iGEM 2020 — Team CSMU_Taiwan

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# Team:CSMU Taiwan/Notebook

Home - Brand

[![](https://static.igem.org/mediawiki/2020/1/1f/T--CSMU_Taiwan--logo_light_darkdark.png) CSMU 2020](https://2020.igem.org/Team%3ACSMU_Taiwan)

* Team

  [**Team members**](https://2020.igem.org/Team%3ACSMU_Taiwan/Team)
  [**attributions**](https://2020.igem.org/Team%3ACSMU_Taiwan/Attributions)
  [**Collaborations**](https://2020.igem.org/Team%3ACSMU_Taiwan/Collaborations)
  [**Partnership**](https://2020.igem.org/Team%3ACSMU_Taiwan/Partnership)
* Project

  [**Description**](https://2020.igem.org/Team%3ACSMU_Taiwan/Description)
  [**Engineering Success**](https://2020.igem.org/Team%3ACSMU_Taiwan/Engineering)
  [**Model**](https://2020.igem.org/Team%3ACSMU_Taiwan/Model)
  [**Measurement**](https://2020.igem.org/Team%3ACSMU_Taiwan/Measurement)
  [**Proof of Concept**](https://2020.igem.org/Team%3ACSMU_Taiwan/Proof_Of_Concept)
  [**Proposed Implementation**](https://2020.igem.org/Team%3ACSMU_Taiwan/Implementation)
  [**Protocol**](https://2020.igem.org/Team%3ACSMU_Taiwan/Protocol)
  [**Notebook**](https://2020.igem.org/Team%3ACSMU_Taiwan/Notebook)
* Parts

  [**Parts Overview**](https://2020.igem.org/Team%3ACSMU_Taiwan/Parts)
  [**Contribution**](https://2020.igem.org/Team%3ACSMU_Taiwan/Contribution)
  [**Improve**](https://2020.igem.org/Team%3ACSMU_Taiwan/Improve)
* [safety](https://2020.igem.org/Team%3ACSMU_Taiwan/Safety)
* Human Practices

  [**Human Practice**](https://2020.igem.org/Team%3ACSMU_Taiwan/Human_Practices)
  [**Science Communication**](https://2020.igem.org/Team%3ACSMU_Taiwan/Education)
  [**sustainable**](https://2020.igem.org/Team%3ACSMU_Taiwan/Sustainable)
  [**Entrepreneurship**](https://2020.igem.org/Team%3ACSMU_Taiwan/Entrepreneurship)
* [awards](https://2020.igem.org/Team%3ACSMU_Taiwan/Awards)

## Notebook

## Here lies the diary of our journey......

## May

##### [Evaluation of protein synthesis kit](#accordion-2 .item-2)

05/20 Transformation, Toehold Test
 05/21 Agar plate preparation, Culturing bacteria, toehold test
 05/22 Plasmid extraction, RE check
 05/25 toehold test
 05/27 toehold test

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/8/89/T--CSMU_Taiwan--Notebook_Evaluation_of_protein_synthesis_kit_.pdf)

##### [Assessment of invertase](#accordion-2 .item-3)

05/14 Transformation

 05/18 Culturing bacteria

 05/19 Bacteria storage, Plasmid Extraction, RE check

 05/20 Transformation, Toehold Test

 05/21 Agar plate preparation, Culturing bacteria, toehold test

 05/22 Plasmid extraction, RE check

 05/25 Culturing Bacteria

 05/27 Plasmid Extraction

 05/28 Transformation

 05/29 Culturing bacteria

 05/30 Plasmid Extraction, RE Check

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/9/95/T--CSMU_Taiwan--Notebook_Assessment_of_invertase.pdf)

##### [Characterization- The spectroscopy of mRFP under different PH condition](#accordion-2 .item-4)

05/14 Transformation

 05/18 Culturing bacteria

 05/19 Bacteria storage, Plasmid Extraction, RE check

 05/20 Transformation, Toehold Test

 05/21 Agar plate preparation, Culturing bacteria, toehold test 05/22 Plasmid extraction, RE check

 05/25 toehold test, Culturing Bacteria

 05/26 Plasmid Extraction, RE check

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/0/0a/T--CSMU_Taiwan--Characterization-_Notebook_The_spectroscopy_of_mRFP_under_different_PH_condition.pdf)

## June

##### [Evaluation of protein synthesis kit](#accordion-2 .item-5)

06/04 Toehold Test

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/2/27/T--CSMU_Taiwan--June_Notebook_Evaluation_of_protein_synthesis_kit.pdf)

##### [Assessment of invertase](#accordion-2 .item-6)

06/01 Gel Extraction, Clean up, Electrophoresis, Ligation,

 06/24 PCR, PCR Clean Up

 06/29 PCR, PCR Clean Up

 06/30 Toehold Test, mRFP Test, PCR, PCR Clean Up

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/7/7d/T--CSMU_Taiwan--Notebook_Assessment_of_invertase_.pdf)

##### [Characterization- The spectroscopy of mRFP under different PH condition](#accordion-2 .item-7)

06/02 Culturing Bacteria

 06/03 Bacterial Storage

 06/23 Culturing Bacteria

 06/30 Toehold Test, mRFP Test, PCR, PCR Clean Up

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/a/ae/T--CSMU_Taiwan--Notebook_Characterization-June_Notebook_The_spectroscopy_of_mRFP_under_different_PH_condition.pdf)

## July

##### [Assessment of invertase](#accordion-2 .item-9)

07/01 Toehold Test, Invertase test, mRFP pH Test

 07/03 PCR, Invertase test, Toehold Test, mRFP pH Test

 07/10 Invertase test, Toehold Test
 07/13 Invertase Test, PCR

 07/14 Invertase test, Plasmid Extraction

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/8/8f/T--CSMU_Taiwan--July_Notebook_Assessment_of_invertase.pdf)

##### [Combining invertase and toehold switch](#accordion-2 .item-10)

07/02 Toehold test, mRFP pH Test
 07/09 Toehold test
 07/10 Invertase test, Toehold Test

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/5/5e/T--CSMU_Taiwan--July_Notebook_Combining_invertase_and_toehold_switch.pdf)

##### [Sensitivity test of toehold switch](#accordion-2 .item-11)

07/13 Invertase Test, PCR

 07/17 PCR

 07/19 PCR

 07/20 Resuspension

 07/21 Gel Preparation, Transformation

 07/22 Culturing bacteria

 07/23 Plasmid extraction, RE check

 07/24 Ligation, Transformation

 07/27 Transformation, Culturing Bacteria

 07/28 Bacteria Storage, Plasmid Extraction, Culturing Bacteria

 07/29 Bacteria Storage, Plasmid Extraction

 07/31 PCR, Clean Up

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/1/10/T--CSMU_Taiwan--July_Notebook_Sensitivity_test_of_toehold_switch_.pdf)

## August

##### [Sensitivity test of toehold switch](#accordion-2 .item-15)

08/03 Clean Up, Ligation, Transformation
 08/04 Colony PCR, Culturing Bacteria
 08/05 Bacteria Storage, Plasmid Extraction, RE check, RE for Ligation
 08/06 Clean Up, Ligation
 08/07 Culturing
Bacteria

 08/08 Plasmid Extraction, RE check

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/2/29/T--CSMU_Taiwan--Notebook_August-Sensitivity_test_of_toehold_switch.pdf)

##### [Adding regulatory switch on amilCP](#accordion-2 .item-16)

08/20 Resuspension, Transformation
 08/21 Culturing Bacteria
 08/22 Bacteria Storage, Plasmid Extraction
 08/24 AmilCP Test
 08/25 AmilCP Test
 08/26 AmilCP Test, Culturing Bacteria
 08/27
Plasmid Extraction, AmilCP Test

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/b/bb/T--CSMU_Taiwan--Notebook_August-Adding_regulatory_switch_on_amilCP.pdf)

## September

##### [Sensitivity test of toehold switch](#accordion-2 .item-21)

9/4 Toehold test, transformation

 9/5 Toehold switch transformation

 9/6 Culture bacteria

 9/7 Plasmid extraction, Glucose concentration detection

 9/8 Plasmid extraction

 9/9 Culture bacteria

 9/10 Plasmid extraction, Glucose concentration detection

 9/12 Bacteria storage,Plasmid extraction, RE check, gel electrophoresis

 9/19 Sensitivity test

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/7/76/T--CSMU_Taiwan--Notebook_September-Sensitivity_test_of_toehold_switch_.pdf)

##### [Specificity test of toehold switch](#accordion-2 .item-22)

9/22 Specificity test

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/9/9e/T--CSMU_Taiwan--Notebook_September-Specificity_test_of_toehold_switch_.pdf)

##### [Dose dependent test](#accordion-2 .item-23)

9/26 "ZR" toehold test with different amount of trigger
 9/27 "ZRA" toehold test with different amount of trigger

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/2/26/T--CSMU_Taiwan--Notebook_September-Dose_dependent_test_.pdf)

## October

##### [Dose dependent test](#accordion-2 .item-25)

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2020/3/33/T--CSMU_Taiwan--Notebook_October-Dose_dependent_test_.pdf)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CSMU Taiwan/Engineering

Home - Brand

[![](https://static.igem.org/mediawiki/2020/1/1f/T--CSMU_Taiwan--logo_light_darkdark.png) CSMU 2020](https://2020.igem.org/Team%3ACSMU_Taiwan)

* Team

  [**Team members**](https://2020.igem.org/Team%3ACSMU_Taiwan/Team)
  [**attributions**](https://2020.igem.org/Team%3ACSMU_Taiwan/Attributions)
  [**Collaborations**](https://2020.igem.org/Team%3ACSMU_Taiwan/Collaborations)
  [**Partnership**](https://2020.igem.org/Team%3ACSMU_Taiwan/Partnership)
* Project

  [**Description**](https://2020.igem.org/Team%3ACSMU_Taiwan/Description)
  [**Engineering Success**](https://2020.igem.org/Team%3ACSMU_Taiwan/Engineering)
  [**Model**](https://2020.igem.org/Team%3ACSMU_Taiwan/Model)
  [**Measurement**](https://2020.igem.org/Team%3ACSMU_Taiwan/Measurement)
  [**Proof of Concept**](https://2020.igem.org/Team%3ACSMU_Taiwan/Proof_Of_Concept)
  [**Proposed Implementation**](https://2020.igem.org/Team%3ACSMU_Taiwan/Implementation)
  [**Protocol**](https://2020.igem.org/Team%3ACSMU_Taiwan/Protocol)
  [**Notebook**](https://2020.igem.org/Team%3ACSMU_Taiwan/Notebook)
* Parts

  [**Parts Overview**](https://2020.igem.org/Team%3ACSMU_Taiwan/Parts)
  [**Contribution**](https://2020.igem.org/Team%3ACSMU_Taiwan/Contribution)
  [**Improve**](https://2020.igem.org/Team%3ACSMU_Taiwan/Improve)
* [safety](https://2020.igem.org/Team%3ACSMU_Taiwan/Safety)
* Human Practices

  [**Human Practice**](https://2020.igem.org/Team%3ACSMU_Taiwan/Human_Practices)
  [**Science Communication**](https://2020.igem.org/Team%3ACSMU_Taiwan/Education)
  [**sustainable**](https://2020.igem.org/Team%3ACSMU_Taiwan/Sustainable)
  [**Entrepreneurship**](https://2020.igem.org/Team%3ACSMU_Taiwan/Entrepreneurship)
* [awards](https://2020.igem.org/Team%3ACSMU_Taiwan/Awards)

### Index

[Detection Tool](#intro)
[Reporter](#problem)
[miRNA.DOC](#propose)
[Reference](#reference)

## Engineering Success

![](https://static.igem.org/mediawiki/2020/8/8c/T--CSMU_Taiwan--ESCCC1.jpeg)

![](https://static.igem.org/mediawiki/2020/a/ac/T--CSMU_Taiwan--ESCCC2.jpeg)

![](https://static.igem.org/mediawiki/2020/d/de/T--CSMU_Taiwan--REFne2.jpeg)
[![](https://static.igem.org/mediawiki/2020/e/ec/T--CSMU_Taiwan--REFne3.jpeg)](#intro)

![](https://static.igem.org/mediawiki/2020/6/65/T--CSMU_Taiwan--NSCCC4-1.jpeg)
[![](https://static.igem.org/mediawiki/2020/8/87/T--CSMU_Taiwan--NSCCC4-2.jpeg)](#problem)

![](https://static.igem.org/mediawiki/2020/d/da/T--CSMU_Taiwan--NSCCC5.jpeg)
[![](https://static.igem.org/mediawiki/2020/d/d4/T--CSMU_Taiwan--NSCCC5-2.jpeg)](#propose)

For the sake of decreasing the mortality rate and saving medical resources, we aim to improve the current diagnosis procedure of oral cancer. In our project, we dedicate to develop **a non-invasive, accessible, and quantitative system for oral cancer detection**.

### 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,](#reference) [2,](#reference) [3,](#reference) [4](#reference) 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](#reference) Secondly, several OSCC-related miRNAs are secreted in saliva[5](#reference) , making them very useful for non-invasive clinical applications.

To measure the salivary miRNAs, we adopt a systematic ribocomputing devices, **toehold switch**.[6,](#reference) [7](#reference) 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](#reference) which is a ready-to-use modular education kit. This banana toehold switch sfGFP [BBa\_K3431050](http://parts.igem.org/Part%3ABBa_K3431050) can be triggered by the RNA of banana and expresses sfGFP [BBa\_K3431031](http://parts.igem.org/Part%3ABBa_K3431031) as its reporter.

![](https://static.igem.org/mediawiki/2020/7/71/T--CSMU_Taiwan--ES-Figure_1.jpeg)

Figure. 1. Banana toehold switch sfGFP. (A) Composite part: pT7-BTS-sfGFP-trT7/pSB1C3 ([BBa\_K3431050](http://parts.igem.org/Part%3ABBa_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](https://2020.igem.org/Team%3ACSMU_Taiwan/Protocol) and
[Measurement](https://2020.igem.org/Team%3ACSMU_Taiwan/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.

![](https://static.igem.org/mediawiki/2020/7/72/T--CSMU_Taiwan--ES-Fig.2.png)

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.

### Reporter: Invertase

### Research

To remove the equipment limitation on our detection tool, we did some paper research to find out the best reporter protein for our project. Invertase, also called β-D-fructofuranosidase, is an enzyme for the hydrolysis of sucrose into glucose and fructose. It is commonly used as reporter proteins since its product, glucose, can be easily detected with a **personal glucose meter (PGM)**,[9](#reference) which has a high utilization rate in the public. According to the previous research, ***Thermotoga maritima* Invertase** (invertase from *Thermotoga maritima*) (TmINV) has been proven to have high activity and thermo-stability compared to the commonly used commercial yeast invertase.[10](#reference) Thus, we chose it to be our reporter protein.

### Design & Build

To ensure the measurement quality, the measurement standards needed to be controlled. Thus, we designed an invertase activity experiment to find the best conditions for our measurement. Moreover, we also tested the banana toehold switch invertase, which is the previous banana toehold switch but replaced sfGFP with invertase, to ensure the combination of the toehold switch and the invertase is feasible.
We used the IDT synthesis offer to build the plasmids, and carried out a series of tests on the invertase [BBa\_K3431015](http://parts.igem.org/Part%3ABBa_K3431015%20) and the banana toehold switch invertase [BBa\_K3431051](http://parts.igem.org/Part%3ABBa_K3431051%20).

![](https://static.igem.org/mediawiki/2020/a/a0/T--CSMU_Taiwan--ES-Figure.3.jpeg
)

Figure 3. Invertase and banana toehold switch invertase. (A) Composite part: pT7-RBS-Inv-trT7/pSB1C3 ([BBa\_K3431015](http://parts.igem.org/Part%3ABBa_K3431015%20)), (B) Composite part: pT7-BTS-Inv-trT7/pSB1C3 ([BBa\_K3431051](http://parts.igem.org/Part%3ABBa_K3431051%20)).

### Test & Improve

We used both models and experiments to test the activity of invertase. For the **modeling**, we used MATLAB to create a model for kinetic investigations of the enzymatic reactions. To see the detailed modeling result, please check the [Model](https://2020.igem.org/Team%3ACSMU_Taiwan/Model#invertase). As for the **wet lab experiments**, we produced the invertase with the PURExpress protein synthesis kit. Then we measured its reaction velocity under different sucrose concentrations. The invertase enzymatic reaction was executed at 55℃, which is the best activity temperature for commercial yeast invertase commonly used in PGM-based reaction.[9, 10](#reference) The result of the model and the experiment is shown below. (Fig.4).
![](https://static.igem.org/mediawiki/2020/b/b9/T--CSMU_Taiwan--ES-Fig.4.png)

Figure 4. The initial velocity of the invertase enzymatic reaction under different sucrose concentrations. The green line refers to the regression curve of experimental data, and the blue line refers to the invertase activity model.

The initial velocity of the reaction increases as the concentration of the substrate, sucrose, rises. The trend of experimental data fitted our model.

As for the banana toehold switch invertase test, the experimental method is quite similar to the previous ones. The plasmids would be transcribed and translated with the protein synthesis kit at 37℃ for 2 hours, and in this experiment, we would then add 5μl of 0.5M sucrose and measured the glucose concentration with the glucose meter (Bionime Rightest™ GM550) every 10 minutes at 55℃ for an hour.

![](https://static.igem.org/mediawiki/2020/5/5c/T--CSMU_Taiwan--ES-Fig.5.png
)

Figure 5. The glucose concentration in 60 minutes invertase enzymatic reaction. The green line refers to the conditions with banana triggers, while the blue line refers to the conditions without a banana trigger.

As shown above, there was a significant difference in glucose production between **the ON state** and **the OFF state**. The ON state refers to the conditions with banana triggers, while the OFF state means that there was no trigger in the environment. In conclusion, changing the reporter sequence would not affect the regulatory function of the toehold switch. Likewise, the toehold switch would not destroy the activity of the following invertase. As a result, our idea of **combining the toehold switch with the invertase** was proven to be practicable.

### miRNA.DOC

### Research

The goal of our project is to construct **the toehold switches that can detect the oral cancer-related miRNAs, and express the invertase**. Therefore, **how to design a toehold switch** is an important issue for us.

 According to the research about toehold switch,[6, 7, 11, 12, 13, 14](#reference) there are several basic principles for sequence design:

1. The **TBS (trigger binding site)** should be exactly complementary to its specific RNA trigger.

2. The optimal length of the **loop** is 11-12 nt.

3. The **RBS (ribosome binding site)** should be within the loop region and be 5-6 nt before the start codon.

4. The sequence of **the hairpin and the linker added after the start codon** must not code for a stop codon, as they would prematurely terminate the translation of the gene of interest. Besides, the length of it should also be the multiple of three, avoiding frameshift mutation.

Besides the paper research, we also consulted with Alexander A. Green, an expert on the toehold switch technology. He recommended us to use the conserved loop sequence in his recent paper,[15](#reference) and he also explained different kinds of modeling methods for us. To see the detailed content, check the
[Human Practice](https://2020.igem.org/Team%3ACSMU_Taiwan/Human_Practices#propose).

### Design & Build

We adopted two kinds of models to help us design the sequences of the toehold switches. One is **NUPACK analysis**, which can predict the MFE (minimum free energy) RNA structure at 37℃ and the free energy of the secondary structure. Another one is **Vienna binding**, which can predict the total free energy of binding and the opening energy for different positions in the sequence. To see the detailed model results, check the
 [Model](https://2020.igem.org/Team%3ACSMU_Taiwan/Model#nupack).

Overall, we designed **21 different toehold switches**, including 9 toeholds targeting **miR-21**, 4 toeholds targeting **miR-31**, and the other 8 toeholds targeting **miR-146**. The detailed information about each toehold switch is listed below (Tab.2).

|  |  |  |
| --- | --- | --- |
| miRNA trigger | Toehold Switch | Part Information |
| miR-21 | pp21\_B | [BBa\_K3431001](http://parts.igem.org/Part%3ABBa_K3431001) |
| op21\_A | [BBa\_K3431005](http://parts.igem.org/Part%3ABBa_K3431005) |
| op21\_B | [BBa\_K3431033](http://parts.igem.org/Part%3ABBa_K3431033) |
| oz21\_A | [BBa\_K3431006](http://parts.igem.org/Part%3ABBa_K3431006) |
| oz21\_B | [BBa\_K3431034](http://parts.igem.org/Part%3ABBa_K3431034) |
| zp21\_A | [BBa\_K3431002](http://parts.igem.org/Part%3ABBa_K3431002) |
| zp21\_B | [BBa\_K3431003](http://parts.igem.org/Part%3ABBa_K3431003) |
| zz21\_A | [BBa\_K3431004](http://parts.igem.org/Part%3ABBa_K3431004) |
| zz21\_B | [BBa\_K3431032](http://parts.igem.org/Part%3ABBa_K3431032) |
| miR-31 | zr31 | [BBa\_K3431007](http://parts.igem.org/Part%3ABBa_K3431007) |
| zz31 | [BBa\_K3431008](http://parts.igem.org/Part%3ABBa_K3431008) |
| or31 | [BBa\_K3431009](http://parts.igem.org/Part%3ABBa_K3431009) |
| oz31 | [BBa\_K3431010](http://parts.igem.org/Part%3ABBa_K3431010) |
| miR-146 | zr146\_A | [BBa\_K3431011](http://parts.igem.org/Part%3ABBa_K3431011) |
| zr146\_B | [BBa\_K3431035](http://parts.igem.org/Part%3ABBa_K3431035) |
| zz146\_A | [BBa\_K3431012](http://parts.igem.org/Part%3ABBa_K3431012) |
| zz146\_B | [BBa\_K3431036](http://parts.igem.org/Part%3ABBa_K3431036) |
| or146\_A | [BBa\_K3431013](http://parts.igem.org/Part%3ABBa_K3431013) |
| or146\_B | [BBa\_K3431037](http://parts.igem.org/Part%3ABBa_K3431037) |
| oz146\_A | [BBa\_K3431014](http://parts.igem.org/Part%3ABBa_K3431014) |
| oz146\_B | [BBa\_K3431038](http://parts.igem.org/Part%3ABBa_K3431038) |

Table 2. Toehold switch overview

We used the IDT synthesis offer to construct our 21 toehold switches. For each toehold, we needed to ligate the regulatory toehold section with the invertase reporter sequence. Firstly, we ordered the primers (Tab.3) containing restriction sites and used [BBa\_K3431051](http://parts.igem.org/Part%3ABBa_K3431051%20) (banana toehold switch invertase) as the template, building the PCR product of the invertase fragment with restriction sites at both ends. (Fig. 6A) Secondly, we used the restriction enzymes and the ligases to build our final toehold switch plasmids. (Fig. 6B)
.

|  |  |
| --- | --- |
| Primer | Sequence |
| Invertase\_XbaI\_Forward | 5'- GGCGCGTCTAGAATGTTCAAGCCGAATTATCA -3' |
| T7 terminator\_PstI\_Reverse | 5'- AGAGGACTGCAGCAAAAAACCCCTCAAGACCCGTTTA -3' |

Table 3. Invertase primers

[![](https://static.igem.org/mediawiki/2020/6/6b/T--CSMU_Taiwan--ES-Fig.6-new.jpeg
)](https://static.igem.org/mediawiki/2020/6/6b/T--CSMU_Taiwan--ES-Fig.6-new.jpeg)

Figure 6. Gene cloning of the toehold switch regulated invertase. (A) Using PCR to produce the target insert, which includes invertase and T7 terminator sequences. The forward primer contained XbaI and overlapped with the 5’ end of the invertase; while the reverse primer contained PstI and was complementary to the 3’ end of the T7 terminator. (B) Lane 1 to 8 are the toehold switch vectors digested with XbaI and PstI, whose length is about 2000 bp. Lane 9 is the Insert containing invertase and T7 terminator, whose length is 1358 bp. (C) Ligate the invertase sequence with the toehold switches we designed.

|  |  |  |
| --- | --- | --- |
| miRNA Trigger | Toehold Switch Invertase | Part Information |
| miR-21 | pp21\_B | [BBa\_K3431016](http://parts.igem.org/Part%3ABBa_K3431016) |
| op21\_A | [BBa\_K3431021](http://parts.igem.org/Part%3ABBa_K3431021) |
| op21\_B | [BBa\_K3431042](http://parts.igem.org/Part%3ABBa_K3431042) |
| oz21\_A | [BBa\_K3431022](http://parts.igem.org/Part%3ABBa_K3431022) |
| oz21\_B | [BBa\_K3431043](http://parts.igem.org/Part%3ABBa_K3431043) |
| zp21\_A | [BBa\_K3431017](http://parts.igem.org/Part%3ABBa_K3431017) |
| zp21\_B | [BBa\_K3431018](http://parts.igem.org/Part%3ABBa_K3431018) |
| zz21\_A | [BBa\_K3431020](http://parts.igem.org/Part%3ABBa_K3431020) |
| zz21\_B | [BBa\_K3431041](http://parts.igem.org/Part%3ABBa_K3431041) |
| miR-31 | zr31 | [BBa\_K3431023](http://parts.igem.org/Part%3ABBa_K3431023) |
| zz31 | [BBa\_K3431024](http://parts.igem.org/Part%3ABBa_K3431024) |
| or31 | [BBa\_K3431025](http://parts.igem.org/Part%3ABBa_K3431025) |
| oz31 | [BBa\_K3431026](http://parts.igem.org/Part%3ABBa_K3431026) |
| miR-146 | zr146\_A | [BBa\_K3431027](http://parts.igem.org/Part%3ABBa_K3431027) |
| zr146\_B | [BBa\_K3431044](http://parts.igem.org/Part%3ABBa_K3431044) |
| zz146\_A | [BBa\_K3431028](http://parts.igem.org/Part%3ABBa_K3431028) |
| zz146\_B | [BBa\_K3431045](http://parts.igem.org/Part%3ABBa_K3431045) |
| or146\_A | [BBa\_K3431029](http://parts.igem.org/Part%3ABBa_K3431029) |
| or146\_B | [BBa\_K3431046](http://parts.igem.org/Part%3ABBa_K3431046) |
| oz146\_A | [BBa\_K3431030](http://parts.igem.org/Part%3ABBa_K3431030) |
| oz146\_B | [BBa\_K3431047](http://parts.igem.org/Part%3ABBa_K3431047) |

Table 4. Toehold switch invertase overview

### Test & Improve

For each kind of oral cancer-related miRNA, we have designed several toehold switches to detect it. With a series of experiments, we could select the best one for each miRNA and further tested its functionality. Here are our selection criteria for toehold switch:

1. **High ON/OFF ratio.** The protein expression should be high in the ON state (with trigger) and low in the OFF state (without trigger).

- **High sensitivity.** The ultimate glucose signal should be high enough so that a small number of miRNAs can also be detected.

- **High specificity.** The toehold structure should only be opened by its specific trigger. In other words, the ON/OFF ratio of the non-specific miRNAs should be close to 1.

We used the same experimental method as the above banana toehold switch invertase test. The plasmids were transcribed and translated with the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs) at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose, and measured the glucose concentration with Bionime Rightest™ GM550 glucose meter after 30 minutes of enzymatic reaction time.

[![](https://static.igem.org/mediawiki/parts/c/c1/T--CSMU_Taiwan--ES-Figure.7.jpeg)](https://static.igem.org/mediawiki/2020/c/c1/T--CSMU_Taiwan--ES-Figure.7.jpeg)

Figure 7. The sensitivity and ON/OFF ratio test of the toehold switches for miR-21, miR-31, miR-146. The blue bars refer to the OFF state (not added with miRNA trigger); and the green bars refer to the ON state (added with miRNA trigger). The ON/OFF ratio of each toehold switch was listed below. (A) Tests of the toehold switches for miR-21, (B) Tests of the toehold switches for miR-31, (C) Tests of the toehold switches for miR-146.

The above figure shows the sensitivity and the ON/OFF ratio of each toehold switch. As seen, the toehold switches for miR-21 did not meet our expectation, since some sensitivities were too low, and some ON/OFF ratios were lower than 1. Therefore, we abandoned them in the following tests. As for the toehold switches for miR-31 and miR-146, most of them had great sensitivities and high ON/OFF ratios. Thus, we conducted a further specificity test for them.

 We used miR-191 and miR-233 for our negative selection, which are highly expressed in saliva[3](#reference), to see whether our toehold switches would be accidentally turned on by the unrelated miRNA.

[![](https://static.igem.org/mediawiki/2020/4/4f/T--CSMU_Taiwan--ES-Fig.8.png)](https://static.igem.org/mediawiki/2020/4/4f/T--CSMU_Taiwan--ES-Fig.8.png)

Figure 8. The specificity test of the toehold switches for miR-31, miR-146. The blue bars refer to the OFF state (not added with miRNA trigger). The green bars refer to the ON state (added with the specific miRNA trigger). The yellow bars refer to the toehold switches with non-related RNA (miR-191). The pink bars refer to the toehold switches with non-related RNA (miR-223). The ON/OFF ratio of each toehold switch was listed above the bars. (A) Tests of the toehold switches for miR-31, (B) ON/OFF ratios of the toehold switches for miR-31, (C) Tests of the toehold switches for miR-146, (D) ON/OFF ratios of the toehold switches for miR-146.

As shown in Fig. 8, most toehold switches have high specificity, as their ON/OFF ratios of the non-related miRNAs, miR-191 and miR-223, were close to 1.

In conclusion, most of the toehold switches had great functionality. The results indicated that we successfully developed a new invertase toehold switch system to detect miRNAs.

After a systematic evaluation with ON/OFF ratio, sensitivity, and specificity, we found that **zr31** [BBa\_K3431023](http://parts.igem.org/Part%3ABBa_K3431023%20) and **zr146\_A** [BBa\_K3431027](http://parts.igem.org/Part%3ABBa_K3431027%20) are the best toehold switches for miR-31 and miR-146 detection. Therefore, we decided to conduct a further examination on these two toehold switches. We measured the glucose concentration under different amount of the miRNA trigger.

![](https://static.igem.org/mediawiki/2020/7/75/T--CSMU_Taiwan--ES-Fig.9.png
)

Figure 9. The glucose production of zr31 and zr146\_A in the environment with different amounts of miRNA. (A) The glucose-trigger relation of zr31, (B) The glucose-trigger relation of zr146\_A.

As shown above, the glucose concentration rises as the triggers increase, suggesting the positive correlation. With the regression curve formulas, we can measure the amount of the miRNA from the glucose meter readouts.

For future work, we are planning to use our toehold switches to measure **the miRNAs in the real saliva**. With the comprehensive experiments and the data analysis, we can build a model for oral cancer. Understanding the correlation between the possibility of oral cancer, the amount of salivary miRNA, the invertase expression, and glucose production will help us develop **a quantitative system for oral cancer detection**.

### Reference

1. Min, A., Zhu, C., Peng, S., Rajthala, S., Costea, D. E., & Sapkota, D. (2015). MicroRNAs as Important Players and Biomarkers in Oral Carcinogenesis. BioMed research international, 2015, 186904. https://doi.org/10.1155/2015/186904
2. Zahran, F., Ghalwash, D., Shaker, O., Al-Johani, K., & Scully, C. (2015). Salivary microRNAs in oral cancer. Oral diseases, 21(6), 739–747. https://doi.org/10.1111/odi.12340
3. Momen-Heravi, F., Trachtenberg, A. J., Kuo, W. P., & Cheng, Y. S. (2014). Genomewide Study of Salivary MicroRNAs for Detection of Oral Cancer. Journal of Dental Research, 93(7\_suppl), 86S-93S. https://doi.org/10.1177/0022034514531018
4. Liu, C. J., Lin, S. C., Yang, C. C., Cheng, H. W., & Chang, K. W. (2012). Exploiting salivary miR-31 as a clinical biomarker of oral squamous cell carcinoma. Head & neck, 34(2), 219–224. https://doi.org/10.1002/hed.21713
5. Park, N. J., Zhou, H., Elashoff, D., Henson, B. S., Kastratovic, D. A., Abemayor, E., & Wong, D. T. (2009). Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection. Clinical cancer research : an official journal of the American Association for Cancer Research, 15(17), 5473–5477. https://doi.org/10.1158/1078-0432.CCR-09-0736
6. Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold switches: de-novo-designed regulators of gene expression. Cell, 159(4), 925–939. https://doi.org/10.1016/j.cell.2014.10.002
7. Green, A. A., Kim, J., Ma, D., Silver, P. A., Collins, J. J., & Yin, P. (2017). Complex cellular logic computation using ribocomputing devices. Nature, 548(7665), 117–121.
8. Huang, A., Nguyen, P. Q., Stark, J. C., Takahashi, M. K., Donghia, N., Ferrante, T., Dy, A. J., Hsu, K. J., Dubner, R. S., Pardee, K., Jewett, M. C., & Collins, J. J. (2018). BioBits™ Explorer: A modular synthetic biology education kit. Science advances, 4(8), eaat5105. https://doi.org/10.1126/sciadv.aat5105
9. Xiang, Y., & Lu, Y. (2011). Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets. Nature chemistry, 3(9), 697–703. https://doi.org/10.1038/nchem.1092
10. Du, Y., Hughes, R. A., Bhadra, S., Jiang, Y. S., Ellington, A. D., & Li, B. (2015). A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers. Scientific reports, 5, 11039. https://doi.org/10.1038/srep11039
11. Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N. M., Bosch, I., Dudley, D. M., O'Connor, D. H., Gehrke, L., & Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell, 165(5), 1255–1266. https://doi.org/10.1016/j.cell.2016.04.059
12. Chappell, J., Westbrook, A., Verosloff, M., & Lucks, J. B. (2017). Computational design of small transcription activating RNAs for versatile and dynamic gene regulation. Nature communications, 8(1), 1051. https://doi.org/10.1038/s41467-017-01082-6
13. Sadat Mousavi, P., Smith, S. J., Chen, J. B., Karlikow, M., Tinafar, A., Robinson, C., Liu, W., Ma, D., Green, A. A., Kelley, S. O., & Pardee, K. (2020). A multiplexed, electrochemical interface for gene-circuit-based sensors. Nature chemistry, 12(1), 48–55. https://doi.org/10.1038/s41557-019-0366-y
14. Hong, F., Ma, D., Wu, K., Mina, L. A., Luiten, R. C., Liu, Y., Yan, H., & Green, A. A. (2020). Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators. Cell, 180(5), 1018–1032.e16. https://doi.org/10.1016/j.cell.2020.02.011

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CSMU Taiwan/Experiments

Home - Brand

[![](https://static.igem.org/mediawiki/2020/1/1f/T--CSMU_Taiwan--logo_light_darkdark.png) CSMU 2020](https://2020.igem.org/Team%3ACSMU_Taiwan)

* Team

  [**Team members**](https://2020.igem.org/Team%3ACSMU_Taiwan/Team)
  [**attributions**](https://2020.igem.org/Team%3ACSMU_Taiwan/Attributions)
  [**Collaborations**](https://2020.igem.org/Team%3ACSMU_Taiwan/Collaborations)
  [**Partnership**](https://2020.igem.org/Team%3ACSMU_Taiwan/Partnership)
* Project

  [**Description**](https://2020.igem.org/Team%3ACSMU_Taiwan/Description)
  [**Engineering Success**](https://2020.igem.org/Team%3ACSMU_Taiwan/Engineering)
  [**Model**](https://2020.igem.org/Team%3ACSMU_Taiwan/Model)
  [**Measurement**](https://2020.igem.org/Team%3ACSMU_Taiwan/Measurement)
  [**Proof of Concept**](https://2020.igem.org/Team%3ACSMU_Taiwan/Proof_Of_Concept)
  [**Proposed Implementation**](https://2020.igem.org/Team%3ACSMU_Taiwan/Implementation)
  [**Protocol**](https://2020.igem.org/Team%3ACSMU_Taiwan/Protocol)
  [**Notebook**](https://2020.igem.org/Team%3ACSMU_Taiwan/Notebook)
* Parts

  [**Parts Overview**](https://2020.igem.org/Team%3ACSMU_Taiwan/Parts)
  [**Contribution**](https://2020.igem.org/Team%3ACSMU_Taiwan/Contribution)
  [**Improve**](https://2020.igem.org/Team%3ACSMU_Taiwan/Improve)
* [safety](https://2020.igem.org/Team%3ACSMU_Taiwan/Safety)
* Human Practices

  [**Human Practice**](https://2020.igem.org/Team%3ACSMU_Taiwan/Human_Practices)
  [**Science Communication**](https://2020.igem.org/Team%3ACSMU_Taiwan/Education)
  [**sustainable**](https://2020.igem.org/Team%3ACSMU_Taiwan/Sustainable)
  [**Entrepreneurship**](https://2020.igem.org/Team%3ACSMU_Taiwan/Entrepreneurship)

## Protocol

### Here are some of our protocols for the success of miRNA.DOC! Feel free to click on the images for full details documented on protocol.io!

### Contribution / Characterization

[![](https://static.igem.org/mediawiki/2020/f/f5/T--CSMU_Taiwan--notebook_for_characterization.png)](https://www.protocols.io/view/characterization-binjkdcn)

### Transformation

[![](https://static.igem.org/mediawiki/2020/e/e9/T--CSMU_Taiwan--note_book_for_transformation_and_plasmid_extaction.png)](https://www.protocols.io/view/transformation-bg4ajyse)

### Long-Term Storage of Bacteria

[![](https://static.igem.org/mediawiki/2020/1/1b/T--CSMU_Taiwan--note_book_for_Long-Term_Storage_of_Bacteria_and_Restriction_enzyme_%26_gel_electrophoresis.png)](https://www.protocols.io/view/long-term-storage-of-bacteria-bh48j8zw)

### Bacterial Culture for Plasmid Extraction

[![](https://static.igem.org/mediawiki/2020/e/e9/T--CSMU_Taiwan--note_book_for_transformation_and_plasmid_extaction.png)](https://www.protocols.io/view/plasmid-extraction-bh5bj82n)

### Restriction enzyme & gel electrophoresis

[![](https://static.igem.org/mediawiki/2020/9/9e/T--CSMU_Taiwan--note_book_for_Restriction_enzyme_%26_gel_electrophoresis_and_Banana_sensor_sfGFP_test.png)](https://www.protocols.io/view/restriction-enzyme-amp-gel-electrophoresis-bh67j9hn)

### Banana sensor sfGFP test

[![](https://static.igem.org/mediawiki/2020/9/9e/T--CSMU_Taiwan--note_book_for_Restriction_enzyme_%26_gel_electrophoresis_and_Banana_sensor_sfGFP_test.png)](https://www.protocols.io/view/banana-sensor-sfgfp-test-bh66j9he)

### Sensitivity and specificity test of toehold

[![](https://static.igem.org/mediawiki/2020/4/4e/T--CSMU_Taiwan--Protocol_for_Specificity_test_of_toehold_.png)](https://www.protocols.io/view/sensitivity-and-specificity-test-of-toehold-bn4emgte)

### Improve

[![](https://static.igem.org/mediawiki/2020/f/f4/T--CSMU_Taiwan--Protocol_for_improve_.png)](https://www.protocols.io/view/improve-bnw2mfge?fbclid=IwAR1Rjdz0m9_VuuqFuuaySblFuFWFLirIWQwcqyCT12m8BRdSIkGtGECBURo)

### Dose Dependent Test

[![](https://static.igem.org/mediawiki/2020/f/f4/T--CSMU_Taiwan--Protocol_for_improve_.png)](https://www.protocols.io/view/dose-dependent-test-bn34mgqw)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CSMU Taiwan/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)