Difference between revisions of "Team:CSMU Taiwan/Poster"

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<div class="text"><p><b>Reporter protein selection</b><br>
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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)[10],  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.[11]  Thus, we chose it to be our reporter protein.<br><br>
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<b>Invertase activity test</b><br>
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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. 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 result of the model and the experiment is shown below.
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Figurex. 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.
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The initial velocity of the reaction increases as the concentration of the substrate, sucrose, rises. The trend of experimental data fitted our model.</p>
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Revision as of 07:47, 9 November 2020

Poster: CSMU_Taiwan



Poster Template

This poster template will let you create an interactive poster! The poster is divided in two parts: the visual overview on the left and the documentation on the right. The visual overview is broken down into sections that the user can click on. When a user clicks on a poster section in the visual overview, the documentation on the right will display the text and graphics associated with this section of the poster. You can find the documentation on how to use this template, as well as an example here: https://2020.igem.org/Competition/Deliverables/Poster

miRNA.DOC: A novel detection method for Oral Cancer

Presented by team CSMU_Taiwan 2020


Cheng-Yang, Ma1, Yi-Ching, Chen1, Hung-Yu, Chen1, Huan-Jui, Chang1, Dai-Rou, Lee1, Hung-Liang, Pai1, Tzu-Hsuan, Hsiao1, Matilda, Key1, Cheng-Ruei, Yang1, Hsin-Jung, Lee2, Kuan-Lin, Chen2, Ting-Yu, Lin2, Ting-Yu, Lin2, Shao-Chi, Lo2, Ho-Lo, Huang2, Shen-Lin, Chen2, Kuo-Chen, Huang2, Dr. Yu-Fan, Liu3


1iGEM Student Team Member, 2iGEM Team Advisor, 3iGEM Team PI


Abstract

Inspection and palpation are the two main ways for doctors to determine whether they would take a biopsy test or not when doctors suspect that their patients get oral cancer.1 Inspection and palpation may have detection bias and largely depends on the experience and judgment of the doctor. Therefore, iGEM CSMU_Taiwan developed a new oral cancer detection method, miRNA.DOC, to deal with this problem. We adapted the "Toehold Switch" technique and a glucometer to create a novel detection device.2 Those toehold switches we designed would detect miRNAs in human saliva and it will allow the reporter protein, invertase, to be translated. Invertase would break down sucrose into glucose and fructose. After that, the glucometer would be used to measure the concentration of glucose and present quantitative data for the patients' oral condition. We successfully found the best two toehold switches, zr31 and zr146_A, from all the 21 toehold switches we design after testing their ON/OFF ratios, sensitivities, and specificities. Then we measured the glucose concentration under different amounts of the miRNA triggers and verified the positive correlation between the glucose concentration and the amount of those triggers. With the regression curve formulas, we can measure the amount of the miRNA from the glucometer readouts. With miRNA.DOC, we hope to provide a quantitative, non-invasive, and accessible method for oral cancer detection. By using this product, patients can be diagnosed earlier, recover sooner, and move one step closer to good health.

Introduction
Introduce your project and your team's goals.
Toehold Mechanism

Toehold switch is a lock that can repress the expression of the reporter protein. Correspondingly, our targeted RNA sequence serves as a key to open the lock.

Toehold switch is mainly composed of three parts, the trigger binding site (TBS) that is complementary to a target RNA sequence, a hairpin loop containing the ribosomal binding site (RBS), and a linker sequence that connects the toehold structure with the protein-coding sequence. [2]

When the targeted RNA sequence binds to the trigger binding sites, it unwinds the lower and upper stem. The RBS and start codon are exposed. Thus, the ribosome can attach to the start codon and translate the reporter protein sequence.

Biomarker
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Toehold Design
Initially, we designed our toehold switches based off of Wang et al.'s (2019)[8]. They have designed a toehold switch for miRNA 21 which is also one of the miRNAs that we plan to detect. However, we were still having trouble with designing our own version of the toehold switch as the analysis with NUPACK showed unoptimal results. Therefore, we consulted with Alexander Green from Arizona State University and he advised us to design the toehold switch based off of one of his more recent studies on toehold switch for ZIKA virus (2016)[9].
We split the design of toehold switches into three sections:
  1. Trigger binding site
    The trigger binding site (TBS) was solely designed upon reverse translating the sequence.
  2. Loop
    We looked at one of Green’s[9] and Wang’s paper for our choice of design. In the paper, he designed two seperate loop structures and tested their effects on the ON/OFF ratio.
  3. Linker
    We adapted linkers from Green, Wang, and our randomization.
After they were put together, we ran NUPACK to ensure the spontaneous formation of the secondary structure at 37 degrees Celsius and Vienna RNA Package to predict the interaction between our trigger and the toehold switch. Figure 1. This is the result from NUPACK for one of our designs, it shows spontaneous formation and proper secondary structure for the loop.
Figure 2. This is the result from Vienna RNA package for the same design as the figure above. The black line in the figure indicates the amount of energy required to open the secondary structures of the TBS. The line red indicates the amount of energy required to open the secondary structure after the binding of the trigger.
Reporter

Reporter protein selection
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)[10], 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.[11] Thus, we chose it to be our reporter protein.

Invertase activity test
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. 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 result of the model and the experiment is shown below.

Figurex. 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.

Experiment
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).
  2. High sensitivity. The ultimate glucose signal should be high enough so that a small number of miRNAs can also be detected.
  3. 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.
The plasmids of the toehold switches would be transcribed and translated with the PURExpress® In Vitro Protein Synthesis Kit (New England Biolabs) at 37℃ for 2 hours. Afterward, we would add 5μl of 0.5M sucrose, and measured the glucose concentration with Bionime Rightest™ GM550 glucose meter after 30 minutes of enzymatic reaction time.
In our experiments, the ON state refers to the conditions with the miRNA triggers; while the OFF state means that there was no miRNA in the environment. We calculated the ON/OFF ratio of the toehold switch, which is defined below:
By measuring the ON/OFF ratios and the glucose concentration values in the ON state, we could find out the most sensitive toehold for microRNA detection.
Moreover, we used miR-191 and miR-233 for our negative selection, which are highly expressed in saliva[12], to see whether our toehold switches would be accidentally turned on by the unrelated miRNA. By comparing the ON/OFF ratios of the specific microRNA with which of the non-related ones, we could find out the most specific toehold switch.
With a systematic evaluation with ON/OFF ratio, sensitivity, and specificity, we could select the best toehold switches for detecting miR-21, miR-31, and miR-146.
For the best toehold switches, we further conducted a quantitative examination on them. We measured the glucose concentrations under different amounts of the miRNA trigger. Understanding the correlation between the two, we can measure the amount of miRNA from the glucose meter readouts and further build up a quantitative system for oral cancer detection.
Result
To select the best toehold switches for detecting miR-21, miR-31, and miR-146, we have carried out a functional test for each toehold switch.
The figure shows the glucose productions of the toehold switches in different states. The blue bar refers to the OFF state (not added with miRNA); the green bar refers to the ON state (added with the specific trigger); the yellow bar refers to the state with non-related RNAs (added with miR-191); the pink bar refers to the state with non-related RNAs (added with miR-223). The ON/OFF ratio of each toehold switch with target microRNAs were listed below. As shown, most of the toeholds had high ON/OFF ratios and great sensitivities, as the glucose concentrations in the ON state are much higher than that in the OFF state. However, the results of the toehold switches for miR-21 detection were different from our expectation, thus we abandon them in further experiments. As for the specificity, most of the ON/OFF ratios with the non-related microRNAs were close to 1, which means the toehold switches had great specificity to their own target microRNAs.
After a systematic evaluation with ON/OFF ratio, sensitivity, and specificity, we found that zr31 (BBa_K3431023) and zr146_A (BBa_K3431027) are the best toehold switches for miR-31 and miR-146 detection. Therefore, we further conducted a quantitative examination on them.
The glucose production of zr31 and zr146_A in the environment with different amounts of miRNA. As shown, the glucose concentration rises as the amount of the trigger increases, suggesting a positive correlation.
Future
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Human Practices
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