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<img align="center" src="https://static.igem.org/mediawiki/2016/7/78/Pencil-typography-black-design.jpg" alt="Banner Image" style="width:100%;height:450px"> | <img align="center" src="https://static.igem.org/mediawiki/2016/7/78/Pencil-typography-black-design.jpg" alt="Banner Image" style="width:100%;height:450px"> | ||
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− | <h4 align="center"> | + | <h4 align="center"> With proper <a href="https://2016.igem.org/Team:WPI_Worcester/Description"> motivation and background</a> for using the CRISPR/dCas9 and the editing enzymes APOBEC or ADAR to target and edit mRNA, designing effective plasmids and reporters was the first step in ensuring that we would be able to accurately show this proof of concept. </h4> |
<h1 align="center"> Reporters </h1> | <h1 align="center"> Reporters </h1> | ||
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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/2016/6/69/TunabilityTrans.png" alt = "tunability" /> | <img align="center" src="https://static.igem.org/mediawiki/2016/6/69/TunabilityTrans.png" alt = "tunability" /> | ||
− | <h4 align="center"> One of the | + | <h4 align="center"> One of the most common suggestions we received when conducting our <a href="https://2016.igem.org/Team:WPI_Worcester/Integrated_Practices"> integrated human practices</a> was to design a tunable system. We don't want the enzymes to be editing at all time, especially if something was to go wrong. Regulation is the purpose of the rtTA gene in this plasmid. rtTA, which stands for reverse tetracycline trans activator, is constitutively expressed. It acts as a repressor binding to the tetracycline responsive element (TRE) promoter and preventing transcription of the editing enzyme and dCas9. However, when doxycycline is added to the system it will bind to the rtTA, removing it from the promoter and allowing transcription. Editing should not occur unless we introduce doxycycline into the cells. </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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Latest revision as of 20:29, 19 October 2016