Difference between revisions of "Part:BBa K3905008:Design"

 
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===Design Notes===
 
===Design Notes===
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We had to ensure that the toehold switch's sequence did not bind to the linker sequence deforming the structure, that the start codon was not exposed in the 'off' state and that the RBS held the correct formation in the 'on' state.
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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 13:13, 4 October 2021


T7 / gen2 517-5p Toehold Switch / Luciferase / Double Terminator


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 41
    Illegal XbaI site found at 79
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 41
    Illegal XbaI site found at 79
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 41
    Illegal XbaI site found at 79
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 922


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.

References