iGEM SORBONNE
NAWI
Results
Our progress
Through our NAWI project we wanted to create a new innovative food source to feed humanity. To achieve this goal, we designed a plasmid to genetically modify the genome of the green algae C. reinhardtii. This plasmid is composed of a replication origin, gypsy insulator sequences, an inducible promoter, the THB1 gene, the Su(Hw) gene which codes for a protein working with gypsy and a hygromycin resistance gene. All these parts can be standardized for the MoClo kit for C. reinhardtii. The MoClo (Modular Cloning) kit allows an easy and modular building of plasmids.
We therefore started to standardize all these parts by adding fusion sites at their terminals by following the MoClo kit model (for level 0).
Crozet and al., 2018, A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii
We started with the creation of the PSAD promoter part in the A2-B1 position. For this we did a PCR on a level 0 plasmid from the lab with pPSAD in A1-B1 position, thanks to floating tail primers.
Example with mVenus in B2-B5 position
We started by performing a PCR on p0-35, a level 0 plasmid with mVenus in B2 from the lab, in order to get it in B2-B5 to suit our design, thanks to floating tail primers (cf. protocol PCR)
Agarose gel result for PCR from p0-35.
As expected we obtained a 835nt PCR product, which was then extracted and purified using a kit from NEBⓇ.
This purified insert was used in a MoClo reaction in order to get mVenus in B2-B5 in a level 0 plasmid (p0-mVenus).In order to check if the MoClo reaction worked, we digested the obtained product with BsaI and we then made an agarose gel to see the results.
Agarose gel result from the digestion of the product of the MoClo reaction with BsaI.
As expected we obtained a 815nt product, which corresponds to mVenus, the insert. And a product of 2057nt, which corresponds to the universal plasmid, the backbone.
During our experimental period, we also had the time to make level 0 plasmids with pPSAD in A2-B1 position, and tPSAD in B6.
Unfortunately , we were unable to receive the THB1 gene sequence, and the gypsy insulator sequence from our sponsors. So we were not able to finish our build step…
