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<h1 align="center"> Plasmids </h1> | <h1 align="center"> Plasmids </h1> | ||
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− | <h4 align="center"> To accomplish our experiment we designed three different plasmids. The components of each were cloned in E. coli cells and then miniprepped. All three plasmids were then transfected into our chassis, HEK293T cells. </h4> | + | <h4 align="center"> To accomplish our experiment we designed three different plasmids. The reason that we broke our system into three plasmids is because of the size of the components. The coding region for dCas9 on its own is about 4,000 base pairs. The components of each plasmid were cloned in E. coli cells and then miniprepped. All three plasmids were then transfected into our chassis, HEK293T cells. </h4> |
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<h3 align="center"> <b> dCas9 Plasmid </b> </h3> | <h3 align="center"> <b> dCas9 Plasmid </b> </h3> | ||
<img align="center" src="https://static.igem.org/mediawiki/2016/1/11/DcasPlasmidTrans.png" alt = "dCas9Plasmid" /> | <img align="center" src="https://static.igem.org/mediawiki/2016/1/11/DcasPlasmidTrans.png" alt = "dCas9Plasmid" /> | ||
− | <h4 align="center"> This is our first plasmid. It contains one of the editing enzymes, either ADAR1, ADAR2, or APOBEC1, fused to dCas9. The enzyme and dCas9 have 1-3 repeats of an XTEN linker to allow the enzyme to edit a certain distance away from dCas9. Downstream on the same plasmid is the rtTA gene and promoter. </h4> | + | <h4 align="center"> This is our first plasmid. It contains one of the editing enzymes, either ADAR1, ADAR2, or APOBEC1, fused to the N terminus of dCas9. The enzyme and dCas9 have 1-3 repeats of an XTEN linker to allow the enzyme to edit a certain distance away from dCas9. Downstream on the same plasmid is the rtTA gene and promoter. </h4> |
<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 key parts of our system is that we wanted it to be tunable. 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 | + | <h4 align="center"> One of the key parts of our system is that we wanted it to be tunable. 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 type of RFP and is constitutively expressed. This is our transfection marker and shows that the plasmids have sucessfully entered the cells. </h4> | + | <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 sucessfully entered the cells. </h4> |
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Revision as of 21:11, 14 October 2016