Part:BBa_K2254001

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Usage and Biology

The toehold trigger cloning tool is a part used by Hong Kong-CUHK 2017 team for convenient cloning and validation of toehold switch that detect specific sequence of RNA. After designing the trigger of a toehold switch in silico, user can insert the trigger into the part by Eco31I digestion followed by ligation.

To obtain the trigger insert, user can mix 2 DNA oligos (about 60nt): one oligo contains forward trigger sequence with AGGG at 5’ end, and one oligo contains reverse complement trigger sequence with AGTA at 5’ end. To allow convenient screening of correct clone, double digestion by Eco31I will remove the GFP gerenrator (I20260), and insertion of trigger will result in colonies that don’t express GFP, whereas ligation of single digested plasmid will give GFP colonies.

When the toehold switch hairpin is linearized by its orthogonal trigger RNA, RBS will be exposed, allowing the translation of downstream mRFP reporter gene.

Characterisation

Experimental:
Characterisation

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 39
  Illegal NheI site found at 62
  
• 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 27
  Illegal BsaI.rc site found at 738
  

Functional Parameters

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