iGEM 2020 — Team KUAS_Korea

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# Team:KUAS Korea/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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# Team:KUAS Korea/Engineering

# [KUAS](/Team%3AKUAS_Korea)

* [Home](/Team%3AKUAS_Korea)
* Team
  + [Team members](/Team%3AKUAS_Korea/Team)
  + [Collaborations](/Team%3AKUAS_Korea/Collaborations)
  + [Attributions](/Team%3AKUAS_Korea/Attributions)
  + [Official Team Profile](//igem.org/Team.cgi?team_id=3421)
* Project
  + [Project Description](/Team%3AKUAS_Korea/Description)
  + [Design](/Team%3AKUAS_Korea/Design)
  + [Experiments](/Team%3AKUAS_Korea/Experiments)
  + [Results](/Team%3AKUAS_Korea/Results)
  + [Safety](/Team%3AKUAS_Korea/Safety)
  + [Model](/Team%3AKUAS_Korea/Model)
  + [Engineering](/Team%3AKUAS_Korea/Engineering)
  + [Implementation](/Team%3AKUAS_Korea/Implementation)
  + [Contribution](/Team%3AKUAS_Korea/Contribution)
* Human Practices
  + [Integrated Practices](/Team%3AKUAS_Korea/Human_Practices)
  + [Public Engagement & Science Communication](/Team%3AKUAS_Korea/Education)
* Additional Project
  + [Additional Project](/Team%3AKUAS_Korea/CCP)

# Engineering principles

## Introduction

![](https://static.igem.org/mediawiki/2020/7/74/T--KUAS_Korea--Troubleshooting.jpeg)

Synthetic biology is a biology field where engineering principles are applied. Design-Build-Test-Learn is a basic engineering cycle.

This year, we couldn't get into our lab due to COVID-19. So our activities were mostly done by literature research and designing experiments that we are going to do if we can get into our lab. We would like to share some of the struggles we went through for future teams.

We could hardly get into our lab. Engineering principles we followed were based on researching literature, making assumptions and compromises. But it was a great experience since even discussions between us can proceed ahead.

What we did this year to achieve 'Engineering principles'. We archived DBTL cycle we went through in this menu and planned our future experiments in [[Link]](https://2020.igem.org/Team%3AKUAS_Korea/Experiments)

## DBTL cycle

### Brainstorming

![](https://static.igem.org/mediawiki/2020/8/84/T--KUAS_Korea--BS.png)

Description : We brainstormed for several weeks, creating a mind-map. We converged into 5 key ideas and proceeded to develop them. The ideas were
- Stent made by ELM (Engineered Living Materials)
- 3D bio printer
- DNA Cryptography
- Jaundice Patients' temperature detecting patch

Learn : To select a single topic among the candidates, we set two criteria for selection. 1) Does the topic motivate us? 2) Can we produce a meaningful output with the topic by the end of the project?
- Stent made by ELM (Engineered Living Materials : stents are known to have downsides such as causing blood clots, allergy and hypersensitivity. To deal with this problem, the prerequisites including biocompatibility of a stent was extremely challenging to achieve. When we consulted our PI about this topic, he gave us a feedback that we would only able to partially handle this issue and suggested an experiment that tests whether the biomaterial of our choice can be well expressed in a strain and registering it as a part on the registry of iGEM. We felt that this was not our original intentions about this topic and came to the agreement that this topic doesn’t suits our motivation. But the project is extened to an additional project as a small project - CCP. CCP is a program in Korea University which support research projects. We attended the program with this topic, **[[Link]](https://2020.igem.org/Team%3AKUAS_Korea/CCP)**
- 3D bio printer : Designing a 3D bio printing system using phototaxic microbes was our original idea. However, we realized that this project heavily relies on it is a mechanical engineering aspect. Our members are mostly based on biotechnology so agreed that it would be hard for us to get a meaningful result.
- DNA Cryptography : Designing a DNA Cryptographic mechanism was also one of our candidates. We considered constructing logic gates with nucleic acids and then using them to emulate cryptographic mechanisms. However, The complex mathmatical concepts that has to be understood was to vast for us to understand within the given amount of time. Thus, we agreed that it would be hard to achieve a meaningful result and that it doesn’t matches our motivation
- Jaundice Patients’ temperature detecting patch : So we chose this topic for our topic of this year

### Jaundice Patients

Imagine : We measure the temperature of jaundice patients by a thermopatch. The jaundice patients are treated phototherapy. At that time, their temperature must be monitored. How about using a temperature detecting patch for the situation?

Design : To do the phototherapy, a blue light is used. If the patch detects a certain temperature, it makes some fluorescence that is activated by the light.

Build : we made brief explanation about our imagination and design.

Test : we did an interview with Pediatrician **[[Link]](https://2020.igem.org/Team%3AKUAS_Korea/Human_Practices)**

Learn : monitoring body temperature of jaundice patients is a matter of life and death. So reliability matters. And the pediatrician said there is already a reliable product.

Improve : we agreed to change our project target

### COVID-19 fever detecting patch

Imagine : COVID-19 occurred. Due to the virus, entering buildings in Korea University, the body temperatures of visitors are monitored. If they are OK, patches are distributed. What if the patches can detect the temperature as well?

Design : We designed a sticker that receives human body temperature as an input, processes the degree of body temperature, and outputs light.

Build : The input is thermal diffusion. How to process and output?

Test : Thermal diffusion was modeled in matlab using the heat transfer equation.

\( {{\delta T} \over {\delta t}} = k \nabla ^2 T \)

![](https://static.igem.org/mediawiki/2020/5/55/T--KUAS_Korea--heat.png)

[Fig 1. Heat distribution across the patch after 5 minutes attached to human body]

Learn : We found that the heat from human body transfers occurs well.

Improve : We decided to focus on processing and output

### Jilin China

Imagine : While looking for a thermosensor-related project at iGEM, we came to the Jilin China project (https://2018.igem.org/Team:Jilin\_China). So we decided to introduce this system on our patch system

Design : We designed a patch system that operates in the following stages.
1) Jilin China part expresses mediator protein by heat.
2) The mediator protein triggers the process of inducing the expression of a fluorescent protein or a luciferase.
3) Light generation due to fluorescence or luciferase expression is output.

Build&Test : due to the COVID-19 situation, we couldn’t build and test what we designed. But we did literature research and brainstorming

Learn : It has been shown that various molecules and conditions necessary for cell free expression such as dNTP, amino acids, polymerase, ribosomes, and energy source are required.
Considering that our products will be dispensed to the general public, this sophisticated system can hardly be used.

Improve : Literature research was conducted to see if there is a system that requires fewer molecules.

### RNA Aptamer

Imagine : While searching for biological systems that can emit various lights, we discovered `RNA aptamer’ and we found a review paper on this.
 **[Genetically encoded light-up RNA aptamers and their applications for imaging and biosensing, Puchakayala Swetha et al., Journal of Materials Chemistry B, 2020.]**

Design : We looked at several systems introduced in the review paper based on 1) Molecules used, 2) Compatibility with our system, 3) Reputation (reliability).

Build : Each team member was assigned a system to be investigated.

Test : Split members gathered at the meeting time introduced investigated system and rated each system according to the set criteria. As a result, the CHA system was chosen as the most appropriate system for us to use.

Learn : The number of molecules used was reduced to three RNA aptamers and the fluorophore DFHBI-1T.

Improve : Our system came to use the CHA system.

### Collaboration with Jilin China

Imagine : Although the CHA system exhibits fluorescence, the paper does not tell us how to control the behavior according to temperature.

Design : We decided to introduce a system designed by Jilin China for the behavior according to temperature. Jilin china tried to control the operating temperature by making a hairpin with various loop sequences between complementary SD sequences.
However, our sequence was so long that when a complementary sequence was attached and made hairpin structure, the predicted melting temperature was too high.

Build&Test : We sent a mail to Jilin China and asked how they designed the loop sequence, and how they chose RNA candidate. And we also asked for opinions as the experienced on attaching a sequence that is only partially complementary to our target sequence.

Learn : RBS sequence of Jilin china’s system was short enough to make a hairpin structure with appropriate melting temperature. Also, the thermodynamics including the melting temperature predicted by pre-experiment modeling had only 37% accuracy. So we don’t have to fear failures.

Improve : We attached an arbitrary sequence to the end of our target sequence. New nucleotides are consisted of 3-4 base pairs in length complemented to the sequence in the middle of the target sequence.

### Attaching part

Imagine : We thought that adding an additional sequence to the target sequence would create an additional hairpin structure and control its behavior according to temperature.

Design : The target sequence is composed as follows.
Target sequence : GACATCTACCAACAGTATCGCTC

The underlined sequence is that binds to H1 and H2. And the other part is that binds only to H1. If the elongated base pairs are complement to the end of the underlined sequence, then it must regulate the affinity between Target sequence and H1.

Build : 3, 4 base pairs are added as below.

|  |  |
| --- | --- |
| Modified Sequences with 3 elongated base pairs | |
| C1 | CTGGACATCTACCAACAGTATCGCTC |
| C2 | TGTGACATCTACCAACAGTATCGCTC |
| C3 | GTTGACATCTACCAACAGTATCGCTC |
| Modified Sequences with 4 elongated base pairs | |
| C4 | CTGTGACATCTACCAACAGTATCGCTC |
| C5 | TGTTGACATCTACCAACAGTATCGCTC |
| C6 | GTTGGACATCTACCAACAGTATCGCTC |

Test : In order to test the behaviors of the new parts, we need to find proper software or equations.

### DNA aptamer

Imagine : Since our CHA system operates based on RNA, we predicted the short lifespan problem due to RNA degradation. To solve this problem, there was an opinion to leave the target sequence as RNA and use a DNA aptamer instead of RNA for subsequent CHA system hairpin sequences.

Design : There were many papers about the use of DNA to aptamer research, so we decided to check about this. A new CHA system composed of DNA aptamers was designed and its advantages and disadvantages were predicted and compared with the existing RNA CHA system.

Build & Test : We investigated how the CHA system works with DNA through the paper.

Learn : Through the paper, we were able to find the sequence of the CHA system that works with DNA, and the method of operation of fluorescence is Fluorophore-Quencher method, not Chemical Fluorophore. When designing a DNA CHA system through discussion, it has the advantage of solving the short-lived problem due to RNA degradation, but since it is a completely different sequence from the RNA sequence we currently designed, the system must be redesigned. There was a disadvantage that the experiment had to be based because there was no information on whether Target RNA – DNA CHA system connection was possible. In addition, even if we changed the CHA system to DNA, we can’t know how long the lifespan problem would be solved because Target sequence is still RNA and it is the key in the operation of the entire system.

Improve : We decided to keep the current RNA CHA system and record the discussion above.

Literature : (1) Real-Time Detection of Isothermal Amplification Reactions with
Thermostable Catalytic Hairpin Assembly, Yu (Sherry) Jiang et al., Journal of the american chemical society, 2013. (2) Fast and quantitative differentiation of single-base mismatched DNAby initial reaction rate of catalytic hairpin assembly, Chenxi Li et al., Biosensors and Bioelectronics, 2014. (3) Catalytic Hairpin Assembly Actuated DNA Nanotweezer for Logic Gate Building and Sensitive Enzyme-Free Biosensing of MicroRNAs, Dandan Li et al., Analytical chemistry, 2016.

### RNA production

Imagine : Since we have to experiment with the CHA method, we have to get the pure RNA we need for the experiment.

Design : Since we haven't done any method on how to get pure RNA, we did some research to determine how we will experiment.

Build & Test : In the reference paper, when experimenting in vitro, RNA was synthesized and tested through in vitro transcription, and when experimenting in vivo, RNA gene was transformed and tested. In addition, RNA could be ordered and synthesized directly.

Learn : As a result of investigating the cost of the above methods, in vitro transcription was selected because we are doing in vitro experiments and synthesizing RNA was expensive.

Improvement : There are several types of in vitro transcription, among which the in vitro transcription protocol using T7 RNA polymerase was selected. Therefore, the T7 promotor sequence was attached in front of the designed RNA sequence, and the sequence was changed to DNA.

### Hairpin - van't hoff equation

Imagine : We can hardly get into our lab. So we need to do some modeling to predict the thermal behavior of our designed parts

Design : If we use van’t hoff equation, it is assumed that we can predict the fraction of certain secondary structures of our RNAs.

![](https://static.igem.org/mediawiki/2020/0/02/T--KUAS_Korea--equation.png)

Build : I tried to build the graph from van’t hoff equation. K is the ratio that is the fraction of the folded RNA divided by the fraction of the unfolded RNA. f is the fraction of the folded RNA.

![](https://static.igem.org/mediawiki/2020/1/10/T--KUAS_Korea--equation2.png)

Learn : We found that at least one experimental value is needed to draw a graph using van't hoff equation. To know F, we must know K\_1 at T\_1.

Improve : We needed to find a new method that doesn’t require any experiment. Furthermore, even though we found an initial value from an experiment and plot the van’t hoff graph. We need to do a modeling for hybridization. So the method we designed here is not sufficient.

### Hybridization

Imagine : So what we need to do is hybridization modeling not secondary structure modeling.

Details : In Mfold, there is a hybridization of two different strand modeling. [[Link]](https://2020.igem.org/Team%3AKUAS_Korea/Model)

### Experiment Design

Imagine : It was thought that results similar to the modeling values described above would be produced by experiments.

Design & Build: To confirm this, time-scale experiments for each temperature per target C [standard C, Modified Cs] were designed. And graphs will be drawn with the fluorescence intensity for each temperature of each target C. [[Link]](https://2020.igem.org/Team%3AKUAS_Korea/Experiments)

Test : Planning to do.

### Body temperature measurement

Imagine : In order to observe the color change of the sticker, it must be attached to the outside that is easily visible. Currently, a thermometer that measures body temperature is a method of measuring the temperature inside the body that is mostly covered.

Design : We investigated the locations where body temperature could be measured, and determined which locations could represent body temperature well and could be easily observed.

Build & Test : For the skin temperature to represent the core temperature, the location of the skin to be measured must be near the major vessel or core tissue. For this reason, the locations of the skin commonly used to measure body temperature are the mouth, ears, armpits, and rectum. In the case of the neck, carotid artery, a major vessel, exists under the skin. In addition, two papers could be found saying that the body temperature can be calculated when measuring the skin temperature of the neck.

Learn : Therefore, based on the results of the above two papers, it can be said that the core temperature can be measured through the neck.

Improvement : Our sticker needs to be observed from the outside, and the neck is exposed outside the other positions mentioned above. Therefore, we decided the position to attach the sticker to the neck.

Literatures : (1) NIHR, “Non-contact infrared thermometers”, Horizon Scan Report 0025, November 2013, (2) Denise Landon et al, “12 ways to take a temperature”, www.NursingMadeIncrediblyEasy.com, (3) A Did You Know Poster by Diane L Carroll, PhD, RN, and Donna Furlong, RN, (4) http://www.exodontia.info/Cranial-Temporal-Giant\_Cell\_Arteritis.html, (5) Venkatesh Selvaraj and Pughal Vendan Gnanaprakasam, "Evaluation of skin temperature over carotid artery for temperature monitoring in comparison to nasopharyngeal temperature in adults under general anesthesia”, Anesth Essays Res. 2016 May-Aug; 10(2): 291–296., (6) Håkan Geijer, Ruzan Udumyan, Georg Lohse, and Ylva Nilsagård, “Temperature measurements with a temporal scanner: systematic review and meta-analysis”, BMJ Open. 2016; 6(3): e009509., (7) C K Suhail, Nandini Dave, Raylene Dias, and Madhu Garasia, “Accuracy of skin temperature over carotid artery in estimation of core temperature in infants and young children during general anaesthesia”, Indian J Anaesth. 2018 Jun; 62(6): 418–423., (8) Pradeep Buddharaju, Ioannis Pavlidis, and Chinmay Manohar, “Face Recognition Beyond the Visible Spectrum”, Advances in Biometrics, pp 157-180

### KUAS

Korea University

In-Geol Choi

South Korea

**Tel:** +82 10 3103 7332

**Email:** kuaskorea@gmail.com

#### Sponsors

[![CSBL](https://static.igem.org/mediawiki/2020/1/13/T--KUAS_Korea--Transparent_CSBL.png)](http://compbio.korea.ac.kr/)CSBL at Korea University

![WingstaBio](https://static.igem.org/mediawiki/2020/4/40/T--KUAS_Korea--Transparent_Wingstabio.png)Wingstabio 이중철 (Joong-Chul Lee)

![DC Medical](https://static.igem.org/mediawiki/2020/6/6b/T--KUAS_Korea--Transparent_Decimal.png)DC Medical

이덕한 (TheKhan Lee)

![](https://static.igem.org/mediawiki/2020/6/67/T--KUAS_Korea--Transparent_CTL.png)CTL

![KUSSO](https://static.igem.org/mediawiki/2020/8/89/T--KUAS_Korea--Transparent_KUSSO.png)KUSSO

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

# Team:KUAS Korea/Experiments

# [KUAS](/Team%3AKUAS_Korea)

* [Home](/Team%3AKUAS_Korea)
* Team
  + [Team members](/Team%3AKUAS_Korea/Team)
  + [Collaborations](/Team%3AKUAS_Korea/Collaborations)
  + [Attributions](/Team%3AKUAS_Korea/Attributions)
  + [Official Team Profile](//igem.org/Team.cgi?team_id=3421)
* Project
  + [Project Description](/Team%3AKUAS_Korea/Description)
  + [Design](/Team%3AKUAS_Korea/Design)
  + [Experiments](/Team%3AKUAS_Korea/Experiments)
  + [Results](/Team%3AKUAS_Korea/Results)
  + [Safety](/Team%3AKUAS_Korea/Safety)
  + [Model](/Team%3AKUAS_Korea/Model)
  + [Engineering](/Team%3AKUAS_Korea/Engineering)
  + [Implementation](/Team%3AKUAS_Korea/Implementation)
  + [Contribution](/Team%3AKUAS_Korea/Contribution)
* Human Practices
  + [Integrated Practices](/Team%3AKUAS_Korea/Human_Practices)
  + [Public Engagement & Science Communication](/Team%3AKUAS_Korea/Education)
* Additional Project
  + [Additional Project](/Team%3AKUAS_Korea/CCP)

# Experiments

## Experiments

### 1. Preparation

#### 1.1 DNA order

Since in vitro transcription is to be performed using T7 RNA polymerase, a duplex dsDNA template is prepared by attaching the T7 promotor sequence (shown in red) in front of the RNA sequence to be used. DNA was ordered through IDT. The sequences are as follows.

**Standard CHA sequence**
H1 : TAATACGACTCACTATAG GAGCGATACTGTTGGTAGATGTCGCCATGTCTTGCGACATCTACCAACAGCGAGACAACAGAGAGACGGTCGGGTCCACAGTTTCC
H2 : TAATACGACTCACTATAG GGAAACTGTGTCGAGTAGAGTGTGGGCTCTCTGTTGTCTCGAGATGTCGCAAGACATGGCGACATCTACCAACAGCCATGTCTTG
Standard Target C : TAATACGACTCACTATAG GACATCTACCAACAGTATCGCTC
 **Modified target C(to be used)**
C1 : TAATACGACTCACTATAG CTGGACATCTACCAACAGTATCGCTC
C2 : TAATACGACTCACTATAG TGTGACATCTACCAACAGTATCGCTC
C3 : TAATACGACTCACTATAG GTTGACATCTACCAACAGTATCGCTC
C4 : TAATACGACTCACTATAG CTGTGACATCTACCAACAGTATCGCTC
C5 : TAATACGACTCACTATAG TGTTGACATCTACCAACAGTATCGCTC
C6 : TAATACGACTCACTATAG GTTGGACATCTACCAACAGTATCGCTC
Broccoli sequence(Control) : TAATACGACTCACTATAG GAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTC

#### 1.2 RNA preparation

**In vitro transcription with T7 RNA polymerase**
In vitro transcription using T7 RNA polymerase is performed to synthesize RNAs (RNA aptamers [H1, H2, Thermosensing RNAs], target RNA sequence).
In vitro T7 transcription is the synthesis of RNAs using a T7 promoter and purified enzyme. It is the standard method of making up to several mg of RNAs longer than about 20nts with relatively high quality as compared to solid phase synthesis.

### 2. Activity test [Time-scale fluorescence assay]

Using the previously made RNAs (H1, H2, Thermosensing RNAs, target RNA sequence) and Fluorophore (DFHBI-1T), we verify that the CHA system used by our team works well. Also, we verify that the system we designed works by detecting the target temperature of 37.5℃ well.
In all experiments below, H1 and H2 are used at 250nM according to the referenced paper. DFBHI-1T is used at 5 μM according to the paper. Also all experiments are repeated 3 times to calculate the average.

#### 2.1 Conventional CHA system verification

Using standard target C, we are going to confirm that the Standard CHA system works well. Experiment with the same conditions as the paper referenced, and check whether the data appears the same.

#### 2.2 Fluorescence intensity according to temperature

Through a time-scale experiment for each temperature per target C[standard C, Modified Cs], the time for each fluorescence intensity to reach equilibrium is obtained, and at this point, a graph is drawn with the fluorescence intensity for each temperature of each target C.
Through the graph, we check the temperature that the fluorescence intensity starts increasing, and compared the results with that of modeling [[Link]](https://2020.igem.org/Team%3AKUAS_Korea/Model).
A sample without target C was used as a negative control, and a sample containing only broccoli sequence and a fluorophore was used as a positive control.
Measurements are made for each target C every 10 minutes, from 30℃ to 40℃ at the intervals of 2℃.

## Protocol

### 1. In vitro transcription with T7 RNA polymerase

**Materials**
Template DNA
Transcription buffer and other components
1X buffer (Make 10X stock, store at -20°C.)
- 50 mM Tris-HCl, pH 7.5
- 15 mM MgCl2
- 5 mM dithiothreitol (DTT)
- 2 mM spermidine
10X NTPs (Store at -20°C.)
- 20 mM each of ATP, CTP, GTP, and UTP
Inorganic pyrophosphatase (optional, 0.1 U/μL stock solution in H2O, store at -20°C.)
T7 RNA polymerase
**Method - Transcription reaction**
1. For a 100 μL reaction ("preparative scale") Put the following components in the EP tube:
- 10 μL 10X transcription buffer
- 10 μL 10X NTPs
- x μL DNA template (DNA template amount should be 5–10 pmol)
- 5 μL inorganic pyrophosphatase (0.1 U/μL): 0.005 U/μL final concentration
- y μL T7 RNAP (25 U/μL final concentration)
- Add 4DW to 100 μL
2. Incubate reaction at 37 °C for 2 hr.
3. Add 10U of RNase-free DNase I and incubate at 37 °C, 30 min.
4. Add 5 μL of 500 mM EDTA to stop the reaction.
5. Clean-up/process the RNA.

### 2. RNA Purification by Preparative Polyacrylamide Gel Electrophoresis

Since the length of the RNA to be purified is less than 100nt, a 20% acrylamide gel is used.
![](https://static.igem.org/mediawiki/2020/7/77/T--KUAS_Korea--acrylamide.png)

Sambrook J, et al. (2001)

**Materials**
40% acrylamide/bisacrylamide (19:1)
40% acrylamide/bisacrylamide (29:1)
Urea
Tris base
Boric acid (H3BO3)
EDTA
Potassium hydroxide (KOH)
Ammonium persulfate (APS)
N,N,N’,N’ -tetramethylethylenediamine (TEMED)
Formamide
Sodium dodecyl sulfate (SDS)
Bromophenol blue
Xylene cyanol
Ammonium acetate (NH4OAc)
Sodium acetate (NaOAc)
Ethanol
Solutions & buffers
Step 1 10x TBE
- Tris base 890mM 108g
- EDTA, pH 8.0 20mM 40ml (stock 0.5M)
- Boric acid 890mM 55g
- Dissolve Tris and boric acid in ~750 ml of deionized water. Add EDTA. Adjust final volume to 1 L with water. There is no need to adjust the pH of this solution
Native gel mix
- TBE 1x 40ml (stock 10x)
- Acrylamide/bis-acrylamide (29:1) X% (X/40)400ml (stock 40%)
- Ammonium persulfate 0.08% 3.2ml (stock 10%)
- Add deionized water to 400 ml
Denaturing gel mix
- TBE 1x 40ml (stock 10x)
- Acrylamide/bis-acrylamide (19:1) X% (X/40)400ml (stock 40%)
- Urea 6.5M 260ml (stock 10M)
- Ammonium persulfate 0.08% 3.2ml (stock 10%)
- Add deionized water to 400 ml
Step 2 2x Denaturing loading buffer
- Formamide 95% 9.5ml (stock 100%)
- EDTA-KOH, pH 8.0 18mM 360μl (stock 500mM)
- SDS 0.025% 25μl (stock 10%)
- Bromophenol blue 0.05% 5mg
- Xylene cyanol 0.05% 5mg
- Add water to 10ml
5x Nondenaturing loading buffer
- TBE 5x 5ml (stock 10x)
- Glycerol 20% 2ml (stock 100%)
- Bromophenol blue 0.05% 5m
- Xylene cyanol 0.05% 5mg
- Add water to 10ml
Running buffer
- TBE 1x 100ml (stock 10x)
- Deionized water 900ml
Step 4 Elution Solution
- NH4OAc 500mM 250ml (stock 2M)
- EDTA-KOH, pH 8.0 1mM 2ml (stock 500mM)
- Add water to 1L
70% Ethanol (v/v)
- Ethanol 70% 7.3ml (stock 96%)
- Water 2.7ml
**Method**
![](https://static.igem.org/mediawiki/2020/9/98/T--KUAS_Korea--preparation.png)

Petrov A, et al. (2013)

**STEP 1 - PREPARING THE GEL**
1.1 Treat gel plates with a siliconizing agent.
1.2 Assemble the gel plates with 1.5mm spacers.
1.3 Prepare the appropriate gel mixture (for native or denaturing gels). The percentage of acrylamide depends on the sizes of the RNA molecules you wish to resolve.
1.4 Add 160μl of TEMED for every 400 ml of the gel mixture to start polymerization. Quickly mix the solution (without introducing air bubbles) and pour the gel. Insert the desired comb and allow the gel to polymerize.
1.5 Mount the gel plates onto the gel running apparatus. Add 1x TBE to both the upper and lower reservoirs. Remove the comb and rinse the wells with 1x TBE using a micropipettor fitted with a gel-loading tip.
1.6 For denaturing gels larger than 20 x 20 cm, clamp an aluminum plate to the front side of the gel plate.
1.7 Prerun denaturing gels at 45–65 V cm^-1 for 30–60 min to equilibrate and preheat the gel. Skip this step for the native gels.

**STEP 2 – RUNNING THE GEL**
2.1 Mix the RNA sample with the appropriate loading buffer. If running a denaturing gel, add equal volumes RNA sample and 2x denaturing loading buffer. If running a native gel, add 1 volume of 5x nondenaturing loading buffer to 4 volumes of RNA sample.
2.2 Heat the samples for the denaturing gel at 94 C for 5 min.
2.3 Rinse the wells extensively with 1x TBE using micropipettor fitted with a gel-loading tip. Load the samples into the wells.
2.4 Run a denaturing gel at 45–65 V cm^-1; run a native gel at 10–25 V cm^-1.
2.5 Use the mobility of the tracking dyes on the gel to determine when to stop running the gel.

**STEP 3 - VISUALIZING THE RNA**
3.1 Remove the gel plates from the gel running apparatus. Remove the spacers. Use a metal spatula to pry open the top glass plate of the gel.
3.2 Cover the gel with plastic wrap. Carefully flip the gel plate so that the side with the gel covered in plastic wrap is facing down. Remove the glass plate. Wrap the gel in plastic wrap.
3.3 Place the gel on top of a fluorescent plate and shine UV light on it.
3.4 The RNA will be visible as dark spots on a brightly fluorescing background.

**STEP 4 - RNA EXTRACTION USING THE ‘CRUSH AND SOAK’ METHOD**
4.1 Cut out the band of interest using a new, clean razor blade.
4.2 Weigh a 50 -ml conical tube and note the weight of the empty tube. Transfer the gel slice into the tube and crush the gel using a disposable pipette.
4.3 Weigh the tube containing the crushed gel slice and calculate the weight of the gel. Add ~2 volumes of Elution Solution (v/w) to the gel.
4.4 Incubate on a tube on a nutator at room temperature for 3 h.
4.5 Centrifuge sample at 5000 x g for 1 min. Collect the supernatant; avoid picking up gel debris. Repeat the centrifugation two more times.
4.6 Add 0.1 volume of 3 M NaOAc, pH 5.2, and 3 volumes of ethanol. Incubate at -20 ℃ for 1 h to precipitate the RNA.
4.7 Collect the RNA by centrifugation at 15 000 rpm for 15 min at 4 ℃. Decant the supernatant and wash the pellet with 70% ethanol.
4.8 Collect the RNA by centrifugation at 15 000 rpm for 15 min at 4 ℃. Decant the supernatant and air-dry the pellet. 4A.9 Resuspend the RNA pellet in water or a desired buffer.
4.9 Resuspend the RNA pellet in water or a desired buffer.

### 3. Determine the concentration of RNA

RNA concentration measurement is performed using a Nanodrop spectrophotometer.
Protocol
1. Enter Nanodrop spectrophotometer on the PC and select “nucleic acids”
2. Pipette 1-2μl of elution buffer (or D.W) onto the measurement pedestal. [Blank]
3. Absorb the elution buffer with dry, lint-free laboratory wipe.
4. Pipette 1-2μl of sample directly onto the measurement using the software on the PC.
5. Lower the sampling arm and initiate a spectral measurement using the software on the PC.
6. When the measurement is complete, raise the sampling arm and wipe the sample from both the upper and lower pedestals using a dry, lint-free laboratory wipe.
7. data analysis - 260/280 ratio of ~1.8 is generally accepted as “pure” for DNA and a ratio of ~2.0 is generally accepted as “pure” for RNA. For any DNA sample with a 260/280 ratio more than 1.8 indicates the presence of RNA as contamination.

### 4. Storing RNA

Always store RNA at a neutral pH with some amount of EDTA. Recommend one is TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). Store RNA at -20°C.

### 5. Time scale fluorescence assay

All the in vitro fluorescence measurements are conducted in a PTI fluorimeter (Horiba, New Jersey, NJ).
RNA molecules are pre-heated at 95°C for 3 min and slowly cooled down to 25°C at the rate of -3°C/min.
Fluorescence assays and RNA assembly reactions are conducted in a buffer consisting of 10mM Tris, 5mM MgCl2, 100mM KCl, and 10mM NaCl at pH=7.5.
250nM H1 and H2 are used for these measurement.
All the reactions are initiated by adding H1 to the mixture and the samples have been incubated for 2 h before taking the measurements.
The kinetic assays are conducted by exciting at 480 nm and collecting the fluorescence data at 500 – 550 nm.
All the data are plotted using the Python software.

## References

1) Cazenave C, Uhlenbeck OC. RNA template-directed RNA synthesis by T7 RNA polymerase. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6972-6.
2) Alexey Petrov, Tinghe Wu, Elisabetta Viani Puglisi, Joseph D. Puglisi, “RNA Purification by Preparative Polyacrylamide Gel Electrophoresis”, Methods in Enzymology, Volume 530.
3) Aruni P. K. K. Karunanayake Mudiyanselage, Qikun Yu, Mark A. Leon-Duque, Bin Zhao, Rigumula Wu, and Mingxu You, “Genetically Encoded Catalytic Hairpin Assembly for Sensitive RNA Imaging in Live Cells”, Journal of the American Chemical Society, 2018, 140, 8739−8745.
4)https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FCAD%2Fmanuals%2Fts-nanodrop-nucleicacid-olv-r2.pdf&title=TmFub0Ryb3AgTnVjbGVpYyBBY2lkIEhhbmRib29r

### KUAS

Korea University

In-Geol Choi

South Korea

**Tel:** +82 10 3103 7332

**Email:** kuaskorea@gmail.com

#### Sponsors

[![CSBL](https://static.igem.org/mediawiki/2020/1/13/T--KUAS_Korea--Transparent_CSBL.png)](http://compbio.korea.ac.kr/)CSBL at Korea University

![WingstaBio](https://static.igem.org/mediawiki/2020/4/40/T--KUAS_Korea--Transparent_Wingstabio.png)Wingstabio 이중철 (Joong-Chul Lee)

![DC Medical](https://static.igem.org/mediawiki/2020/6/6b/T--KUAS_Korea--Transparent_Decimal.png)DC Medical

이덕한 (TheKhan Lee)

![](https://static.igem.org/mediawiki/2020/6/67/T--KUAS_Korea--Transparent_CTL.png)CTL

![KUSSO](https://static.igem.org/mediawiki/2020/8/89/T--KUAS_Korea--Transparent_KUSSO.png)KUSSO

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

# Team:KUAS Korea/Results

# [KUAS](/Team%3AKUAS_Korea)

* [Home](/Team%3AKUAS_Korea)
* Team
  + [Team members](/Team%3AKUAS_Korea/Team)
  + [Collaborations](/Team%3AKUAS_Korea/Collaborations)
  + [Attributions](/Team%3AKUAS_Korea/Attributions)
  + [Official Team Profile](//igem.org/Team.cgi?team_id=3421)
* Project
  + [Project Description](/Team%3AKUAS_Korea/Description)
  + [Design](/Team%3AKUAS_Korea/Design)
  + [Experiments](/Team%3AKUAS_Korea/Experiments)
  + [Results](/Team%3AKUAS_Korea/Results)
  + [Safety](/Team%3AKUAS_Korea/Safety)
  + [Model](/Team%3AKUAS_Korea/Model)
  + [Engineering](/Team%3AKUAS_Korea/Engineering)
  + [Implementation](/Team%3AKUAS_Korea/Implementation)
  + [Contribution](/Team%3AKUAS_Korea/Contribution)
* Human Practices
  + [Integrated Practices](/Team%3AKUAS_Korea/Human_Practices)
  + [Public Engagement & Science Communication](/Team%3AKUAS_Korea/Education)
* Additional Project
  + [Additional Project](/Team%3AKUAS_Korea/CCP)

# Results

## Introduction

We couldn't get into our lab this year. What we did can be divided into two sections. First, we modeled and predicted behaviors of systems in our final product. Second, we designed our future experiments.

## Patch prediction model

|  |  |
| --- | --- |
| Original Sequences in the Reference Paper | |
| H1 | GAGCGATACTGTTGGTAGATGTCGCCATGTCTTGCGACATCTACCAACAGCGAGACAACAGAGAGACGGTCGGGTCCACAGTTTCC |
| H2 | GGAAACTGTGTCGAGTAGAGTGTGGGCTCTCTGTTGTCTCGAGATGTCGCAAGACATGGCGACATCTACCAACAGCCATGTCTTG |
| C | GACATCTACCAACAGTATCGCTC |
| Modified Sequences with 3 elongated base pairs | |
| C1 | CTGGACATCTACCAACAGTATCGCTC |
| C2 | TGTGACATCTACCAACAGTATCGCTC |
| C3 | GTTGACATCTACCAACAGTATCGCTC |
| Modified Sequences with 4 elongated base pairs | |
| C4 | CTGTGACATCTACCAACAGTATCGCTC |
| C5 | TGTTGACATCTACCAACAGTATCGCTC |
| C6 | GTTGGACATCTACCAACAGTATCGCTC |

The table above is the sequences we designed. We added 3-4 nucleotides to the original C sequence that are complementary to a region with in itself so that it would bind to itself and form a loop.

We modeled 4 things and got results of them. (1) Heat transfer and distribution (2) the behavior of the system according to temperature (3) the behavior of the system according to time (4) visibility

#### (1) Heat transfer and distribution

![](https://static.igem.org/mediawiki/2020/5/55/T--KUAS_Korea--heat.png)

The figure above is the heat distribution we modeled.

#### (2) The behavior of the system according to temperature

|  |  |  |
| --- | --- | --- |
| Modified Sequences with elongated base pairs | | Threshold Temperature (°C) |
| C1 | CTGGACATCTACCAACAGTATCGCTC | 51 |
| C2 | TGTGACATCTACCAACAGTATCGCTC | 34 |
| C3 | GTTGACATCTACCAACAGTATCGCTC | 40 |
| C4 | CTGTGACATCTACCAACAGTATCGCTC | 34 |
| C5 | TGTTGACATCTACCAACAGTATCGCTC | 42 |
| C6 | GTTGGACATCTACCAACAGTATCGCTC | Not found |

The table above is the threshold temperatures where the patch starts to be activated.

#### (3) The behavior of the system according to time

It is assumed that it takes about 25 minutes to fully activate the patch system.

#### (4) visibility

|  |  |
| --- | --- |
| Name | Molar concentration (M) |
| H1 | \( 4.08 \times 10^{-06} \) |
| H2 | \( 4.08 \times 10^{-06} \) |
| Cs | \( 4.08 \times 10^{-09} \) |
| DFHBI-1T | \( 8.15 \times 10^{-05} \) |

The table above is the molar concentrations needed to make the patch visible when it is activated.

Details are described in model section. **[[Link]](https://2020.igem.org/Team%3AKUAS_Korea/Model)**

## Experiment design

We designed our future experiment plan.

Details are described in experiments section. **[[Link]](https://2020.igem.org/Team%3AKUAS_Korea/Experiments)**

### KUAS

Korea University

In-Geol Choi

South Korea

**Tel:** +82 10 3103 7332

**Email:** kuaskorea@gmail.com

#### Sponsors

[![CSBL](https://static.igem.org/mediawiki/2020/1/13/T--KUAS_Korea--Transparent_CSBL.png)](http://compbio.korea.ac.kr/)CSBL at Korea University

![WingstaBio](https://static.igem.org/mediawiki/2020/4/40/T--KUAS_Korea--Transparent_Wingstabio.png)Wingstabio 이중철 (Joong-Chul Lee)

![DC Medical](https://static.igem.org/mediawiki/2020/6/6b/T--KUAS_Korea--Transparent_Decimal.png)DC Medical

이덕한 (TheKhan Lee)

![](https://static.igem.org/mediawiki/2020/6/67/T--KUAS_Korea--Transparent_CTL.png)CTL

![KUSSO](https://static.igem.org/mediawiki/2020/8/89/T--KUAS_Korea--Transparent_KUSSO.png)KUSSO