Part:BBa_K5106006

Toehold switch C for detection of hsa-miR-484 with T7 promoter and terminator, and LacZ reporter

MS toehold C (BBa_K5106003) 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_K5106003), 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.

Figure 1: Cell-free expression (PURExpress) of toehold switch constructs in 2 μL reaction volumes. To the samples in the top row, no trigger miRNA was added. To the samples in the bottom row, 0.5 μL miRNA was added. From left to right: negative control; toehold switch 1 (not used); toehold switch 2(not used); MS-specific toehold switch A; MS-specific toehold switch B; MS-specific toehold switch C; toehold switch AND gate; positive control. A red box is place around the samples of MS toehold switch C, discussed on this part. For more details of this experiment, take a look on the website of miRADAR (WageningenUR 2024).

After two hours, 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 conversion of Red-β-D-Galactopyranoside to chlorophenol red by β-galactosidase. For the part containing MS toehold switch C, no colour change wasobserved (Figure 1), indicating that the toehold switch cannot be activated by hsa-miR-484.

Quantitative in vitro test

In addition to this qualitative test, we performed a quantitative measurement to evaluate the performance of the toehold switch. For this 1 μL trigger miRNA (5μM) was added to 5 μL PURExpress containing ~30 ng of DNA template. The tubes were incubated at 37 °C for two hours. Every 20 minutes, a 0.5 μL sample was taken and diluted in 75 μL nuclease free-water. We measured the absorption at 570 nm to measure the product (chlorophenol red), and 415 nm to measure the substrate (Red-β-D-Galactopyranoside) of β-galactosidase, which is translated after activation of the toehold switch.

Figure 2: Left) Absorption of Chlorphenol Red at 570 nm over time. Right) Absorption of Red-β-D-Galactopyranoside at 415 nm over time. Cell-free reactions (PURExpress) of toehold switch A were assembled in a 5 μL volume. At several timepoints, a 0.5 μL sample was taken and diluted in nuclease-free water, after which the absorption was measured. The solid lines indicate the samples that the samples included 1 μL trigger miRNA (5 μM), dashed lines indicate the samples that no trigger miRNA was added. Reactions were performed in triplicate. Error bars represent the standard deviation. For more details of this experiment, take a look on the website of miRADAR (WageningenUR 2024).

The quantitative measurement (Figure 2) showed similar results as the qualitative test (Figure 2). In both measurements, no change in absorbance over time was observed, and no difference between the presence or absence of trigger RNA was observed either. This indicates that this toehold switch indeed does not work. Toehold switch C was designed in a different way as the other toeholds designed by miRADAR (BBa_K5106004 and BBa_K5106005). These other toeholds, designed using our toehold designer model, did show an output signal. It is therefore advised to use this tool for design of miRNA toeholds switches.

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]

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.