T7-RBS-lacZ_Synthetic Toehold Switch TSM_156
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
We followed the Series B design blueprint proposed in the Zika paper . 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.
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.
We ordered the original plasmid with the Zika-Sensor  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'-GGACTTTAGAACAGAGGAGATAAAGATGAGCACGAATTTAAACCTGGCGGCAGCGCAAAAG-3'
- 2. 5'-GCGAATTAATACGACTCACTATAGGGTATCCATTGCTAGAGAACACCCTACAAATTCGTGCTGGACTTTAGAACAGAGGAGATAAAGATG-3'
- 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.