Part:BBa_K1586003:Experience

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Team Berlin diagnostX characterised the switch in vitro. The switch showed no activity.

Because the sensor was not available for order through iGEM, we ordered the sensor and its respective trigger RNA with an appended T7-Promotor for our experiments. The ordered DNAs were:

• Sensor: 5'-GCGAATTAATACGACTCACTATAGGGGGACCCCGGCCTGACGAATATAAGTCGTTTAAAAAAAAAAATATAATTAAAGAGGAGAAACGAATGATATTCGTCTTAAGTGGTAGTGGTAGTGGTGAATGCGAATGCATAATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAAGCTAGTTATTGCTCAGCGG-3'
• Trigger: 5'-GCGAATTAATACGACTCACTATAGGGATGGCAGAGATGAAGACCGATGCCGCTACCCTCGCGCAGGAGGCAGGTAATTTCGAGCGGATCTCCGGCGACCTGAAAACCCAGATCGACCAGGTGGAGTCGACGGCAGGTTCGTTGCAGGGCCAGTGGCGCGGCGCGGCGGGGACGGCCGCCCAGGCCGCGGTGGTGCGCTTCCAAGAAGCAGCCAATAAGCAGAAGCAGGAACTCGACGAGATCTCGACGAATATTCGTCAGGCCGGCGTCCAATACTCGAGGGCCGACGAGGAGCAGCAGCAGGCGCTGTCCTCGCAAATGGGCTTCTGAGCTAGTTATTGCTCAGCGG-3'

We adapted our standard cell free expression protocol to work with GFP. Each well contained 4µl of reaction volume which consisted of:

• 3µl cell free expression system (PURExpress, NEB) with RNA or water
  • the composition of the cell free expression system followed the proportions recommended by the manufacturer (51% Solution A, 39% Solution B, 5% RNAsin, 5% H2O (as replacement for the Chlorophenol Red-β-D-galactopyranoside)
  • either RNA (concentrated for a final concentration of 3000nM in the total volume of 4µl) or an equal volume of nuclease free water was added to the cell free expression system.
• 1µl DNA with a concentration of 12nM which makes for a final concentration of 3nM DNA per 4µl reaction
• as control we used GFP with a T7_Promotor appended in front

Characterisation of BBa K1586003 in vitro

UNIQ3991612ec8bb9d5f-partinfo-00000000-QINU UNIQ3991612ec8bb9d5f-partinfo-00000001-QINU