Composite

Part:BBa_K2206009

Designed by: Sammy Lovat   Group: iGEM17_CLSB-UK   (2017-10-02)
Revision as of 23:31, 30 October 2017 by SLovat (Talk | contribs)

Toehold switch for hsa-mir-27b-3p with luciferase and inducible promoter

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-27b-3p (the trigger RNA). The toehold switch is activated by hsa-miR-27b-3p and regulates production of luciferase. The luminescence intensity from luciferase is proportional to the number of toehold switches activated (as the more switches activated, the greater the amount of luciferase is produced), thus indicating the levels of hsa-miR-27b-3p 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-27b-3p.

The transcripts produced by this part are toxic to E. coli. We recommend the use of an inducible non-leaky promoter to allow for amplification in E. coli. For reference, we found Bba_K808000 was too leaky.

This part is too large to be inserted into the pSB1C3 plasmid backbone in one ligation step. To insert it into the plasmid, the individual parts that make up this construct must be ligated into the plasmid backbone separately.

This part contains a strong RBS sequence.

NUPACK Structure Analysis

Start codonRBSTrigger Binding SiteFree energy of secondary structure: -18.80 kcal/molACGUMFE structure at 37.0°CTrigger Binding SitemiRNAStartcodonRBSFree energy of secondary structure: -46.98 kcal/molACGUMFE structure at 37.0°C

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 2096


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Parameters
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