Difference between revisions of "Team:MIT/project"
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| + | <h2> REPORTERS </h2> | ||
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| + | After designing our 2-exon mKate HBG reporter, which only showed mKate knockdown, we needed a positive reporter. We designed two 3-exon reporters, a split mKate reporter and a dual fluorescence reporter: | ||
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| + | <h3> 3-exon mKate Fluorescent Reporter </h3> | ||
| + | <p> The reporter was designed to contain mKate exon one, HBG Intron two, the REST-4 included exon from our disease model, HBG Intron one, and mKate exon two.[FIGURE] The mKate exons and the HBG Intron two were taken from the 2-exon mKate reporter. The REST-4 exon was chosen because it contains a stop codon, and because we wanted our reporter to mirror our disease model. The HBG Intron one was chosen based on the literature. [Source]. A g-block was designed with the REST exon, HBG intron 2 and complementary sticky ends to be added to the 2-exon mKate HBG reporter between HBG Intron 2 and mKate exon two. </p> | ||
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| + | <p>If our guide system is not present in the cell, mKate exon one, the REST exon, and mKate exon two will be spliced together. During translation, the stop codon in the REST exon will cause the ribosome to fall off, resulting in an incomplete mKate and therefore a knockdown of red fluorescence. When our guide system is introduced to the cells, it will cover the 5’ splice site on HBG Intron 2, causing the spliceosome to skip over the REST exon. The cell will then be able to translate mKate as normal, and we expect to see a rise in red fluorescence. This reporter therefore achieves our goal of being a positive reporter. </p> | ||
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| + | |||
| + | <h3> 3-exon Dual Fluorescent Reporter </h3> | ||
| + | <p>In order to fully demonstrate the functionality of our guide system, we designed a reporter that, instead of showing an increase or decrease of a single color, fluoresced a different color in the presence of our system. Our reporter contains a fluorescent conserved region, HBG Intron two, a yellow fluorescence exon, HBG Intron one, and a blue fluorescence exon. The Yellow Fluorescent Protein (YFP) and the Blue Fluorescent Protein (BFP) used in our lab have a 200 bp conserved region between them, which was ordered a g-block with complementary sticky ends to HBG Intron 2. The YFP and BFP only differ by approximately 50 bp in their non-conserved regions. In order to design a g-block that contained both YFP and BFP, the third base of every amino acid was wobbled in the YFP gene. (The wobble base was chosen based on human codon usage). The second g-block also included HBG Intron one between the two fluorescent exons, and complementary sticky ends with HBG Intron two. </p> | ||
| + | |||
| + | <p>If our guide system is not present in the cell, the conserved region, YFP and BFP will be spliced together. During translation, the stop codon at the end of YFP will cause the ribosome to fall off, resulting in a complete YFP and therefore yellow fluorescence will be made. When our guide system is introduced to the cells, it will cover the 5’ splice site on HBG Intron 2, causing the spliceosome to skip over the YFP exon. The conserved region and BFP will be spliced together, resulting in a complete BFP, and therefore blue fluorescence will be made. </p> | ||
Revision as of 01:22, 29 October 2017
Experimental Design
Explanation of all experiments we performed.
REPORTERS
After designing our 2-exon mKate HBG reporter, which only showed mKate knockdown, we needed a positive reporter. We designed two 3-exon reporters, a split mKate reporter and a dual fluorescence reporter:3-exon mKate Fluorescent Reporter
The reporter was designed to contain mKate exon one, HBG Intron two, the REST-4 included exon from our disease model, HBG Intron one, and mKate exon two.[FIGURE] The mKate exons and the HBG Intron two were taken from the 2-exon mKate reporter. The REST-4 exon was chosen because it contains a stop codon, and because we wanted our reporter to mirror our disease model. The HBG Intron one was chosen based on the literature. [Source]. A g-block was designed with the REST exon, HBG intron 2 and complementary sticky ends to be added to the 2-exon mKate HBG reporter between HBG Intron 2 and mKate exon two.
If our guide system is not present in the cell, mKate exon one, the REST exon, and mKate exon two will be spliced together. During translation, the stop codon in the REST exon will cause the ribosome to fall off, resulting in an incomplete mKate and therefore a knockdown of red fluorescence. When our guide system is introduced to the cells, it will cover the 5’ splice site on HBG Intron 2, causing the spliceosome to skip over the REST exon. The cell will then be able to translate mKate as normal, and we expect to see a rise in red fluorescence. This reporter therefore achieves our goal of being a positive reporter.
3-exon Dual Fluorescent Reporter
In order to fully demonstrate the functionality of our guide system, we designed a reporter that, instead of showing an increase or decrease of a single color, fluoresced a different color in the presence of our system. Our reporter contains a fluorescent conserved region, HBG Intron two, a yellow fluorescence exon, HBG Intron one, and a blue fluorescence exon. The Yellow Fluorescent Protein (YFP) and the Blue Fluorescent Protein (BFP) used in our lab have a 200 bp conserved region between them, which was ordered a g-block with complementary sticky ends to HBG Intron 2. The YFP and BFP only differ by approximately 50 bp in their non-conserved regions. In order to design a g-block that contained both YFP and BFP, the third base of every amino acid was wobbled in the YFP gene. (The wobble base was chosen based on human codon usage). The second g-block also included HBG Intron one between the two fluorescent exons, and complementary sticky ends with HBG Intron two.
If our guide system is not present in the cell, the conserved region, YFP and BFP will be spliced together. During translation, the stop codon at the end of YFP will cause the ribosome to fall off, resulting in a complete YFP and therefore yellow fluorescence will be made. When our guide system is introduced to the cells, it will cover the 5’ splice site on HBG Intron 2, causing the spliceosome to skip over the YFP exon. The conserved region and BFP will be spliced together, resulting in a complete BFP, and therefore blue fluorescence will be made.
