Difference between revisions of "Team:Bordeaux/Results"
| Line 154: | Line 154: | ||
- To finish CRISPR methylase constructions<br><br> | - To finish CRISPR methylase constructions<br><br> | ||
- To test the three stategies<br> <br> | - To test the three stategies<br> <br> | ||
| + | - Because of the worm’s capacity to absorb nucleic acids from digested bacteria, contamination can mislead us. To avoid this, our worms will be exclusively fed with dmc- bacterial strains (DNA cytosine methyltransferase) which don’t have the methylated Cytosine epigenetic mark.<br> <br> | ||
<b>Bio-informatics part</b><br><br> | <b>Bio-informatics part</b><br><br> | ||
- To prepare a better protocol for bio-informatics experiments</td> | - To prepare a better protocol for bio-informatics experiments</td> | ||
Revision as of 12:12, 19 October 2016
We built a plasmid containing DSIP under control of U6 promoter. This plasmid has been injected in several worms with another plasmid containing DsRed protein in order to compare injected worms to worms used as control. Worms have been observed by fluorescence microscopy. We tried to create different constructions by changing the promoter used. Except for pRPS27, all promoters own one or several illegal restriction sites (White Collar-1 and White Collar-2 also own illegal restriction sites). However, pRPS27 amplification never worked and mutagenesis of others promoters was hard to do. So, we decided to work on an other promoter: U6. We created two constructions: This second construction permit to us to verify, thanks to GFP, that induced part works even if NLP-22 doesn't have any effect on Caenorhabditis elegans. The creation of our Crispr methylase required a lot of parts to assemble. Each amplification was successful. After that, we tested both cloning using restriction enzymes and Gibson assembly cloning but these techniques never worked. So, we had to think about another way but we ran out of time to complete the experiments. We thought about Golden Gate cloning but it would have taken to buy new primers to create overlapping sequences. In progress
Goals
1. Studying DSIP's effect on Caenorhabditis elegans
➔ No differences was observed between the two types of worms. DSIP doesn't seem to have any effect on Caenorhabditis elegans.
2. Creating a photo-inducible system controlling Caenorhabditis elegans
Inducer part
Initial construction was to assemble WC1, P2A and WC2 under control of U6 promoter. By lack of time, a simpler construction was realized:
Induced part
➔ By lack of time, we didn't finish all of our constructions. So we were not able to validate the photo-inducible system.
3. Creating a Crispr/methylase in order to knock out sleep and allowing the study of Caenorhabditis elegans's epigenetic
➔ By lack of time, we didn't finish our constructions. So we were not able to validate our Crispr methylase.
4. Adding experimental results to the bio-informatics model
Results
Perspectives
Biobricks created in psB1C3
Sleep part
- No effect of DSIP on Caenorhabditis elegans
- Photo-inducible system not finished
EpiCRISPR part
- Crispr methylase not finished
Bioinformatics part
- Non concluant resultsSleep part
- To finish photo-inducible system’s constructions
- To create better primers for pRPS-27 amplification
EpiCRISPR part
- To finish CRISPR methylase constructions
- To test the three stategies
- Because of the worm’s capacity to absorb nucleic acids from digested bacteria, contamination can mislead us. To avoid this, our worms will be exclusively fed with dmc- bacterial strains (DNA cytosine methyltransferase) which don’t have the methylated Cytosine epigenetic mark.
Bio-informatics part
- To prepare a better protocol for bio-informatics experimentspU6
pXBP1
pFRQ
NLP-22
pFRQ::GFP
P2A::GFP
