Part:BBa_K5106004
Toehold switch A for detection of hsa-miR-484 with T7 promoter and terminator, and LacZ reporter
MS toehold A (BBa_K5106001 under control of a T7 promoter and terminator, regulating expression of a LacZ reporter gene, in order to test the toehold functionality in vitro in a cell-free PURExpress system.
Usage and Biology
This composite part consists of the basic toehold switch part (BBa_K5106001), under control of a T7 promoter and terminator, and the LacZ reporter gene. To test the whether the designed toehold switch works, a plasmid expressing this composite part was made, and expressed in a PURExpress cell-free system. This is a reconstituted form of cell-free expression that works by isolating the necessary components, such as ribosomes and amino acids, purifying them, and assembling them into a reaction mixture.1
Qualitative in vitro test
To first check whether the toehold switch was activated by the trigger miRNA hsa-miR-484 (BBa_K5106000), we performed a test in 2 μL PURExpress, with ~25 ng plasmid containing the toehold switch (this composite part). Besides other toehold switches during this test, a negative control, containing no DNA template, and a positive control, containing only lacZ under the control of the T7 promoter and terminator, were included as well. In addition, 0.5 μL miRNA hsa-miR-484 (5 μM) was added to part of the samples. The tubes were incubated at 37 °C for two hours.
miRADAR (WageningenUR 2024).
After two ours, β-galactosidase (LacZ) performed as expected, since no colour change from yellow to purple was observed in the negative control, whereas the colour change was observed in the positive control (Figure 1). This change is caused by For the part containing MS toehold switch A, a clear colour change can also be observed (Figure 1), indicating that the toehold switch can indeed be activated by hsa-miR-484, resulting in further translation of the downstream protein coding region.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
1. Shimizu, Yoshihiro, Akio Inoue, Yukihide Tomari, Tsutomu Suzuki, Takashi Yokogawa, Kazuya Nishikawa, and Takuya Ueda. ‘Cell-Free Translation Reconstituted with Purified Components’. Nature Biotechnology 19, no. 8 (August 2001): 751–55. https://doi.org/10.1038/90802.
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