Difference between revisions of "Part:BBa K3328009"
 
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binding to toehold switch (Bba_K3328008) to express the downstream gene
 
binding to toehold switch (Bba_K3328008) to express the downstream gene
  
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The stability of RNA is a key factor affecting the mechanism of its interaction. Therefore, we hope to optimize the ON/OFF ratio of the toehold switch by improving the stability of trigger RNA. After consulting many pieces of literature, we learned that adding hairpin structure at the 5 'end of RNA could prevent the degradation mediated by RNase, thus improving the stability of RNA. Therefore, we tried to add the hairpin structure at the 5' end of triggers. We used NUPACK and RNAfold to analyze the structure of the trigger with the 5’ end hairpin, and then used NUPACK to analyze the combination of triggers with the 5’ end hairpin and switches.
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===Design===
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Toehold switch is composed of cis-acting element RNA hairpins and trans-acting factor trigger RNA. The binding of a trigger RNA to the toehold sequence allows for a branch migration process, exposing AUG and RBS for translation initiation.
  
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https://2020.igem.org/wiki/images/thumb/b/b2/T--OUC-China--result_fig11.jpg/752px-T--OUC-China--result_fig11.jpg
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===Result===
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<html>
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(A) The secondary structure of 5’ hairpins analyzed using NUPACK. (B) The secondary structure of 5’ hairpins combining with trigger analyzed using NUPACK.
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<head>
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<meta charset="utf-8">
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<body>
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<img src="https://2020.igem.org/wiki/images/7/7e/T--OUC-China--result_fig2_th.jpg" style="width: 80%;">
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</div>
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<p>
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Compared with the blank control (IPTG=0 M, aTc=0 mg/mL), the fluorescence of GFP was low when only the promoter before toehold sequence was turned on (IPTG=0.1 M, aTc=0 mg/mL). This indicates that the toehold has the advantage of low leakage. When the trigger was expressed (IPTG=0.1 M, aTc=0.25 mg/mL), it showed a high GFP fluorescence of up to 32-fold due to the destruction of toehold hairpin structure. Error bar: SD (n=9).
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</p>
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</body>
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</html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 09:26, 27 October 2020


trigger of ON-switch

binding to toehold switch (Bba_K3328008) to express the downstream gene

Design

Toehold switch is composed of cis-acting element RNA hairpins and trans-acting factor trigger RNA. The binding of a trigger RNA to the toehold sequence allows for a branch migration process, exposing AUG and RBS for translation initiation.

Result

Compared with the blank control (IPTG=0 M, aTc=0 mg/mL), the fluorescence of GFP was low when only the promoter before toehold sequence was turned on (IPTG=0.1 M, aTc=0 mg/mL). This indicates that the toehold has the advantage of low leakage. When the trigger was expressed (IPTG=0.1 M, aTc=0.25 mg/mL), it showed a high GFP fluorescence of up to 32-fold due to the destruction of toehold hairpin structure. Error bar: SD (n=9).

Sequence and Features


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