Other
TSM_62

Part:BBa_K2482001:Design

Designed by: Jonathan Hiss   Group: iGEM17_Berlin_diagnostX   (2017-10-27)


T7-RBS-lacZ_Synthetic Toehold Switch TSM_62


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]


Design Notes

We followed the Series B design blueprint proposed in the Zika paper [1]. This design has the following characteristics:

  • a 36-nt long recognition site of which the first 25 nucleotides are unpaired (toehold domain)
  • a complementary 11-nt long bottom stem
  • a complementary 5-nt long top stem
  • 12-nt long hairpin loop containing the RBS (ribosomal binding site) and connecting the bottom & top stem
  • 3-nt long bulge which contains the start-codon for the translation of the reporter protein (confined between the top and bottom stem)
  • 22-nt long linking sequence, witch a variable & an invariable domain, attaching the reporter protein sequence (LacZ) to the bottom stem thus providing an open reading frame in combination with the 11-nt long bottom stem.

The sequence from bulge to bulge is fixed, while the sequence of the bottom stem is determined by the toehold-sensor target. In the linker region only the first of the 22 nucleotides are variable.

Predicted secondary structure (nupack.org)

We ranked the likelihood of success for every target-specific sensor using bioinformatic methods. Out of those candidates we selected the final sequence by in vitro screening.

Source

We ordered the original plasmid with the Zika-Sensor [1] off Addgene (ZIKV_Sensor_27B_LacZ, 75006) and changed the sensor sequence in front of the LacZ with an extension PCR using the following primers:

  • 1. 5'-GGACTTTAGAACAGAGGAGATAAAGATGCAAATTGAGTCAAACCTGGCGGCAGCGCAAAAG-3'
  • 2. 5'-GCGAATTAATACGACTCACTATAGGGAACACCCTACAAATTCGTGCTCTCCGACTCAATTTGGGACTTTAGAACAGAGGAGATAAAGATG-3'

References

  1. Pardee K, Green Alexander A, Takahashi Melissa K, Braff D, Lambert G, Lee Jeong W, et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell. 2016;165(5):1255-66.