Difference between revisions of "Part:BBa K3905004: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.
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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.
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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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Latest revision as of 10:59, 30 September 2021


gen2 517-5p Toehold Switch


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 15
    Illegal XbaI site found at 53
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 15
    Illegal XbaI site found at 53
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 15
    Illegal XbaI site found at 53
  • 1000
    COMPATIBLE WITH RFC[1000]


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

Synthetic. Designed on NUPACK

References