Regulatory

Part:BBa_K2206012

Designed by: Sammy Lovat   Group: iGEM17_CLSB-UK   (2017-10-26)
Revision as of 00:04, 31 October 2017 by SLovat (Talk | contribs)


Toehold switch for hsa-miR-15b-5p (second series)

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.

The trigger sequence can be split into different RNA molecules. Colocalization of the split trigger parts results in a complete trigger RNA that is capable of activating the toehold switch.

BBa_K2206013 and hsa-miR-15b-5p contain roughly half of the trigger sequence each. Colocalization of the split trigger RNA sequences results in a functional trigger RNA, capable of activating the toehold switch. The formation of the duplex occurs through a highly specific binding step which results in single-base mismatch specificity. This part codes for a toehold switch that has a region complementary to the complete trigger RNA sequence formed of BBa_K2206013 and hsa-miR-15b-5p Therefore, this part can be used to regulate the expression of any protein in response to presence of both BBa_K2206013 and hsa-miR-15b-5p.

For more information, see the section on second series of toehold switches on the design page of CLSB-UK's 2017 wiki - http://2017.igem.org/Team:CLSB-UK/Design.

We recommend using this part to regulate the expression of a reporter protein. This allows for quantification of hsa-miR-15b-5p. Our simulations show that this part would be better suited for hsa-miR-15b-5p quantification than our original toehold switch BBa_K2206000 due to its increased specificity and on state activity.

We found that the transcripts produced by our other toehold switch parts were toxic to E.coli. We therefore recommend using this part with a non-leaky promoter. For reference, we found the part BBa_K808000 to be too leaky.

This part contains a strong RBS sequence.

NUPACK Structure Analysis

anti-miRNAmiRNAFree energy of secondary structure: -18.57 kcal/molMFE structure at 35.0°CACGU Free energy of secondary structure: -15.27 kcal/molMFE structure at 35.0°CACGUTrigger Binding SiteStart CodonRBS Free energy of secondary structure: -68.61 kcal/molMFE structure at 35.0°CACGUStart CodonTrigger Binding Siteanti-miRNAmiRNARBS

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None