Difference between revisions of "Part:BBa K3905012:Design"

 
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===Design Notes===
 
===Design Notes===
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Had to ensure that only half the bases were bound with the complementary miRNA 517-5p and the other half would remain unbound to serve as a trigger. Needed to have a base trigger different to that of the anti-MiRNA 210-3p to ensure that these miRNAs wouldn't bind to each other. Had to ensure that the anti-Mirna alone wouldn't trigger the toehold switch.
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At first, we designed our switches base by base, checking the minimum free energy (MFE) structure, using NUPACK, to ensure that our switch had a strong hairpin structure in the ‘off’ state and was properly unfolded in its ‘on’ state. As well as preventing the occurrences of secondary binding in the trigger site or in the linker. We were successful in designing multiple switches that had MFE structures fulfilling this criteria, but multiple bases had low probabilities of being in their respective positions. Therefore, we used the NUPACK API to generate a Python program to test a randomly generated list of one-hundred-thousand linker regions (which did not contain any stop or start codons) and simulate 1000 Boltzmann samples for each, showing which switch had the highest probability of fulfilling our requirements.
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===Source===
 
===Source===

Latest revision as of 13:15, 4 October 2021


T7 / gen3 210-3p-517-5p AND-Gate / Luciferase / Double Terminator / T7 / gen3 210-3p-517-5p AND-Gate


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 108
    Illegal SapI.rc site found at 932


Design Notes

At first, we designed our switches base by base, checking the minimum free energy (MFE) structure, using NUPACK, to ensure that our switch had a strong hairpin structure in the ‘off’ state and was properly unfolded in its ‘on’ state. As well as preventing the occurrences of secondary binding in the trigger site or in the linker. We were successful in designing multiple switches that had MFE structures fulfilling this criteria, but multiple bases had low probabilities of being in their respective positions. Therefore, we used the NUPACK API to generate a Python program to test a randomly generated list of one-hundred-thousand linker regions (which did not contain any stop or start codons) and simulate 1000 Boltzmann samples for each, showing which switch had the highest probability of fulfilling our requirements.

Source

This is synthetic, designed in silico, using NUPACK.

References