Regulatory

Part:BBa_K2206001

Designed by: Sammy Lovat   Group: iGEM17_CLSB-UK   (2017-10-02)
Revision as of 10:26, 7 October 2021 by Ng849 (Talk | contribs)

Toehold switch for hsa-miR-27b-3p

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). In the presence of hsa-miR-27b-3p, translation of the downstream protein coding region occurs.

This part can be used to regulate the expression of any protein in response to the levels of hsa-miR-27b-3p present in a system.

The transcripts 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 was too leaky.

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

Information contributed by City of London UK (2021)

ToeholdTools.png

This toehold switch was characterized in silico using the ToeholdTools project that our team developed. See https://github.com/lkn849/thtools for more information.

Metadata:

  • Group: City of London UK 2021
  • Author: Lucas Ng
  • Summary: Used our software ToeholdTools to investigate the target miRNA specificity and activation of this part.

Raw data:

This contribution was autogenerated by the script contrib.py, available at https://github.com/lkn849/thtools/tree/master/registry.

This switch was designed to detect the miRNA hsa-miR-27b-3p at a temperature of 37. We tested it against every Homo sapiens RNA in miRBase and our analysis shows that it is best used to detect hsa-miR-6752-5p.

With hsa-miR-6752-5p at 37°C, the switch has a specificity of 20 ± 100 % and an activation of 15 ± 5 %. These values represent 95% confidence limits (z=1.96).

The temperature–activation–specificity relationship is shown here:

BBa K2206001 graph.png

Error bars represent the standard error (SE). The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's SE.

Caveats:

  • As per the above, we cannot confirm that this switch detects the desired RNA sequence.
  • Although at the indented usage temperature of {celsius}°C this switch best detected {inferred_target_name}, this RNA did not appear as a best target for any of separate tests taken at the temperatures tested in the above graph ({temperature_range}). Therefore, we cannot confirm the reliability of this switch.

We do not recommend this part for future usage.

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]


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