Difference between revisions of "Part:BBa K4245210:Design"

 
 
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===Design Notes===
 
===Design Notes===
 
T7 regulated miRNA1-broccoli-spacer
 
T7 regulated miRNA1-broccoli-spacer
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As we were testing Rolling Circle Transcription (RCT) with our hsa-miR-1-3p RCT Padlock Probe (<partinfo>BBa_K4245201</partinfo>), we were unable to obtain significant fluorescence results (see Fig. 1).
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[[File:BBa_K4245210_RCT_with_10_uM_dye.jpg|thumb|center|500px|<i>Figure 1.  Non-significant fluorescence results from running RCT with hsa-miR-1-3p RCT Padlock Probe (BBa_K4245121). </i>]]
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After consulting with Dr. Jaffrey, we realized that the folding of the spacer and aptamer sequences might have affected the fluorescence of our RCT reactions. We decided to check this by using a folding simulator (see Fig. 2)
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[[File:BBa_K4245210_Padlock_Folding_Using_RNAfold.jpg|thumb|center|500px|<i>Figure 2.  The folding of the transcript sequence, predicted using RNAfold</i>]]
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In order to determine the effect of the folding on fluorescence alone, and eliminate the factors introduced by the RCT reaction, we designed <partinfo>BBa_K4245210</partinfo>, the T7 regulated miRNA1-broccoli-spacer part.
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This part produces the sequence for <partinfo>BBa_K4245006</partinfo> the hsa-miR-1-3p, <partinfo>BBa_K3380153</partinfo>, the Broccoli fluorescent RNA aptamer, and <partinfo>BBa_K4245160</partinfo>, the Padlock spacer sequence, which is analogous to one repeat in the transcript of a successful RCT reaction.
  
  

Latest revision as of 14:36, 10 October 2022


T7 regulated miRNA1-broccoli-spacer


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 137
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

T7 regulated miRNA1-broccoli-spacer
As we were testing Rolling Circle Transcription (RCT) with our hsa-miR-1-3p RCT Padlock Probe (BBa_K4245201), we were unable to obtain significant fluorescence results (see Fig. 1).

Figure 1. Non-significant fluorescence results from running RCT with hsa-miR-1-3p RCT Padlock Probe (BBa_K4245121).


After consulting with Dr. Jaffrey, we realized that the folding of the spacer and aptamer sequences might have affected the fluorescence of our RCT reactions. We decided to check this by using a folding simulator (see Fig. 2)

Figure 2. The folding of the transcript sequence, predicted using RNAfold


In order to determine the effect of the folding on fluorescence alone, and eliminate the factors introduced by the RCT reaction, we designed BBa_K4245210, the T7 regulated miRNA1-broccoli-spacer part.
This part produces the sequence for BBa_K4245006 the hsa-miR-1-3p, BBa_K3380153, the Broccoli fluorescent RNA aptamer, and BBa_K4245160, the Padlock spacer sequence, which is analogous to one repeat in the transcript of a successful RCT reaction.


Source

T7 regulated miRNA1-broccoli-spacer

References