Part:BBa_K5106010

Toehold AND gate, T7 promoter and terminator, LacZ reporter

Basic toehold AND gate (BBa_K5106007 under a T7 promoter and terminator, regulating LacZ reporter gene, to test the basic construct in vitro in a cell-free PURExpress system.

Usage and Biology

This composite part consists of the basic toehold AND gate (BBa_K5106007), under control of a T7 promoter and terminator, and the LacZ reporter gene. To test the whether the designed toehold AND gate 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 toehold switch AND gate, 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 first qualitative test (Figure 1), the AND gate showed a clear colour change when both trigger RNAs were added, indicating the gate can regulate translation based on the presence of the two trigger RNAs. We also observed leakiness, since the tube without trigger RNA shows an orange colour, instead of the yellow we see in the negative control.

Quantitative in vitro test

To determine how the AND gate functions over time, we performed a quantitative in vitro measurement on samples with no trigger RNA, with either one of the two trigger RNAs and with both triggers present (Figure 2). For this, 1 μL trigger RNA (5 μM) 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 of the diluted samples using a plate reader.

Figure 2: Absorption of Chlorophenol Red at 570 nm over time. Cell-free reactions (PURExpress) of the AND gate 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. Solid lines indicate that the samples included 1 μL trigger RNA (5 μM), dashed lines indicate that no trigger RNA was added. T1 & T2 – both trigger RNAs were added; T1 – only trigger RNA 1 was added; T2 – only trigger RNA 2 was added; No trigger – no trigger 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).

In this measurement, we observe that the toehold switch AND gate shows an increase in absorption over time in the presence of both triggers, indicating that the AND gate was indeed activated by RNA. However, we also observed notable leakiness in the system with all the samples producing the same level of absorption after 180 minutes. However, the difference between the presence and absence of trigger RNAs between 80 and 120 minutes was still considerable. This indicates that after two hours, the toehold switch AND gate is less applicable to a diagnostic test due to increased chances of false positives. The source of leakiness requires further research. This data was also fitted to our continuous ODE model to look at the dynamics of the system. This result, coupled to the quantitative measurement of the MS-specific toehold switches, also indicates that it may be necessary to read the test output directly after incubation to ensure an accurate test result. In the final test, the output is based on the colour change so no imagers are necessary. To fully characterise the toehold switch and AND gate output over time, we could investigate alternatives to absorption in the future, such as UV-Vis or colourimetry, to visualise the differences between the presence and absence of trigger (mi)RNA more clearly. 

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]