Difference between revisions of "Team:MIT/2mk-HBG"
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<h1 style="color:#f20253; text-align: center; font-size: 40px; line-height: 40px;">Experiments with 2 Exon mKate HBG Reporter</h1> | <h1 style="color:#f20253; text-align: center; font-size: 40px; line-height: 40px;">Experiments with 2 Exon mKate HBG Reporter</h1> | ||
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<p>We designed a reporter similar to the mKate FF4 reporter, except the intron has been changed.</p> | <p>We designed a reporter similar to the mKate FF4 reporter, except the intron has been changed.</p> | ||
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| − | <img src="https://static.igem.org/mediawiki/parts/0/03/2ex.png"> | + | <p>The mKate HBG reporter features the red fluorescent mKate gene split into two exons, with human beta globin (HBG) intron 2 in between these two exons. We used this split mKate 2-exon construct as a reporter construct that we used to determine if our dCas13 or Ms2 systems were successful in splicing out the HBG Intron 2. We used HBG Intron 2 for reasons outlined in our <a href= "https://2017.igem.org/Team:MIT/project">intron design </a> discussion. Upstream of these sequences is a either the constitutive hEF1a promoter or the DOX controlled TRE-tight promoter. </p> |
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| + | <center><img src="https://static.igem.org/mediawiki/parts/0/03/2ex.png"></center> | ||
<p>The figure above represents our construct. The sequence in between the exons represents HBG intron 2. The 3' splice site will be targeted by the guide-dCas13a complex (or guide-Ms2 complex). In the absence of our system, the intron will be spliced out and result in mRNA transcript with both exons included, and will lead to the creation of a red fluorescent protein. In the presence of our system, the RNA binding protein should block the splice site, and the second exon will be spliced out along with the intron. The fluorescent protein wouldn't be produced, and we should see less red fluorescence. </p> | <p>The figure above represents our construct. The sequence in between the exons represents HBG intron 2. The 3' splice site will be targeted by the guide-dCas13a complex (or guide-Ms2 complex). In the absence of our system, the intron will be spliced out and result in mRNA transcript with both exons included, and will lead to the creation of a red fluorescent protein. In the presence of our system, the RNA binding protein should block the splice site, and the second exon will be spliced out along with the intron. The fluorescent protein wouldn't be produced, and we should see less red fluorescence. </p> | ||
Revision as of 10:56, 1 November 2017
Experiments with 2 Exon mKate HBG Reporter
We designed a reporter similar to the mKate FF4 reporter, except the intron has been changed.
The mKate HBG reporter features the red fluorescent mKate gene split into two exons, with human beta globin (HBG) intron 2 in between these two exons. We used this split mKate 2-exon construct as a reporter construct that we used to determine if our dCas13 or Ms2 systems were successful in splicing out the HBG Intron 2. We used HBG Intron 2 for reasons outlined in our intron design discussion. Upstream of these sequences is a either the constitutive hEF1a promoter or the DOX controlled TRE-tight promoter.
The figure above represents our construct. The sequence in between the exons represents HBG intron 2. The 3' splice site will be targeted by the guide-dCas13a complex (or guide-Ms2 complex). In the absence of our system, the intron will be spliced out and result in mRNA transcript with both exons included, and will lead to the creation of a red fluorescent protein. In the presence of our system, the RNA binding protein should block the splice site, and the second exon will be spliced out along with the intron. The fluorescent protein wouldn't be produced, and we should see less red fluorescence.
