iGEM 2021 — Team KUAS_Korea

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# 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)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:KUAS Korea/Engineering

* KUAS\_Korea
* [Home](https://2021.igem.org/Team%3AKUAS_Korea)
* [Team](https://2021.igem.org/Team%3AKUAS_Korea/Team2)
* Collaborations
  + [Collaborations](https://2021.igem.org/Team%3AKUAS_Korea/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3AKUAS_Korea/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3AKUAS_Korea/Description)
  + [Design](https://2021.igem.org/Team%3AKUAS_Korea/Design)
  + [Experiments](https://2021.igem.org/Team%3AKUAS_Korea/Experiments)
  + [Model](https://2021.igem.org/Team%3AKUAS_Korea/Model)
  + [Engineering](https://2021.igem.org/Team%3AKUAS_Korea/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AKUAS_Korea/Implementation)
  + [Contribution](https://2021.igem.org/Team%3AKUAS_Korea/Contribution)
* Human Practices
  + [Human Practices](https://2021.igem.org/Team%3AKUAS_Korea/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AKUAS_Korea/Communication)
  + [Sustainable](https://2021.igem.org/Team%3AKUAS_Korea/Sustainable)

![](https://static.igem.org/mediawiki/2021/a/a2/T--KUAS_KOREA--frog_banner_2.png)

# Engineering Success

### Kill switch design

As a safety measure, we have designed a kill switch which is activated when the cell detaches from the colony. This mechanism will prevent the cells from surviving when they detach from the frog's mucus layer. This composite part contains the luxR,luxI system, cI inverter, and a MazE,MazF toxin-antitoxin system.

[BBa\_K3837011](http://parts.igem.org/Part%3ABBa_K3837011)

According to this design, when cells exist in high densities, there should be enough HSL to induce the expression of cI repressors that would repress the expression of MazF toxin. On the other hand, for the detached cell, HSL must be in concentrations which is not enough to induce cI repressor expression. Without the repressor MazF toxin will increase and eventually kill the cell.

However, While designing our quorum sensing based kill switch, we were unsure of which combination of promoters and RBS to use for LuxR and LuxI expression. Therefore, we decided to create quorum sensing models with different combination of promoters(J23100,J23106) and RBS(B0034,B0032), and revise the design with the one with the most promising results. We created two different models with Matlab for two different situations. The first model considers a situation when a single cell has detached from the colony and presumes that external concentration of HSL is 0. In this model, HSL/LuxR dimers must be low enough to prevent the expression of cI repressors. The second model assumes a colony of cells in a closed 1mL of space. HSL produced by the cells will accumulate within this space and eventually will start to flow back into the cell and induce expression. The results for the model must show enough HSL to induce cI production. Results can be seen in our [Model page](https://2021.igem.org/Team%3AKUAS_Korea/Model)

The only construct that showed a significant difference of HSL/LuxR dimers at the two models contained J23100 and R0034 for both luxR and luxI. Through this modeling process, we were able to finalize the design which is expected to show the most promising results.

![](https://static.igem.org/mediawiki/2021/1/11/T--KUAS_Korea--_final_kill_swtich.png)

### Disabling CadC DBD

The dimerization of LYK5 is independent of chitin or CERK1 presence. Therefore, to prevent spontaneous gene experssion by the dimerization of DBDs, LYK5 domains have to be fused to CadC with a nonfunctional DBD. Luckily, we found a research paper which characterized the binding mechanism of CadC DBDs. Schlundt A et. al. identified Aa residues involved in both specific and non specific DNA binding.

![](https://static.igem.org/mediawiki/2021/1/19/T--KUAS_Korea--_CadC_residues.png)

Figure source: Schlundt A et. al. Structure-function analysis of the DNA-binding domain of a transmembrane transcriptional activator. Sci Rep. 2017 Apr 21;7(1):1051. doi: 10.1038/s41598-017-01031-9. PMID: 28432336; PMCID: PMC5430869. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Diagram (d) in the figure above shows the interaction of Aa residues and the DNA. Red dotted lines indicate specific binding while the black dotted lines indicate non-specific binding. Among the essential Aa residues Thr69 does not directly bind to DNA but stabilizes his66 and arg50 which are involved in DNA binding. Thus we decided to mutate Thr69 to inhibit DNA binding.

![](https://static.igem.org/mediawiki/2021/f/fe/T--KUAS_Korea--_CadC_mut_activity.png)

Figure source: Schlundt A et. al. Structure-function analysis of the DNA-binding domain of a transmembrane transcriptional activator. Sci Rep. 2017 Apr 21;7(1):1051. doi: 10.1038/s41598-017-01031-9. PMID: 28432336; PMCID: PMC5430869. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Experimental results from the diagram above shows that T69A mutation completely disables gene expression from the promoter cadBA. So we substituted thr69 with ala so that the dimer does not have cadBA activation in our part. Though we couldn't test this part this year, we hope our rational design process turns out to be successful.

KUAS

Korea University

In-Geol Choi

South Korea

Tel: +82 10 3103 7332

Email: kuaskorea@gmail.com

Sponsor and Special thanks for

[![](https://static.igem.org/mediawiki/2021/7/7a/T--KUAS_KOREA--tiger.jpg)](http://compbio.korea.ac.kr/csbl/index.html)

CSBL at Korea University

Sponsors

![](https://static.igem.org/mediawiki/2021/0/0f/T--KUAS_KOREA--sponsor_safety_grant.jpg)

![](https://static.igem.org/mediawiki/2021/9/90/T--KUAS_KOREA--sponsor_impact_grant.jpg)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:KUAS Korea/Experiments

* KUAS\_Korea
* [Home](https://2021.igem.org/Team%3AKUAS_Korea)
* [Team](https://2021.igem.org/Team%3AKUAS_Korea/Team2)
* Collaborations
  + [Collaborations](https://2021.igem.org/Team%3AKUAS_Korea/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3AKUAS_Korea/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3AKUAS_Korea/Description)
  + [Design](https://2021.igem.org/Team%3AKUAS_Korea/Design)
  + [Experiments](https://2021.igem.org/Team%3AKUAS_Korea/Experiments)
  + [Model](https://2021.igem.org/Team%3AKUAS_Korea/Model)
  + [Engineering](https://2021.igem.org/Team%3AKUAS_Korea/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AKUAS_Korea/Implementation)
  + [Contribution](https://2021.igem.org/Team%3AKUAS_Korea/Contribution)
* Human Practices
  + [Human Practices](https://2021.igem.org/Team%3AKUAS_Korea/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AKUAS_Korea/Communication)
  + [Sustainable](https://2021.igem.org/Team%3AKUAS_Korea/Sustainable)

![](https://static.igem.org/mediawiki/2021/a/a2/T--KUAS_KOREA--frog_banner_2.png)

# Experiments

## Violacein AND gate

The original design for the AND gate was to use an igem part T7 polymerase AND gate (BBa\_K568004). However this part along with the Vio operon would result a part that is almost 15kb. Our PI noted us of the difficulties of transforming a part of such size an suggested a different AND gate design. His AND gate design was a split vio operon which had few vio genes under control of one promoter and the rest of the genes under the control of a different promoter. Thus, the full violacein pathway is constituted only when both of the promoters are activated.

As described in the Description page, we decided to use light as one of the inputs for violacein production. For the second input, for simplification and reliability, the IPTG inducible lac promoter was used.
Gibson assembly was used to connect two addgene plasmids:

[Violacein `addgene' vector](https://www.addgene.org/73440/)

[Violacein `addgene' vector](https://www.addgene.org/43796/)

The final construct is as follows.

![final_pathway](https://user-images.githubusercontent.com/87188354/137825930-f057ebca-0942-45a7-8f6b-6c5e437be36e.png)

Though it would be the best to include the full violacein pathway, the gibson process would be a lot more complicated. Thus, our PI suggested a proof of concept plasmid which has vioA under the control of pDawn system and vioB and vioE under the control of the IPTG inducible system.

Due to time constraints and the fact that there were no experienced wet lab member in our team, our wet lab instructors Sera Kang and Xiaoyue conducted the assembly and transformation proccess.

The protocols for the experiments can be seen below.

## Protocol

### A. Prepare pDawn vector with enzyme cut for Gibson Assembly(GA)

- plasmid enzyme cut → check gel → inactivation → clean up

#### 1. Mix all below chemical substances to make total 50 ㎕ solution.

|  |  |
| --- | --- |
| **Total** | **50 ㎕** |
| pDawn | 40 ㎕ |
| NdeI | 1 ㎕ |
| NotI-HF | 1 ㎕ |
| Cutsmart(10x) | 5 ㎕ |
| DW | 3 ㎕ |

#### 2. Let mixture in 37℃ for 50 minutes.

#### 3. Check gel with uncut.

#### 4. Put mixture in 65℃ for 20 minutes.

#### 5. Clean up

- 1-1, 1-3 -> 50 ㎕ elution

- 2-1, 2-2, 2-3 -> 100 ㎕ elution

### B. Prepare insert sequence "vio ABE" PCR for GA

#### 1. Mix all below chemical substances

|  |  |
| --- | --- |
| **Total** | **25 ㎕** |
| template(1/10) | 2 ㎕ |
| reaction buffer | 5 ㎕ |
| dNTP | 2 ㎕ |
| Primer -Forward | 1 ㎕ |
| Primer -Reverse | 1 ㎕ |
| Q5 polymerase | 0.2 ㎕ |
| DW | 13.8 ㎕ |

\* plasmid: pETM6-G6-vioABE-4A6-vioC-3A2-vioD

\* primer

(1) vioA(1.4 kb) : vioA\_F(55℃), vioA\_R(60℃)

(2) vioAB(4.7 kb) : vioA\_F(55℃), vioAB\_R(53℃)

(3) vioAB(vioABE)(3.5 kb) : vioA\_F(55℃), vioABE\_mid\_R(56℃)

(4) vioBE(vioABE)(2.1 kb) : vioABE\_mid\_F(56℃), vioABE\_R(54℃)

(5) vioABE(5.6 kb) : vioA\_F(55℃), vioABE\_R(54℃)

#### 2. Turn PCR.

![PCR_process](https://user-images.githubusercontent.com/79410957/138125700-d86c41f4-21d6-4c67-b16d-1c0c3b51d61d.png)

- (1) 56℃ / 58℃ / 60℃ in 1 minute.

- (2), (5) 53℃ / 55℃ / 57℃ in 3 minutes.

- (3) 54℃ / 56℃ / 58℃ in 2 minutes.

- (4) 53℃ / 55℃ / 57℃ in 1 and a half minute.

- (1), (4) 53℃ / 56℃ / 60℃ in 1 and a half minute.

=> Mix and put DPN 1.5 ㎕ each of them.

- (2), (3), (5) 53℃ / 56℃ / 58℃ in 3 minutes.

=> (5) showed 2 bands, so we redo with a little higher temperature.

- (5) again 58℃ / 60℃ / 62℃ in 3 minutes.

=> It showed 2 bands again, and we figured out that vioA\_F has overlapped sequence so that it resulted in 2 bands.

![result_band_vioA_BE](https://user-images.githubusercontent.com/79410957/138127890-c71e1eeb-b9ee-48e0-94e6-8e22556ea161.png)

Result band of vioA and vioBE

![result_band_vioAB](https://user-images.githubusercontent.com/79410957/138127912-a6787096-09cf-43ad-81f4-1cce91052a83.png)

Result band of vioAB

### C. Combine vector and insert in order to do GA

#### 1. vioABE

|  |  |  |
| --- | --- | --- |
| **pDawn(vector)** | **vioAB(vioABE)** | **vioBE(vioABE)** |
| 7.1 kb | 3.5 kb | 2.1 kb |
| 21 ng/㎕ | 47 ng/㎕ | 65 ng/㎕ |
| 0.0045 pmol | 0.02 pmol | 0.047 pmol |

(1) Mix vioAB and vioBE with 2 : 1 ratio.

(2)

|  |  |  |
| --- | --- | --- |
|  | 1:2 | 1:3 |
| vector | 1/5 | 1.5/5 |

(3-1)

|  |  |
| --- | --- |
| **Total** | **20 ㎕** |
| pDawn | 5 ㎕ |
| insert | 1 ㎕ |
| HiFi master mix | 10 ㎕ |
| DW | 4 ㎕ |

(3-2) If insert is 0.03 to 0.2 pmol, follow below table. (This can make total amount in a half)

|  |  |
| --- | --- |
| **Total** | **20 ㎕** |
| pDawn | 5 ㎕ |
| insert | 1.5 ㎕ |
| HiFi master mix | 10 ㎕ |
| DW | 3.5 ㎕ |

#### 2. vioA

|  |  |
| --- | --- |
| **pDawn(vector)** | **vioA(insert)** |
| 7.1 kb | 1.4 kb |
| 21 ng/㎕ | 54 ng/㎕ |
| 0.0045 pmol | 0.058 pmol |

(1)

|  |  |  |
| --- | --- | --- |
|  | 1:2 | 1:3 |
| vector | 1.5/6.5 | 1.5/6.5 |

(2-1)

|  |  |
| --- | --- |
| **Total** | **20 ㎕** |
| pDawn | 6.5 ㎕ |
| vioA | 1 ㎕ |
| HiFi master mix | 10 ㎕ |
| DW | 2.5 ㎕ |

(2-2)

|  |  |
| --- | --- |
| **Total** | **20 ㎕** |
| pDawn | 6.5 ㎕ |
| vioA | 1.5 ㎕ |
| HiFi master mix | 10 ㎕ |
| DW | 2 ㎕ |

#### 3. vioAB

|  |  |
| --- | --- |
| **pDawn(vector)** | **vioAB(insert)** |
| 7.1 kb | 4.7 kb |
| 21 ng/㎕ | 30 ng/㎕ |
| 0.0045 pmol | 0.097 pmol |

(1)

|  |  |  |
| --- | --- | --- |
|  | 1:2 | 1:3 |
| vector | 4/4 | 6/4 |

(2-1)

|  |  |
| --- | --- |
| **Total** | **20 ㎕** |
| pDawn | 4 ㎕ |
| vioAB | 4 ㎕ |
| HiFi master mix | 10 ㎕ |
| DW | 2 ㎕ |

(2-2)

|  |  |
| --- | --- |
| **Total** | **20 ㎕** |
| pDawn | 4 ㎕ |
| vioA | 6 ㎕ |
| HiFi master mix | 10 ㎕ |

#### 4. control

|  |  |
| --- | --- |
| **Total** | **10 ㎕** |
| pDawn | 2.5 ㎕ |
| DW | 7.5 ㎕ |

#### 5. Put all in 50℃ for 15 minutes.

#### 6. transformation (DH5α)

### D. vio ABE chemical transformation

#### 1. Mix competent cell 50 ㎕ and plasmid(pDawn\_vio ABE) 2 ㎕.

#### 2. Incubate ice for 30 minutes.

#### 3. Incubate it in 43℃ for 40 seconds.

#### 4. Incubate it in ice for 5 minutes.

#### 5. Mix LB 1ml.

#### 6. Incubate in 37℃ for 1 hour.

#### 7. Centrifuge 13,500 rpm for 1 minute.

#### 8. Discard supernatant 800ul and resuspense leftover.

#### 9. Spread each of 100 ㎕ in LB/KAN plate.

#### 10. Separate each plate with light, and without light. So wrap with tinfoil plate without light.

#### 11. Incubate them in 37℃ through overnight.

## Results

- We made vector and insert sequence that include vio genes into a single plasmid through Gibson assembly.

- By inserting the plasmid into E. coli through transformation, it was possible to create a strain having vioABE that we wanted. This strain was observed to form colonies while growing them in medium.

- However, the results were not promising since there were no visible difference among the two plates. It may be that the light of the incubator was not strong enough to induce a response or maybe the product was not enough to be differentiated with the naked eye. This experiment will need further debugging

- We can get some pictures after transformation that we gained the strain we wanted.

![10.12](https://user-images.githubusercontent.com/79410957/138143507-9a81f908-4393-42da-831d-8ba7b1afc561.jpg)

12 Oct. < immediately after transformation >

![10.14.both](https://user-images.githubusercontent.com/79410957/138143456-fd19abd5-66fe-48e1-9d3f-e52060439c46.jpg)

14 Oct. < 2 days after transformation (both immediately after taking out of refrigerator) >

![10.14.both.open](https://user-images.githubusercontent.com/79410957/138144129-22d8e2e4-2226-442e-82ac-de5c3968c7ca.jpg)

14 Oct. < 2 days after transformation (both plates after removing tin foil from the plate grown without light) >

![10.14.with.light](https://user-images.githubusercontent.com/79410957/138143799-6384aa14-05c4-49af-ab76-1548bdee6c68.jpg)

14 Oct. < 2 days after transformation (plate grown with light) >

![10.14.without.light](https://user-images.githubusercontent.com/79410957/138144013-a1c68920-3b56-4c2e-b1dd-becc0f36171e.jpg)

14 Oct. < 2 days after transformation (plate grown without light) >

![10.18.both](https://user-images.githubusercontent.com/79410957/138144332-1665e426-2aad-47b9-ad47-c567952e39c3.jpg)

18 Oct. < 6 days after transformation (both immediately after taking out of refrigerator) >

![10.18.both.open](https://user-images.githubusercontent.com/79410957/138144433-20d6d046-8c09-4255-aaca-e7f6fecdc090.jpg)

18 Oct. < 6 days after transformation (both plates after removing tin foil from the plate grown without light) >

![10.18.with .light](https://user-images.githubusercontent.com/79410957/138144973-116bc7e2-0c24-42a4-b6bc-28093a0cd514.jpg)

18 Oct. < 6 days after transformation (plate grown with light) >

![10.14.without.light](https://user-images.githubusercontent.com/79410957/138144963-53af02d2-4c75-47c4-b48c-e0538dbf1feb.jpg)

18 Oct. < 6 days after transformation (plate grown without light) >

KUAS

Korea University

In-Geol Choi

South Korea

Tel: +82 10 3103 7332

Email: kuaskorea@gmail.com

Sponsor and Special thanks for

[![](https://static.igem.org/mediawiki/2021/7/7a/T--KUAS_KOREA--tiger.jpg)](http://compbio.korea.ac.kr/csbl/index.html)

CSBL at Korea University

Sponsors

![](https://static.igem.org/mediawiki/2021/0/0f/T--KUAS_KOREA--sponsor_safety_grant.jpg)

![](https://static.igem.org/mediawiki/2021/9/90/T--KUAS_KOREA--sponsor_impact_grant.jpg)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:KUAS Korea/Results

* KUAS\_Korea
* [Home](https://2021.igem.org/Team%3AKUAS_Korea)
* [Team](https://2021.igem.org/Team%3AKUAS_Korea/Team2)
* Collaborations
  + [Collaborations](https://2021.igem.org/Team%3AKUAS_Korea/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3AKUAS_Korea/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3AKUAS_Korea/Description)
  + [Design](https://2021.igem.org/Team%3AKUAS_Korea/Design)
  + [Experiments](https://2021.igem.org/Team%3AKUAS_Korea/Experiments)
  + [Model](https://2021.igem.org/Team%3AKUAS_Korea/Model)
  + [Engineering](https://2021.igem.org/Team%3AKUAS_Korea/Engineering)
  + [Implementation](https://2021.igem.org/Team%3AKUAS_Korea/Implementation)
  + [Contribution](https://2021.igem.org/Team%3AKUAS_Korea/Contribution)
* Human Practices
  + [Human Practices](https://2021.igem.org/Team%3AKUAS_Korea/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AKUAS_Korea/Communication)
  + [Sustainable](https://2021.igem.org/Team%3AKUAS_Korea/Sustainable)

![](https://static.igem.org/mediawiki/2021/a/a2/T--KUAS_KOREA--frog_banner_2.png)

### 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.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)