Part:BBa_K2206002

Toehold switch for hsa-miR-15b-5p with GFPmut3b

Toehold switches are synthetic riboregulators that regulate gene expression post-transcriptionally. Gene expression can be activated in the presence of a cognate single stranded RNA molecule that contains an arbitrary sequence (the trigger RNA). The trigger RNA binds to the switch through base pairing, causing a conformational change that results in translation of the downstream protein coding region.

This part codes for a toehold switch that contains a region that is complementary to the microRNA hsa-miR-15b-5p (the trigger RNA). The toehold switch is activated by hsa-miR-15b-5p and regulates production of GFPmut3b. The fluorescence intensity from GFPmut3b is proportional to the number of toehold switches activated (as the more switches activated, the greater the amount of GFPmut3b is produced), thus indicating the levels of hsa-miR-15b-5p present (as the more microRNA there is, the greater the number of switches activated). This part can therefore be used to quantify the levels of hsa-miR-15b-5p.

The transcripts levels produced by this part are toxic to E. coli. Therefore, we recommend the use of an inducible non-leaky promoter to allow for amplification in E. coli. For reference, we found BBa_K808000 suitable for expressing this part.

This part contains a strong RBS sequence.

NUPACK Structure Analysis

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 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 764