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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> | + | <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> | ||
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<img align="center" src="https://static.igem.org/mediawiki/2016/8/8d/MCherryPlasmidTrans.png" alt = "mCherryPlasmid" /> | <img align="center" src="https://static.igem.org/mediawiki/2016/8/8d/MCherryPlasmidTrans.png" alt = "mCherryPlasmid" /> | ||
− | <h4 align="center"> This plasmid contains our guide RNA and mCherry. The guide RNA is complementary to the target editing site and will help direct the dCas9 there. mCherry is a type of RFP and is constitutively expressed. This is our transfection marker and shows that the plasmids have | + | <h4 align="center"> This plasmid contains our guide RNA and mCherry. The guide RNA is complementary to the target editing site and will help direct the dCas9 there. mCherry is a type of RFP and is constitutively expressed. This is our transfection marker and shows that the plasmids have successfully entered the cells. </h4> |
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Revision as of 13:55, 16 October 2016