Team:Sorbonne U Paris/Experiments

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.

Transformation of bacteria

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

Transplanting a colony into a liquid medium

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

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.

DNA concentration measurement

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

Digestion for quality control of generated DNA

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

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.

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.

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.

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.

For more details, go on our Protocols document :

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