 10µM and then 15µL of plasmid DNA concentrated at 50-100 ng.µL-1.

DNA gel electrophoresis:

After the quality control digestion, the DNA fragments are separated according to their size on an agarose gel. The size of the DNA fragment is evaluated in comparison with a DNA size marker.

Materials :

- Agarose powder (Sigma)
- TAE buffer 0.5X (Tris-acetate 40mM, EDTA 1mM, pH 7.8 from Sigma)
- 1 kb DNA ladder (NEB)
- Gel tank
- Comb
- Microwave oven
- Scales
- Ethidium bromide 10 mg.mL-1

Methods:

1. Preparation of the 1% agarose gel in TAE buffer: 

1.1 Dissolve 0.5g of agarose powder in 50 mL of TAE buffer 0.5X by heating the solution in the microwave. Heat for 30 seconds and swirl the flask until the powder is completely dissolved and the solution is clear.
1.2 Cool the solution (not for too long or it will solidify) on the table or you can allow hot agarose to cool in a water bath set at 60°C for 10min.
1.3 Add 1 μL of Ethidium bromide (EtBr) solution (final concentration:0.5 μg/mL) to the cooled gel.
1.4 Cast the solution on an electrophoresis gel mold, insert the comb and let the gel solidify at room temperature.

2. Gel migration:

2.1 Remove the comb.
2.2 Immerse the gel with 0.5X TAE migration buffer. Cover the gel with 2-3 mm of buffer for best results.
2.3 Load 3 µL of the DNA ladder on a well.
2.4 Load 15 µL DNA samples with the corresponding amount of DNA loading dye (DNA loading dye 1X) Run the gel for 30 minutes at 100V.

3. Gel revelation: At the end of the electrophoresis migration, collect the gel and take a picture of it using a gel reader (GelDocBiorad) under UV light. Adjust the brightness if necessary.

Caution with hot agarose!

Wear protective gloves, lab coat and eyes protection when handling an extremely hot agarose solution and heat the solution in several short intervals—do not let the solution boil for long periods as it may boil out of the flask or cause a loss to water vapor.

Caution with Ethidium Bromide (EtBr)!

Ethidium Bromide is a sensitive fluorescent dye used to detect nucleic acids in agarose gels. It is a probable mutagen and probable carcinogen. Always wear gloves, lab coat and eyes protection when working with ethidium bromide. Wipe the area with a damp cloth after working with ethidium bromide. Also, while wearing gloves after handling ethidium bromide, be careful not to touch and thereby contaminate other surfaces. Discard the gloves and pipet tips in appropriate trash.

Culture of Chlamydomonas:

Materials :

- Tris-Acetate-Phosphate (TAP) medium 65
- Agar (1.6 % m/V),

Bold’s minimal media supplemented with 36 gL-1 NaCl

Toxicity test :

The toxicity test is an important part of our project. Indeed, we designed this test in order to be able to screen our genetically modified algae. It should allow us to determine whether an algae colony expresses our plasmid or not. To do so, we have to determine the natural resistance of Chlamydomonas reinhardtii for each pollutant. We want to experiment by testing wild type culture in different concentrations of these pollutants. Once we determine the deadly concentration for the cells, we will be able to make some culture medium with a high toxicity level for each pollutant. After this, only the transformed cells which express the specific degradation enzymes will be able to grow on the different mediums. Currently, we tested two pollutants, the atrazine and the cyanuric acid. We would like to assess whether the cyanuric acid is toxic for Chlamydomonas reinhardtii since it does not appear to influence the growth of the cells. So we have to determine whether the concentrations were too low or the cells are resistant.

Materials:

- Laminar flow hood
- Algem Pro Photobioreactor System
- 24 x 1mL well plates
- 1L erlenmeyer flask
- TAP medium (https://www.chlamycollection.org/methods/media-recipes/)
- DMSO
- Pollutant (Atrazine/Cyanuric acid)
- Chlamydomonas reinhardtii strain D66

Methods:

1. Put Chlamydomonas reinhardtii in exponential phase (2 millions cells per mL) in TAP (Tris-Acetate Phosphate) medium.
2. Prepare stock solutions for atrazine and cyanuric acid (1g/L). These pollutants have to be diluted into DMSO. The growth ability of the wild-type microalgae was assessed by colorimetry and a toxicity threshold was determined for every pollutant (minimum concentration to inhibit the growth of Chlamydomonas reinhardtii strain D66). 
3. Put 1mL of culture into some 24 1mL well plates with a 1% DMSO control and growing concentrations of each pollutant (0, 1, 2, 5, 10, 25, 50, 75, 100, 250, 500, 1000, 1100, 1250 and 1500 µg/L).
4. At each time point (0h, 6h, 12h, 24h, 48h, 72h, 96h) take a photograph of each plate.

We also performed a toxicity test with the Algem Pro Photobioreactor System. This hightech bioreactor allows us to record in real time the growth of our microalgae with reproducible optical density (OD) mesure while monitoring environmental parameters (pH and temperature). The Algem also enables us to verify our plate experiment on a larger scale (from 1mL wells to 1L standard erlenmeyer flask). Each flask is mixed along the experiment with an automatic hand-swirling like mouvement.

1. Put some Chlamydomonas reinhardtii strain D66 cells at exponential phase (1 millions cells/mL) with 400mL TAP medium into a 1L erlenmeyer flask.
2. Try several atrazine concentrations ( 0, 77, 250 and 500 µg/L).
3. Collect real time data for 50h. Set the environmental parameters at pH=7 and T =28°C. Measure the OD at 740nm (absorbance peak of Chlorophyll a pigment).