Difference between revisions of "Part:BBa K2429035:Experience"

 
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===Applications of BBa_K2429035===
 
===Applications of BBa_K2429035===
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<h1 style="color:#ff0000; text-align: center; font-size: 40px; line-height: 40px;">Experiments with 3 Exon mKate HBG Reporter</h1>
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<p>After designing our 2-exon mKate HBG reporter, which only showed mKate fluorescent knockdown, we needed a positive reporter. We designed two 3-exon reporters, one of which is what we called the <b>3 Exon mKate HBG Reporter.</b> </p>
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<h3> 3-exon mKate Fluorescent Reporter </h3>
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<p> The reporter was designed to contain mKate exon one, HBG Intron two, the REST-4 included exon from our <a href= "http://2017.igem.org/Team:MIT/REST"> disease model,</a> HBG Intron one, and mKate exon two.</p>
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<img src="https://static.igem.org/mediawiki/parts/9/95/3-mk.png">
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<p>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 <a href= "http://2017.igem.org/Team:MIT/project"> chosen </a> based on the literature.  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. </p>
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<img src="https://static.igem.org/mediawiki/parts/b/b7/Case1.png">
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<p>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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<img src="https://static.igem.org/mediawiki/parts/f/f2/Case2.png">
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<h2>Results</h2>
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<h3> Verifying the construct </h3>
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<h4> mKate Titration </h4>
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<p>Before being able to test our guides against our newly designed reporter, we needed to ensure it was exhibiting proper behavior. To that end, we ran an titration on the 3-exon mKate-HBG construct. We expected that in absence of our system, there would be no mKate fluorescence, as the stop codon in the REST exon will be included. If our system is present, we expect that levels of red output will rise with increase in reporter construct. These expected results can be seen below. The different colored lines corrispond to different <a href= "http://2017.igem.org/Team:MIT/HowtoreadaCytoflowgraph"> transfection bins</a> </p>
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<center><img src="https://static.igem.org/mediawiki/2017/6/6e/Expected_output_3-exon_mkate.png" alt="Expected output" style= "width:50%;"></center>
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<p> In our experiment, we varied the amounts of 3-exon mKate-HBG from 10 to 500 ng. We also transfected Guide3 with dCas13a, and ASO2+ with Ms2 at optimized amounts from earlier experiments. (For a detailed explanation of how to plan a mammalian transfection <a href= "https://static.igem.org/mediawiki/2017/6/63/Planning_Mamallian_Transfections_%C2%B7_Benchling.pdf"> click here</a>) </p>
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<center><h4>mKate Titration for 3-exon mKate-HBG</h4></center>
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<center><img src="https://static.igem.org/mediawiki/2017/d/df/3exon_mkate_plain.svg" alt="Figure ASO3vsmkateamt_red" style= "width:50%;"></center>
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<p><i>3-exon mKate-HBG reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the <a href= "http://2017.igem.org/Team:MIT/HowtoreadaCytoflowgraph"> transfection bins</a> of each result.</i></p>
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<p>We expected that the reporter would produce no fluorescence, but our results here show normal output levels for mKate across the reporter concentrations. Additionally, as shown below adding dCas13a and Ms2 have no effect on the red output.</p>
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<center><h4>3-exon mKate-HBG titration with ASO2+ &nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp; &nbsp;&nbsp; 3-exon mKate-HBG titration with Guide3 </h4></center>
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<img src="https://static.igem.org/mediawiki/2017/8/87/3exon_mkate_ms2_ASO2plus_red.svg" alt="Expected output" align= "left" style= "width:49%;"><img src="https://static.igem.org/mediawiki/2017/b/b9/3exon_mkate_cas_guide3_red.svg" alt="Expected output"  align= "right" style= "width:49%;">
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<center><i>DOX Concentrations (5-500uM) vs. the amount of red fluorescence (AU) for ASO1 (left) and plain mKate(right). The color of the line indicate the <a href= "http://2017.igem.org/Team:MIT/HowtoreadaCytoflowgraph"> transfection bins</a> of each result.</i></center>
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<strong>Unfortunately, we were unable to test any of our constructs against this reporter, because the reporter was producing red in the control condition.</strong> '
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<p>Our next step is to debug our reporter. We suspect the problem may be with the intron we chose, or the way we built that intron, because the mKate-ff4 was outputting fine.</p>
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===User Reviews===
 
===User Reviews===

Latest revision as of 18:32, 1 November 2017


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2429035

Experiments with 3 Exon mKate HBG Reporter

After designing our 2-exon mKate HBG reporter, which only showed mKate fluorescent knockdown, we needed a positive reporter. We designed two 3-exon reporters, one of which is what we called the 3 Exon mKate HBG 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.



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. 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.




Results

Verifying the construct

mKate Titration

Before being able to test our guides against our newly designed reporter, we needed to ensure it was exhibiting proper behavior. To that end, we ran an titration on the 3-exon mKate-HBG construct. We expected that in absence of our system, there would be no mKate fluorescence, as the stop codon in the REST exon will be included. If our system is present, we expect that levels of red output will rise with increase in reporter construct. These expected results can be seen below. The different colored lines corrispond to different transfection bins

Expected output

In our experiment, we varied the amounts of 3-exon mKate-HBG from 10 to 500 ng. We also transfected Guide3 with dCas13a, and ASO2+ with Ms2 at optimized amounts from earlier experiments. (For a detailed explanation of how to plan a mammalian transfection click here)

mKate Titration for 3-exon mKate-HBG

Figure ASO3vsmkateamt_red

3-exon mKate-HBG reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bins of each result.

We expected that the reporter would produce no fluorescence, but our results here show normal output levels for mKate across the reporter concentrations. Additionally, as shown below adding dCas13a and Ms2 have no effect on the red output.

3-exon mKate-HBG titration with ASO2+                                          3-exon mKate-HBG titration with Guide3

Expected outputExpected output















DOX Concentrations (5-500uM) vs. the amount of red fluorescence (AU) for ASO1 (left) and plain mKate(right). The color of the line indicate the transfection bins of each result.
Unfortunately, we were unable to test any of our constructs against this reporter, because the reporter was producing red in the control condition. '

Our next step is to debug our reporter. We suspect the problem may be with the intron we chose, or the way we built that intron, because the mKate-ff4 was outputting fine.

User Reviews

UNIQf2b6bda87ed854e4-partinfo-00000001-QINU UNIQf2b6bda87ed854e4-partinfo-00000002-QINU