Assembly of plasmide pL0-294, containing our antioxidant insert, with GoldenGate method and transformation of the assembled plasmid into NEB 10-beta Competent Escherichia coli (High Efficiency), that are plated on LB-agar+Spec+Xgal plates.
Week 2 = from the 19th to the 23th of july
Selection of white transformed bacteria on spectinomycin plates and growth in liquid culture of LB of our selected clones.
Extraction and purification of plasmid DNA.
Digestion with restriction enzyme BsaI and migration on agarose gel to confirm the presence of our insert : amplification by PCR on transformed colonies and migration on agarose gel.
Third attempt to identify if the antiox sequence is present in the plasmid : digestion by BsteII and HindIII.
The plasmid is sent to sequencing.
Assembly of the p1 plasmid, transformation into E. Coli and selection on ampicillin.
PCR reaction and agarose gel electrophoresis on selected bacterial clones.
Extraction and purification of plasmid level 1 with Nucleospin kit.
Assembly of the pM plasmid, transformation into E. Coli and selection on spectinomycin plates.
Week 3 = from the 26th to the 30 of july
Assembly of the pM plasmid, transformation into E. Coli and selection on spectinomycin plates.
Range of toxicity essay with 3 oxidant molecules (H2O2, Rose Bengale and Methyl Viologen) at different concentrations on non transformed Chlamydomonas reinhardtii.
Revelation of dead cells by incubation with Evans Blue.
Extraction of plasmidic DNA to recover final pM and verification by agarose gel electrophoresis after digestion with BbsI.
Digestion of pM-156
Linearization of pM-156 with EcoR
First transformation of Chlamydomonas reinhardtii strain 45-33 using glass marbles with the plasmide pM-156.
Week 4 = from the 2nd to the 6th of August
Prepared TAP broth.
Count of dead and alive cells within the oxidant range on non transformed Chlamydomonas strain determination of the survival rate.
Second oxidant range on non transformed Chlamydomonas reinhardtii, dead cells are colored with Evans blue. We used doses of oxidants to try to obtain survival rates between 20 and 50%.
Count of dead and alive cells within the second oxidant range on non transformed Chlamydomonas strain determination of the survival rate.
Second attempt of transformation of Chlamydomonas reinhardtii strain 45-33, using Onishi protocol.
Week 5 = from the 9th to the 13the of August
Dilution of dry peptides we had ordered.
Several Bradford and BCA (bicinchoninic acid) dosages were made to determine the decapeptide concentration. (LOL)
First antioxidant test with H2O2 and Rose Bengale on non transformed Chlamydomonas reinhardtii in the presence of our 2 peptides.
Week 6 = from the 16th to the 20th of August
Revelation of dead cells by incubation with Evans Blue.
Manganese toxicity range of wild-type Chlamydomonas reinhardtii.
Transformation of Chlamydomonas reinhardtii strain 45-33 attempt 3 with Onishi protocol.
Toxicity and characterisation assay of our 2 peptides to assess whether it’s toxic and understand why it provokes agglomeration.
Plates patch of strain 45-33 on different conditions check its antibiotic resistance : we came to the conclusion that strain 45-33 was resistant to hygromycin, so we couldn’t use it to select our transformed algae.
Highlight of the interaction between Evans Blue and our peptides -which forms wide aggregates
under the microscope.
Week 7 = from the 23th to the 27th of August
Linearization of pM-156
First transformation of Chlamydomonas reinhardtii strain UVM4 with Onishi protocol and spreading on hygromycin plates. Cells seemed to have not survived the electroporation process, so we had to throw them out and try again.
Second transformation of UVM4 with Onishi protocol on hygromycin plates.
Week 8 = from the 30th of August to the 3rd of September
Prepared PBS buffer.
Prepared TAP broth.
Third transformation attempt of Chlamydomonas reinhardtii UVM4 with plasmid pM156 containing the antioxidant gene, with glass marbles.
Week 9 = from the 6th to the 9th of of September
First try of LDH test : we tried to characterize the activity of LDH enzyme after flowing through the Amicon tube. We wanted to use Amicon to get rid of H2O2 before putting the substrates (NADH and pyruvate) and reading the absorbance. The absorbance was too important so we modified the NADH concentration.
Second try of LDH test : this time the absorbance was right but the enzymatic activity detected was about 0,0297 ΔAbs/min, which is quite low. We thought we had lost a lot of enzymes through the Amicon tube.
Third try of the LDH test : we characterize the activity of LDH without Amicon tube and without any ROS to assess how the reaction takes place without any perturbation. The enzymatic activity was around 0,097 ΔAbs/min.
Fourth try of the LDH test : we don’t need the Amicon tube because H2O2 will be diluted in the final volume of tampon and pyruvate. Incubation of LDH with H2O2 10mM reduces the activity of the enzyme just enough for us to work with.
Week 10 = from the 13th to the 17th of September
Selection of 10 clones that grew on hygromycin after transformation with plasmid pM-156.
Extraction of RNAs from the transformed strain of Chlamydomonas reinhardtii UVM4.
Electrophoresis of the total extracted RNAs on an agarose gel.
Week 11 = from the 20th to the 24th of September
Reverse-transcription of total RNAs extracted.
Multiple PCR tests with primers RT-08 and RT-09 to amplify our insert and CBLP primers (housekeeping gene), with different annealing temperature and revelation with agarose gel electrophoresis.
In vivo test on 10 clones of transformed cells UVM4 with H2O2 to see if the expression of the peptides has a protective effect.
Week 12 = from the 27th of September to the 1st of October
PCR of the cDNA with 2 new primers pairs : RT10/RT11 and RT12/RT13.
Shipment of 5x10^6 cells of the clones expressing the antioxidant peptide : 2, 5, 7 and the untransformed UVM4 strain (each sample in triplicate).
Week 13 = 4th to 8th of October
Irradiation of Chlamydomonas reinhardtii shipped at the Belgian nuclear research center
Preparation of 16 TAP-agar 6 wells plates.
Collect of irradiated strain, serial dilution, spread in previously made plates and growth under light.
