Difference between revisions of "Part:BBa K2429051:Experience"

 
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===Applications of BBa_K2429051===
 
===Applications of BBa_K2429051===
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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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<br> </br>
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<br> </br>
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<br> </br>
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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 03:58, 2 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_K2429051

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

UNIQc47375bdcfe2a5b6-partinfo-00000001-QINU UNIQc47375bdcfe2a5b6-partinfo-00000002-QINU