Difference between revisions of "Team:WPI Worcester/Design"
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<h4 align="center"> The overall goal of our project is to prove that single base nucleotide changes can occur in an organism when editing enzymes are coupled with the CRISPR/dCas9 system. Designing effective plasmids and reporters was the first step in ensuring that we would be able to accurately show this proof of concept. </h4> | <h4 align="center"> The overall goal of our project is to prove that single base nucleotide changes can occur in an organism when editing enzymes are coupled with the CRISPR/dCas9 system. Designing effective plasmids and reporters was the first step in ensuring that we would be able to accurately show this proof of concept. </h4> | ||
Revision as of 17:48, 14 October 2016
The overall goal of our project is to prove that single base nucleotide changes can occur in an organism when editing enzymes are coupled with the CRISPR/dCas9 system. Designing effective plasmids and reporters was the first step in ensuring that we would be able to accurately show this proof of concept.
Reporters
We designed 2 reporters in total, one for each editing enzyme. Each reporter is designed for targeting by the CRISPR/dCas9 complex onto its mRNA due to a 5’ untranslated region. They also both used a green fluorescent protein (GFP) as the editing identifier. When expressed in the cell without presence of the editing complexes, the reporters should not produce GFP. Thus, the cells would not fluoresce. Once the editing complex binds and edits the reporters, however, GFP should then be translated and the cells will glow green.
APOBEC Reporter
For our APOBEC experiments we used the following reporter. The reporter is a GFP sequence with the 5’ UTR for the CRISPR/dCas9 to bind to. The only change we made to the GFP sequence was mutating the start codon (ATG) to an ACG codon. The reason for this being that when the ribosome would bind to the reporter’s mRNA, translation should not occur since there is no start codon. Therefore, we should not see any fluorescence in the transfected cells.
Once the CRISPR/dCas9 and APOBEC fusion is added to the cells the CRISPR/dCas9 will be able to bind to the 5’ UTR of the reporter. APOBEC will then make its C to U edit on the ACG codon, making it an AUG codon, which is a start codon in mRNA. Now when the ribosome binds, the GFP should be translated and the cells will fluoresce.
ADAR Reporter
test
