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<h1 align="center"> Reporters </h1> | <h1 align="center"> Reporters </h1> | ||
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− | <h4 align="center"> We designed 2 reporters, one for each editing enzyme. Each reporter is designed to emulate certain point mutations that can occur and cause diseases. The APOBEC reporter imitates a mutation to a start codon and the ADAR reporter imitates a mutation that creates a premature termination codon (PTC). | + | <h4 align="center"> We designed 2 reporters, one for each editing enzyme. Each reporter is designed to emulate certain point mutations that can occur and cause diseases. The APOBEC reporter imitates a mutation to a start codon and the ADAR reporter imitates a mutation that creates a premature termination codon (PTC). Both reporters 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. </h4> |
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<h3 align="center"> <b> APOBEC Reporter </b> </h3> | <h3 align="center"> <b> APOBEC Reporter </b> </h3> | ||
<img align="center" src="https://static.igem.org/mediawiki/2016/d/d8/APOBECreporterTrans.png" alt = "APOBECreporterTrans" /> | <img align="center" src="https://static.igem.org/mediawiki/2016/d/d8/APOBECreporterTrans.png" alt = "APOBECreporterTrans" /> | ||
− | <h4 align="center"> For our APOBEC experiments we used the following reporter. The reporter is a GFP sequence with | + | |
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+ | <h4 align="center"> For our APOBEC experiments we used the following reporter. The reporter is a GFP sequence with a 5’ untranslated region (UTR). The 5' UTR is a portion of a GAPDH sequence 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. </h4> | ||
<img align="center" src="https://static.igem.org/mediawiki/2016/f/fc/APOBECeditTrans.png" alt = "APOBECeditTrans" /> | <img align="center" src="https://static.igem.org/mediawiki/2016/f/fc/APOBECeditTrans.png" alt = "APOBECeditTrans" /> | ||
<h4 align="center"> 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. </h4> | <h4 align="center"> 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. </h4> |
Revision as of 21:28, 14 October 2016