Detection Tool: Toehold Switch
Research
Our team aim to design a modular device for oral cancer detection, and hope that it can have the following features:
- Non-invasive:
the sample collection and the detection procedure should not involve the introduction of instruments into the body.
- Accessible:
the tool can be easily conducted by the general public.
- Quantitative:
the device can provide quantitative data for more precise and objective detection.
Therefore, we thought about finding a biomarker that can be precisely measured and provide quantitative data for oral cancer. Salivary miRNA can be a perfect biomarker for our project.1, 2, 3, 4 Firstly, the miRNA expression has a significant difference between OSCC (oral squamous cell carcinoma) and control tissues. For instance, miR-21, miR-31, and miR-146 are proven to be upregulated in OSCC.1 Secondly, several OSCC-related miRNAs are secreted in saliva5 , making them very useful for non-invasive clinical applications.
To measure the salivary miRNAs, we adopt a systematic ribocomputing devices, toehold switch.6, 7 According to the research, a toehold switch is a secondary structure RNA with a hairpin loop that can regulate translation. Only with the specific RNA trigger can its structure be opened and start the translation of the following reporter.
Based on the research, We used toehold switches as our biosensors, designing a device that can detect oral cancer-related miRNAs as well as generate measurable signals.
Design & Build
Consulting with the previous iGEM team 2019 EPFL, whose project was also about toehold switches, we were suggested to perform some tests on the existing toehold switches. This could help us understand the mechanism of them. Therefore, we ordered a set of banana toehold switch sensor (Biobits),8 which is a ready-to-use modular education kit. This banana toehold switch sfGFP BBa_K3431050 can be triggered by the RNA of banana and expresses sfGFP BBa_K3431031 as its reporter.
Figure. 1. Banana toehold switch sfGFP. (A) Composite part: pT7-BTS-sfGFP-trT7/pSB1C3 (BBa_K3431050), (B) The legend of the basic part elements and the gene names.
To produce reporter proteins in a cell-free system, we used the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs). The kit is comprised of two kinds of reagents: A solution contains tRNA and small components such as amino acids and rNTP, and B solution contains ribosomes and protein components such as T7 RNA polymerase, amino-acyl synthetase, or energy regeneration enzyme. In short, they provide all necessary components needed for in vitro transcription and translation, which are purified from E. coli. Besides the basic components in the kit, the RNase inhibitor (1:40, NEB Murine RNAse Inhibitor or Thermofisher Ribolock) is also recommended to be added, as it can considerably enhance protein production. All the reagents required are listed below (Tab.1). To see the detailed protocol and our experimental adjustment, check the
Protocol and
Measurement .
|
Order
|
Reagent
|
Amount
|
|
1
|
DNase/RNase free water
|
till 5 μl
|
|
2
|
Solution A
|
2 μl
|
|
3
|
Solution B
|
1.5 μl
|
|
4
|
RNase inhibitor
|
0.2 μl
|
|
5
|
Toehold switch
|
50 ng
|
|
6
|
Trigger
|
0 or 50 ng
|
Table 1. Reagent table for PURExpress protein synthesis kit.
Test & Improve
We carried out a functionality test of banana toehold switch sfGFP, measuring the real-time fluorescence of GFP within the 80 minutes protein production process. The result is shown below.
Figure 2. The relative fluorescence of the sfGFP in the 80 minutes protein production process. The relative fluorescence was measured by the Synergy™ H1 Hybrid Multi-Mode Microplate Reader. The green line refers to the conditions with banana triggers, while the blue line refers to the conditions without a banana trigger.
As seen, the sfGFPs would be highly expressed when the banana triggers existed, while they would be inhibited when there was no trigger in the environment. This suggested the regulatory function of the toehold switch.
From the experiment, we understood the basic mechanism of the toehold switch and identified the most optimal condition for testing toehold switches, which would be repeatedly used in our further experiments.
However, we also realized that the process of GFP detection is too inconvenient for the general public, as it requires a plate reader or a spectrometer. As a result, we wanted to replace the sfGFP with another reporter that could be easily measured by anyone.
