iGEM 2021 — Team UTokyo

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

# Team:UTokyo/Notebook

YEAST-AID

## [![](https://static.igem.org/mediawiki/2021/8/84/T--UTokyo--theme--img--project-logo.svg)](https://2021.igem.org/Team%3AUTokyo)

- Project
  * [Description](https://2021.igem.org/Team%3AUTokyo/Description)
  * [Contribution](https://2021.igem.org/Team%3AUTokyo/Contribution)
  * [Engineering](https://2021.igem.org/Team%3AUTokyo/Engineering)
  * [Safety](https://2021.igem.org/Team%3AUTokyo/Safety)
  * [Notebook](https://2021.igem.org/Team%3AUTokyo/Notebook)
  * [Excellence in another area](https://2021.igem.org/Team%3AUTokyo/Excellence_Area)
- Wet Lab
  * [Results](https://2021.igem.org/Team%3AUTokyo/Results)
  * [Experiments](https://2021.igem.org/Team%3AUTokyo/Experiments)
  * [Parts](https://2021.igem.org/Team%3AUTokyo/Parts)
- Dry Lab
  * [Model](https://2021.igem.org/Team%3AUTokyo/Model)
  * [Hardware](https://2021.igem.org/Team%3AUTokyo/Hardware)
  * [Software](https://2021.igem.org/Team%3AUTokyo/Software)
- Human Practices
  * [Integrated Human Practices](https://2021.igem.org/Team%3AUTokyo/Human_Practices)
  * [Implementation](https://2021.igem.org/Team%3AUTokyo/Implementation)
  * [Education Communication](https://2021.igem.org/Team%3AUTokyo/Communication)
- Team
  * [Members](https://2021.igem.org/Team%3AUTokyo/Team)
  * [Attributions](https://2021.igem.org/Team%3AUTokyo/Attributions)
  * [Collaborations](https://2021.igem.org/Team%3AUTokyo/Collaborations)
  * [Partnership](https://2021.igem.org/Team%3AUTokyo/Partnership)
- [Awards](https://2021.igem.org/Team%3AUTokyo/Awards)

- Project
  * [Description](https://2021.igem.org/Team%3AUTokyo/Description)
  * [Contribution](https://2021.igem.org/Team%3AUTokyo/Contribution)
  * [Engineering](https://2021.igem.org/Team%3AUTokyo/Engineering)
  * [Safety](https://2021.igem.org/Team%3AUTokyo/Safety)
  * [Notebook](https://2021.igem.org/Team%3AUTokyo/Notebook)
  * [Excellence in another area](https://2021.igem.org/Team%3AUTokyo/Excellence_Area)
- Wet Lab
  * [Results](https://2021.igem.org/Team%3AUTokyo/Results)
  * [Experiments](https://2021.igem.org/Team%3AUTokyo/Experiments)
  * [Parts](https://2021.igem.org/Team%3AUTokyo/Parts)
- Dry Lab
  * [Model](https://2021.igem.org/Team%3AUTokyo/Model)
  * [Hardware](https://2021.igem.org/Team%3AUTokyo/Hardware)
  * [Software](https://2021.igem.org/Team%3AUTokyo/Software)
- Human Practices
  * [Integrated Human Practices](https://2021.igem.org/Team%3AUTokyo/Human_Practices)
  * [Implementation](https://2021.igem.org/Team%3AUTokyo/Implementation)
  * [Education Communication](https://2021.igem.org/Team%3AUTokyo/Communication)
- Team
  * [Members](https://2021.igem.org/Team%3AUTokyo/Team)
  * [Attributions](https://2021.igem.org/Team%3AUTokyo/Attributions)
  * [Collaborations](https://2021.igem.org/Team%3AUTokyo/Collaborations)
  * [Partnership](https://2021.igem.org/Team%3AUTokyo/Partnership)
- [Awards](https://2021.igem.org/Team%3AUTokyo/Awards)

![](https://static.igem.org/mediawiki/2021/c/c4/T--UTokyo--theme--img--yeast-eyecatch.png)

# Notebook

##### Index

1. [1. Our notebook](#Our-notebook)
   1. [1.1. Week 7/12-7/18](#Week-7-12-7-18)
   2. [1.2. Week 7/19-7/25](#Week-7-19-7-25)
   3. [1.3. Week 7/26-8/1](#Week-7-26-8-1)
   4. [1.4. Week 8/2-8/8](#Week-8-2-8-8)
   5. [1.5. Week 8/18-22](#Week-8-18-22)
   6. [1.6. Week 8/23-8/29](#Week-8-23-8-29)
   7. [1.7. Week 8/30-9/5](#Week-8-30-9-5)
   8. [1.8. Week 9/6-10](#Week-9-6-10)
   9. [1.9. Week 9/13-17](#Week-9-13-17)
   10. [1.10. Week 9/20-24](#Week-9-20-24)
   11. [1.11. Week 9/27-10/1](#Week-9-27-10-1)
2. [2. Data from team Kyoto](#Data-from-team-Kyoto)
   1. [2.1. 10/8-10/9](#10-8-10-9)

## Our notebook

### Week 7/12-7/18

We learned basic experimental techniques: preparation of LB amp medium, the transformation of pRS316 plasmid into *E. coli*, inoculation, and PCR.

![](https://static.igem.org/mediawiki/2021/2/20/T--UTokyo--figures--notebook_pictures.png)

### Week 7/19-7/25

SLiCE and iVEC to splice the PCR products from the previous week to HBD3 and CYC1
Plasmid extraction of HBD3 and CYC1

### Week 7/26-8/1

Yeast experiments were started this week.
Transformation of HBD3 and CYC1 into YPD medium and BMMD medium yeast
Colony PCR on yeast

### Week 8/2-8/8

The assay of the effect of HBD3 secretion on the yeast’s growth
HBD3’s antimicrobial ability on bacteria in a halo assay

### Week 8/18-22

Transformation of pRS316 into yeast

### Week 8/23-8/29

Assay of antimicrobial susceptibility by bacterial liquid absorption method and halo method
Preparation of cell fibers
Assay of Met-responsiveness of Met17 promoter

### Week 8/30-9/5

PCR for pRS316+mCherry cloning
pANB1 cloning
pROX1 cloning
SLiCE of pANB1 and pROX1
Colony PCR for pANB1 and pROX1
Plasmid extraction of pANB1 and pROX1
Transformation of pANB1 and pROX1 into yeast

### Week 9/6-10

Colony PCR for the yeast transformed with pANB1 or pROX1 in the previous week
SLiCE (Linear Vector Backbone: pRS316+mCherry, Insert DNA: pANB1)
Overlap Extension PCR (OE-PCR) for the sake of connecting three VP16
Colony PCR for the E. coli transformed with pANB1
Defensin assay
Cell fiber preparation
OE-PCR for the sake of connecting three VP16
pANB1 plasmid was extracted from E. coli
Yeast transformation with pANB1
OE-PCR for the sake of connecting three VP16
Defensin assay
SLiCE (Linear Vector Backbone: pRS316, Insert DNA: LasI)
Colony PCR for the yeast transformed with pANB1
Colony PCR for the E. coli transformed with LasI

### Week 9/13-17

Operation check of Met17 promotor
Evaluation of oxygen promoter (pANB1)
Defensin assay
OE-PCR for the sake of connecting three VP16
Defensin assay
OE-PCR for the sake of connecting three VP16
OE-PCR for the sake of connecting three VP16

### Week 9/20-24

OE-PCR for the sake of connecting three VP16
SLiCE (Linear Vector Backbone: pRS316+mCherry, Insert DNA: pROX1)
Halo assay
Colony PCR for the E. coli transformed with pROX1
OE-PCR for the sake of connecting three VP16
pROX1 plasmid was extracted from E. coli
Yeast transformation with pROX1

### Week 9/27-10/1

SLiCE (Linear Vector Backbone: pRS316+mCherry, Insert DNA: pROX1)
Defensin assay
OE-PCR for the sake of connecting three VP16
Colony PCR for the E. coli transformed with pROX1
pROX1 plasmid was extracted from E. coli
Yeast transformation with pROX1
OE-PCR for the sake of connecting three VP16
OE-PCR for the sake of connecting three VP16
Colony PCR for the yeast transformed with pROX1
Cell fiber preparation
Evaluation of oxygen promoter (pROX1)
OE-PCR for the sake of connecting three VP16

## Data from team Kyoto

### 10/8-10/9

10/8
Using Def1 DTT, Def1 no DTT, Def1 ppt, CecA, LL37, NOP1, NOP1v ,and pet11a purified by His tag, we did HPLC.
We also tried the disk diffusion method using Defencine3 from the UTokyo Team.
We used the outstanding bacteria in the vase and Bacillus subtilis.
Add 100μL of bacteria solution to 40mL of standard agar medium
Leave them in the incubator 27℃, 1day

10/9
The antimicrobial peptide from UTokyo Team might not seem to have the antimicrobial activity.

![](https://static.igem.org/mediawiki/2021/c/c9/T--UTokyo--figures--notebook_kyoto.png)

![](https://static.igem.org/mediawiki/2021/b/b8/T--UTokyo--theme--img--logo.svg)

### iGEM UTokyo

igem.utokyo2021@gmail.com
[@iGEM\_UTokyo](https://twitter.com/iGEM_UTokyo)

![](https://static.igem.org/mediawiki/2021/7/7b/T--UTokyo--theme--img--sanyo.png)![](https://static.igem.org/mediawiki/2021/e/e7/T--UTokyo--theme--img--goukaku.jpg)

© 2021 iGEM UTokyo

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

# Team:UTokyo/Engineering

YEAST-AID

## [![](https://static.igem.org/mediawiki/2021/8/84/T--UTokyo--theme--img--project-logo.svg)](https://2021.igem.org/Team%3AUTokyo)

- Project
  * [Description](https://2021.igem.org/Team%3AUTokyo/Description)
  * [Contribution](https://2021.igem.org/Team%3AUTokyo/Contribution)
  * [Engineering](https://2021.igem.org/Team%3AUTokyo/Engineering)
  * [Safety](https://2021.igem.org/Team%3AUTokyo/Safety)
  * [Notebook](https://2021.igem.org/Team%3AUTokyo/Notebook)
  * [Excellence in another area](https://2021.igem.org/Team%3AUTokyo/Excellence_Area)
- Wet Lab
  * [Results](https://2021.igem.org/Team%3AUTokyo/Results)
  * [Experiments](https://2021.igem.org/Team%3AUTokyo/Experiments)
  * [Parts](https://2021.igem.org/Team%3AUTokyo/Parts)
- Dry Lab
  * [Model](https://2021.igem.org/Team%3AUTokyo/Model)
  * [Hardware](https://2021.igem.org/Team%3AUTokyo/Hardware)
  * [Software](https://2021.igem.org/Team%3AUTokyo/Software)
- Human Practices
  * [Integrated Human Practices](https://2021.igem.org/Team%3AUTokyo/Human_Practices)
  * [Implementation](https://2021.igem.org/Team%3AUTokyo/Implementation)
  * [Education Communication](https://2021.igem.org/Team%3AUTokyo/Communication)
- Team
  * [Members](https://2021.igem.org/Team%3AUTokyo/Team)
  * [Attributions](https://2021.igem.org/Team%3AUTokyo/Attributions)
  * [Collaborations](https://2021.igem.org/Team%3AUTokyo/Collaborations)
  * [Partnership](https://2021.igem.org/Team%3AUTokyo/Partnership)
- [Awards](https://2021.igem.org/Team%3AUTokyo/Awards)

- Project
  * [Description](https://2021.igem.org/Team%3AUTokyo/Description)
  * [Contribution](https://2021.igem.org/Team%3AUTokyo/Contribution)
  * [Engineering](https://2021.igem.org/Team%3AUTokyo/Engineering)
  * [Safety](https://2021.igem.org/Team%3AUTokyo/Safety)
  * [Notebook](https://2021.igem.org/Team%3AUTokyo/Notebook)
  * [Excellence in another area](https://2021.igem.org/Team%3AUTokyo/Excellence_Area)
- Wet Lab
  * [Results](https://2021.igem.org/Team%3AUTokyo/Results)
  * [Experiments](https://2021.igem.org/Team%3AUTokyo/Experiments)
  * [Parts](https://2021.igem.org/Team%3AUTokyo/Parts)
- Dry Lab
  * [Model](https://2021.igem.org/Team%3AUTokyo/Model)
  * [Hardware](https://2021.igem.org/Team%3AUTokyo/Hardware)
  * [Software](https://2021.igem.org/Team%3AUTokyo/Software)
- Human Practices
  * [Integrated Human Practices](https://2021.igem.org/Team%3AUTokyo/Human_Practices)
  * [Implementation](https://2021.igem.org/Team%3AUTokyo/Implementation)
  * [Education Communication](https://2021.igem.org/Team%3AUTokyo/Communication)
- Team
  * [Members](https://2021.igem.org/Team%3AUTokyo/Team)
  * [Attributions](https://2021.igem.org/Team%3AUTokyo/Attributions)
  * [Collaborations](https://2021.igem.org/Team%3AUTokyo/Collaborations)
  * [Partnership](https://2021.igem.org/Team%3AUTokyo/Partnership)
- [Awards](https://2021.igem.org/Team%3AUTokyo/Awards)

![](https://static.igem.org/mediawiki/2021/c/c4/T--UTokyo--theme--img--yeast-eyecatch.png)

# Engineering

##### Index

1. [1. Engineering success](#Engineering-success)
   1. [1.1. New Parts](#New-Parts)
2. [2. Research and Design](#Research-and-Design)
3. [3. Build and Test](#Build-and-Test)
4. [4. Learn and Design](#Learn-and-Design)
5. [5. Build and Test](#Build-and-Test-1)

## Engineering success

### New Parts

As a result of the engineering design cycle, we have successfully developed the following parts that can detect oxygen.

1. [BBa\_K3804000](http://parts.igem.org/Part%3ABBa_K3804000)
2. [BBa\_K3804001](http://parts.igem.org/Part%3ABBa_K3804001)

In our project, we achieved engineering success by repeating many engineering design cycles, especially in the stage of realizing oxygen detection capability. The following steps illustrate our engineering success.

1. Research and Design
2. Build and Test
3. Learn and Design
4. Build and Test

![](https://static.igem.org/mediawiki/2021/3/33/T--UTokyo--figures--engineering_cycle.png)

## Research and Design

We decided to focus on the oxygen-responsive genes inherent in yeast in order to implement the ability to detect oxygen in yeast. Yeast is well known for its Pasteur effect, and we thought that we could implement it smartly by using the pathway for this effect. Therefore, we first proceeded to research the oxygen-responsive gene cluster in yeast.
In the early stages of our research, we discussed which oxygen-responsive genes to focus on [[4]](#fn:4). Since the protein HAP was found to play a major role in the oxygen response in yeast, further research on HAP revealed that CYC1 is regulated by Hap1 [[1]](#fn:1), we found that CYC1 is regulated by Hap1, and we also obtained the relationship between oxygen concentration and transcription level. The transcription level of CYC1 increased linearly around the threshold of oxygen concentration that we wanted to implement. Therefore, we thought that using the CYC1 promoter, we should not only be able to obtain oxygen responsiveness, but also be able to obtain the desired threshold with regulation.
Since we found that oxygen regulates the activity of Hap1 and Hap1 regulates the transcriptional level of CYC1, we decided to design a plasmid that utilizes this effect as follows. We thought that the strength of the fluorescence emitted by mCherry would be different depending on the oxygen concentration in yeast transfected with this plasmid.
![](https://static.igem.org/mediawiki/2021/1/1d/T--UTokyo--figures--cyc1_reporter_genedesign.png)

## Build and Test

We transformed the designed plasmid into yeast and tested whether the intensity of fluorescence changed in response to oxygen concentration. However, contrary to our expectation, there was no significant difference in fluorescence intensity between anaerobic and aerobic conditions, and mCherry fluorescence was observed in both conditions.

## Learn and Design

Following the results of the first cycle of testing, we investigated why there was no difference in fluorescence intensity depending on oxygen concentration; transcriptional regulation by HAP involves changes in chromatin structure [[2]](#fn:2). We hypothesized that since the CYC1 promoter was introduced into a plasmid, it could not be regulated by HAP and thus did not show oxygen responsiveness. This prediction is consistent with our experimental results showing that the CYC1 promoter in the plasmid worked as a constitutive promoter independent of oxygen.
Therefore, we decided to use a promoter that is not directly regulated by HAP, but instead is regulated by proteins that are coordinated by HAP. Specifically, we focused on ROX1, whose expression is promoted by HAP, and decided to use the ANB1 promoter and ROX1 promoter, which are regulated by ROX1, as oxygen-responsive promoters. These promoters have been found to work on plasmids in previous studies [[3]](#fn:3) [[5]](#fn:5), and we thought we could use them to confer oxygen responsiveness to yeast. We created a plasmid with mCherry attached downstream of these promoters.

![](https://static.igem.org/mediawiki/2021/3/33/T--UTokyo--figures--rox1_reporter_genedesign.png)

## Build and Test

The created plasmid was transformed into yeast and tested. The results showed that the fluorescence of the ANB1 promoter-transformed strain was stronger in aerobic conditions than in anaerobic conditions. This indicates that the ANB1 promoter is also oxygen-responsive in the plasmid, which brings us one step closer to one of the goals of our project, namely to create a device that detects and notifies changes in oxygen concentration when a wound is healing.

---

1. 1.T. Hon et al., “A mechanism of oxygen sensing in yeast. Multiple oxygen-responsive steps in the heme biosynthetic pathway affect Hap1 activity,” *J. Biol. Chem*., vol. 278, no. 50, pp. 50771–50780, Dec. 2003. [↩](#fnref:1)
2. 2.T. Zhang, P. Bu, J. Zeng, and A. Vancura, “Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression,” *J. Biol. Chem*., vol. 292, no. 41, pp. 16942–16954, Oct. 2017. [↩](#fnref:2)
3. 3.Z.-A. Fang et al., “Gene responses to oxygen availability in Kluyveromyces lactis: an insight on the evolution of the oxygen-responding system in yeast,” *PLoS One*, vol. 4, no. 10, p. e7561, Oct. 2009. [↩](#fnref:3)
4. 4.K. E. Kwast, P. V. Burke, and R. O. Poyton, “Oxygen sensing and the transcriptional regulation of oxygen-responsive genes in yeast,” *J. Exp. Biol*., vol. 201, no. Pt 8, pp. 1177–1195, Apr. 1998. [↩](#fnref:4)
5. 5.J. Deckert, A. M. Rodriguez Torres, J. T. Simon, and R. S. Zitomer, “Mutational analysis of Rox1, a DNA-bending repressor of hypoxic genes in Saccharomyces cerevisiae,” *Mol. Cell. Biol*., vol. 15, no. 11, pp. 6109–6117, Nov. 1995. [↩](#fnref:5)

![](https://static.igem.org/mediawiki/2021/b/b8/T--UTokyo--theme--img--logo.svg)

### iGEM UTokyo

igem.utokyo2021@gmail.com
[@iGEM\_UTokyo](https://twitter.com/iGEM_UTokyo)

![](https://static.igem.org/mediawiki/2021/7/7b/T--UTokyo--theme--img--sanyo.png)![](https://static.igem.org/mediawiki/2021/e/e7/T--UTokyo--theme--img--goukaku.jpg)

© 2021 iGEM UTokyo

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

# Team:UTokyo/Experiments

YEAST-AID

## [![](https://static.igem.org/mediawiki/2021/8/84/T--UTokyo--theme--img--project-logo.svg)](https://2021.igem.org/Team%3AUTokyo)

- Project
  * [Description](https://2021.igem.org/Team%3AUTokyo/Description)
  * [Contribution](https://2021.igem.org/Team%3AUTokyo/Contribution)
  * [Engineering](https://2021.igem.org/Team%3AUTokyo/Engineering)
  * [Safety](https://2021.igem.org/Team%3AUTokyo/Safety)
  * [Notebook](https://2021.igem.org/Team%3AUTokyo/Notebook)
  * [Excellence in another area](https://2021.igem.org/Team%3AUTokyo/Excellence_Area)
- Wet Lab
  * [Results](https://2021.igem.org/Team%3AUTokyo/Results)
  * [Experiments](https://2021.igem.org/Team%3AUTokyo/Experiments)
  * [Parts](https://2021.igem.org/Team%3AUTokyo/Parts)
- Dry Lab
  * [Model](https://2021.igem.org/Team%3AUTokyo/Model)
  * [Hardware](https://2021.igem.org/Team%3AUTokyo/Hardware)
  * [Software](https://2021.igem.org/Team%3AUTokyo/Software)
- Human Practices
  * [Integrated Human Practices](https://2021.igem.org/Team%3AUTokyo/Human_Practices)
  * [Implementation](https://2021.igem.org/Team%3AUTokyo/Implementation)
  * [Education Communication](https://2021.igem.org/Team%3AUTokyo/Communication)
- Team
  * [Members](https://2021.igem.org/Team%3AUTokyo/Team)
  * [Attributions](https://2021.igem.org/Team%3AUTokyo/Attributions)
  * [Collaborations](https://2021.igem.org/Team%3AUTokyo/Collaborations)
  * [Partnership](https://2021.igem.org/Team%3AUTokyo/Partnership)
- [Awards](https://2021.igem.org/Team%3AUTokyo/Awards)

- Project
  * [Description](https://2021.igem.org/Team%3AUTokyo/Description)
  * [Contribution](https://2021.igem.org/Team%3AUTokyo/Contribution)
  * [Engineering](https://2021.igem.org/Team%3AUTokyo/Engineering)
  * [Safety](https://2021.igem.org/Team%3AUTokyo/Safety)
  * [Notebook](https://2021.igem.org/Team%3AUTokyo/Notebook)
  * [Excellence in another area](https://2021.igem.org/Team%3AUTokyo/Excellence_Area)
- Wet Lab
  * [Results](https://2021.igem.org/Team%3AUTokyo/Results)
  * [Experiments](https://2021.igem.org/Team%3AUTokyo/Experiments)
  * [Parts](https://2021.igem.org/Team%3AUTokyo/Parts)
- Dry Lab
  * [Model](https://2021.igem.org/Team%3AUTokyo/Model)
  * [Hardware](https://2021.igem.org/Team%3AUTokyo/Hardware)
  * [Software](https://2021.igem.org/Team%3AUTokyo/Software)
- Human Practices
  * [Integrated Human Practices](https://2021.igem.org/Team%3AUTokyo/Human_Practices)
  * [Implementation](https://2021.igem.org/Team%3AUTokyo/Implementation)
  * [Education Communication](https://2021.igem.org/Team%3AUTokyo/Communication)
- Team
  * [Members](https://2021.igem.org/Team%3AUTokyo/Team)
  * [Attributions](https://2021.igem.org/Team%3AUTokyo/Attributions)
  * [Collaborations](https://2021.igem.org/Team%3AUTokyo/Collaborations)
  * [Partnership](https://2021.igem.org/Team%3AUTokyo/Partnership)
- [Awards](https://2021.igem.org/Team%3AUTokyo/Awards)

![](https://static.igem.org/mediawiki/2021/c/c4/T--UTokyo--theme--img--yeast-eyecatch.png)

# Experiments

##### Index

1. [1. Cloning Related](#Cloning-Related)
   1. [1.1. SLiCE](#SLiCE)
   2. [1.2. Transformation](#Transformation)
   3. [1.3. iVEC](#iVEC)
   4. [1.4. PCR](#PCR)
   5. [1.5. Inoculation](#Inoculation)
   6. [1.6. Glycerol stock creation](#Glycerol-stock-creation)
   7. [1.7. Plasmid Extraction](#Plasmid-Extraction)
2. [2. Medium Composition](#Medium-Composition)
3. [3. Yeast Transformation](#Yeast-Transformation)
   1. [3.1. Preculture](#Preculture)
   2. [3.2. Transformation](#Transformation-1)
4. [4. Oxygen promotor](#Oxygen-promotor)
   1. [4.1. Evaluation of the promoter](#Evaluation-of-the-promoter)
5. [5. Defensin evaluation](#Defensin-evaluation)
   1. [5.1. Operation check of Met17 promotor](#Operation-check-of-Met17-promotor)
   2. [5.2. Defensin Assay](#Defensin-Assay)
   3. [5.3. Assay Plate preparation](#Assay-Plate-preparation)
   4. [5.4. Halo Assay](#Halo-Assay)
   5. [5.5. Absorption Method (JIS L 1902:)](#Absorption-Method-JIS-L-1902)
6. [6. Cell Fiber](#Cell-Fiber)
   1. [6.1. Cell fiber preparation](#Cell-fiber-preparation)

## Cloning Related

Cloning of plasmids was conducted in following procedures using *E.coli*.

1. Recombine DNA fragments with SLiCE (Seamless Ligation Cloning Extract) method and culture over a night on a LB Amp agar plate.
2. Make sure that the plasmid was successfully inserted with colony PCR, and then culture over a night in liquid LB Amp.
3. Extract plasmids from cultured *E. coli*.

### SLiCE

| Component | Amount |
| --- | --- |
| Insert DNA (20 ng/μL) | 3 μL |
| Linear vector backbone (20ng/μL) | 3 μL |
| 10x Buffer | 1 μL |
| SLiCE | 1 μL |
| Insert DNA (20ng/μL) | 3 μL |
| Water | up to 10 μL (2 μL) |

1. Components listed above are mixed in a 1.5mL tube, then incubated for 30 min at 37°C.
2. Transformation was conducted using this mixture as a DNA vector.
3. Transformed *E. coli* was spread on the LB agar plate.

### Transformation

| Component | Amount |
| --- | --- |
| JM109 (*E. coli* competent cell) | 1 tube |
| Soc medium | 1 mL |
| DNA vector | Proper quantity |

1. The DNA vector was added to the tube containing JM109 melted on ice and mixed by tapping.
2. The tube was put on ice for 30 minutes.
3. The tube was put on a thermal cycler 45 sec at 42°C, then transferred on ice.
4. After more than two minutes, 1mL of 36°C Soc medium was added to the tube and mixed by pipetting.
5. The incubation was conducted for about 30 minutes at 37°C.

### iVEC

| Component | Amount |
| --- | --- |
| Insert DNA (HBD3 or CYC1, 100 ng/μL) | 1~2 μL |
| Linear vector backbone (PCR product from pRS316) | 1~2 μL |
| *E. coli* competent cell |  |
| LB medium | 1 mL |

1. The vector and the insert DNA were added to the tube containing the competent cell melted on ice and mixed it by pipetting softly.
2. The tube was put on ice for 20 minutes.
3. 1 mL of LB medium was added to the tube and the tube was inverted to mix it.
4. The tube was incubated for 45 minutes at 37°C, then centrifuged (5000 g, room temperature, 1 min).
5. The supernatant was discarded, leaving about only 100 μL.
6. The solution of the competent cell was suspended, spread on the LBAmp agar, and cultured overnight at 37°C.

### PCR

#### PrimeSTAR

Used for PCR which require accuracy, such as duplication of plasmid backbones.

| Component | Amount |
| --- | --- |
| PrimeSTAR HS DNA Poly | 0.5 μL |
| 5x PrimeSTAR Buffer (Mg2+ Plus) | 10 μL |
| dNTP Mixture | 4 μL |
| Forward Primer (10-15 pm) | 1 μL |
| Reverse Primer(10-15pm) | 1μL |
| Water | up to 50 μL |
| DNA template (< 200 ng) | 1.5 μL |

| Phase 1 | Phase 2 | Phase 3 | Phase 4 |
| --- | --- | --- | --- |
| 98°C | Cycle setting：98°C→55°C(Tm value-5°C)→72°C | 72°C | 4°C (15°C) |
| 30 sec | 10 sec→5 sec or 15 sec→1 min/kb | 5 min | ∞ |

1. The mixture of the components listed above are put on a thermal cycler and the reaction was started.
2. After the reaction, 2μL of the products was mixed with loading dye and used for electrophoresis to check the specificity of amplification.

#### GoTaq PCR

Used for detecting expression which does not require accuracy, such as colony PCR.

| Component | Amount |
| --- | --- |
| 2x GoTaq Green Master Mix | 10 µL |
| Water | 10 µL |
| Forward Primer (10-15 pm) | 0.5 µL |
| Reverse Primer (10-15 pm) | 0.5 µL |
| DNA template (liquid or colony) | 0.5 µL or one pick |

| Phase 1 | Phase 2 | Phase 3 | Phase 4 |
| --- | --- | --- | --- |
| 95°C | Cycle setting : 95°C→55°C(Tm value -5°C)→72°C | 72°C | 4°C(15°C) |
| 30 sec | 40 sec→30 sec→1 min/kb | 5 min | ∞ |

1. The mixture of the components listed above are put on a thermal cycler and the reaction was started.
2. After the reaction, 7μL of the products was used for electrophoresis to check the specificity of amplification.

#### Purification of PCR products from agarose gel

Wizard SV Gel and PCR Clean-Up System (Promega) was used.

1. After PCR with PrimeSTAR, a tenth of the amount of 10× loading dye was added to the products.
2. Electrophoresis was conducted for 32μL of the mixture mixed in step 1.
3. The gel around the band was cut off for DNA extraction.
4. DNA was extracted from the piece of agar with Promega A9281.

### Inoculation

1. About 2 mL of LB medium and Ampicillin solution were mixed in a sterilized test tube to set ampicillin concentration to 100 mg / L.
2. A single colony of the bacteria was poked with a toothpick and dissolved to the medium.

### Glycerol stock creation

| Component | Amount |
| --- | --- |
| *E. coli* suspension | 0.7 mL |
| 50 % Glycerol | 0.3 mL |

1. 0.7 mL of *E. coli* suspension was added to 0.3 mL of 50 % Glycerol and mixed by pipetting.
2. The mixture was stored at -20°C.

### Plasmid Extraction

PureYield Plasmid Miniprep System (Promega) was used.

1. The culture solution in which *E. coli* cultured overnight was suspended.
2. Purified the plasmid from the *E. coli* suspension with Promega A1223.

## Medium Composition

| LB medium (agar) | Amount | Final conc. |
| --- | --- | --- |
| LB Powder | 12.5 g | 2.5 % |
| (LB agar) | 7.5 g | 1.5 % |
| Water | up to 500 mL |  |

(1000μL of Amp was added to the medium to make LB Amp (agar).)

| YPD medium | Amount | Final conc. |
| --- | --- | --- |
| Bacto Yeast Extract | 2.5 g | 1 % |
| Peptone | 5.0 g | 2 % |
| Glucose | 5.0 g | 2 % |
| Water | up to 250 mL |  |

| BMMD medium (+ Met) | Amount | Final conc. |
| --- | --- | --- |
| Bacto Yeast Nitrogen Base w/o Amino Acids | 1.68 g | 0.67 % |
| Citric Acid | 1.73 g | 36 mM |
| Sodium Hydrogen phosphate | 4.471 g | 126 mM |
| Glucose | 5 g | 2 % |
| (Methionine) | (37.3 mg) | (1 mM) |
| Water | up to 250 mL |  |

| SC-U medium agar | Amount | Final conc. |
| --- | --- | --- |
| Dropout Mix | 0.5 g |  |
| Bacto Yeast Nitrogen Base w/o Amino | 1.68 g | 0.67 % |
| Glucose | 5 g | 2 % |
| Bacto agar | 5 g | 2 % |
| Water | up to 250 mL |  |

## Yeast Transformation

### Preculture

1. 3 mL of medium was poured into a tube.
2. One of the host yeast colonies was poked with the tip of the micropipette.
3. The tip of the micropipette was soaked and shaken in the medium.
4. The tube was put into the shaker overnight at 30°C.
5. In the next morning, the new 3 medium was prepared and 300μL of yeast solution was poured into it.
6. The yeast was cultured for 3 ~ 4 hours.

### Transformation

Following procedure is conducted in accordance with Frozen-EZ Yeast TransformationⅡ (Zymo Research T2001).

1. After preculture, 1 mL of yeast culture solution was put into a 1.5 mL microtube and centrifuged (3000 rpm, 26°C, 1 min) to separate yeast from the liquid. The supernatant was disposed of.
2. 1mL of Frozen-EZ Sol1(washer solution) was added to the tube. Then the tube was vortexed and centrifuged (3000 rpm, 26°C, 1 min) again. The supernatant was discarded.
3. 2 μL of plasmid solution was added to the tube and the solution was mixed by pipetting and vortex.
4. 500 μL of Frozen-EZ Sol3 was added to the tube and the tube was inverted.
5. The tube was incubated for 45 min at 30°C. During this time, the tube was inverted every 15 minutes.
6. After incubation, 150 μL of yeast solution was spread on a plate without mixing.
7. The plate was cultured for 2 ~ 4 days at 30 °C.

## Oxygen promotor

### Evaluation of the promoter

1. Transformed yeast (pANB1) was cultured until saturation.
2. Two 5 mL syringes filled with 300μL of the yeast medium were shaken at 37 °C overnight.
3. After 4 hours of cultivation, fluorescence of the yeast was observed visually and fluorescence microscopy.

## Defensin evaluation

### Operation check of Met17 promotor

1. 200μL of transformed yeast (HBD3, pRS316) solutions were added to YPD medium, after overnight cultivation in BMMD and BMMD +Met medium.
2. Optical density (OD) of the culture medium was measured using a spectrophotometer at appropriate intervals.

### Defensin Assay

1. Transformed yeast (HBD3, pRS316) was cultured overnight in 6mL of YPD medium. *E. coli* was also cultured overnight.
2. 3mL of yeast solution was used to make cell fiber.
3. The remainder of the yeast solution was poured into a tube and centrifuged (1500rpm, 1min).
4. The supernatant obtained in step 1 was filtered, and the yeast was eliminated totally.
5. 1.5 mL of *E. coli* was double diluted and spread on a LB agar plate.
6. Dressing was immersed into the filtered yeast supernatant (or YPD medium as a control) and put on the plate prepared in step 3.
7. The plate was cultured for 24-48 hours.

### Assay Plate preparation

1. 300μL of *E. coli* solution was diluted 10 times with sterilized saline (0.85 % NaCl).
2. LBAmp agar was heated in a microwave and melted.
3. The plate was allowed to cool to 50°C.
4. 1mL of the liquid made in step 1 was added to the plate, and the mixture was stirred.
5. The plate was left gently until solidified.

### Halo Assay

1. About 30μL of liquid to be evaluated were permeated paper disks.
2. The paper disks were placed on the assay plates at the center.
3. The yeast on the plates were cultured for 24-48 hours at 37°C.
4. For all plates, the diameter of the inhibition circle around the paper disk was measured.

### Absorption Method (JIS L 1902:)

1. *E. coli* was cultured in LBAmp for 3 hours at 200rpm and 37°C, after overnight cultivation.
2. The cultured *E. coli* was diluted 20 times with YPD medium.
3. One of the dressings to be evaluated (about 0.05g of each dressing) was put in a tube and soaked with 120ul of the 20-fold diluted *E. coli* culture solution.
4. 12mL of LBAmp was added to the tube and mixed with the solution in the tube by vortex.
5. 3mL of the liquid in the tube was collected and used to make a 10x dilution series, then stored at 5°C to count the number of viable bacteria at that time.
6. 2mL of remaining *E. coli* solution was collected for another experiment.
7. The remaining *E. coli* was cultured 18-24 hours at 36°C and subjected to the same treatment as in step 5.
8. LBAmp agar was heated in a microwave and allowed to cool to 50°C.
9. About 20mL of LBAmp agar was poured to dishes and mixed with 1mL of the *E. coli* dilution series (see also Assay Plate preparation).
10. The plates were cultured for 24-48 hours at 36°C.
11. The number of colonies on each plate was counted.
12. Antimicrobial activity value (AAV) was calculated.
    AAV = {log(control, viable count after incubation) – log(control, viable count before incubation)} - {log(test, viable count after incubation) – log(test, viable count before incubation)}

## Cell Fiber

### Cell fiber preparation

1. 200μL of transformed yeast solution was added to the proper medium and cultured for 3 hours at 37°C.
2. 1.5wt% of sodium alginate aq and yeast suspension were mixed, and the mixture was inhaled with a syringe fitted with a 0.8mm needle.
3. The tip of the syringe was put in the 50mM CaCl­­2 aq, and the syringe was pushed slowly.

![](https://static.igem.org/mediawiki/2021/b/b8/T--UTokyo--theme--img--logo.svg)

### iGEM UTokyo

igem.utokyo2021@gmail.com
[@iGEM\_UTokyo](https://twitter.com/iGEM_UTokyo)

![](https://static.igem.org/mediawiki/2021/7/7b/T--UTokyo--theme--img--sanyo.png)![](https://static.igem.org/mediawiki/2021/e/e7/T--UTokyo--theme--img--goukaku.jpg)

© 2021 iGEM UTokyo

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# Team:UTokyo/Results

YEAST-AID

## [![](https://static.igem.org/mediawiki/2021/8/84/T--UTokyo--theme--img--project-logo.svg)](https://2021.igem.org/Team%3AUTokyo)

- Project
  * [Description](https://2021.igem.org/Team%3AUTokyo/Description)
  * [Contribution](https://2021.igem.org/Team%3AUTokyo/Contribution)
  * [Engineering](https://2021.igem.org/Team%3AUTokyo/Engineering)
  * [Safety](https://2021.igem.org/Team%3AUTokyo/Safety)
  * [Notebook](https://2021.igem.org/Team%3AUTokyo/Notebook)
  * [Excellence in another area](https://2021.igem.org/Team%3AUTokyo/Excellence_Area)
- Wet Lab
  * [Results](https://2021.igem.org/Team%3AUTokyo/Results)
  * [Experiments](https://2021.igem.org/Team%3AUTokyo/Experiments)
  * [Parts](https://2021.igem.org/Team%3AUTokyo/Parts)
- Dry Lab
  * [Model](https://2021.igem.org/Team%3AUTokyo/Model)
  * [Hardware](https://2021.igem.org/Team%3AUTokyo/Hardware)
  * [Software](https://2021.igem.org/Team%3AUTokyo/Software)
- Human Practices
  * [Integrated Human Practices](https://2021.igem.org/Team%3AUTokyo/Human_Practices)
  * [Implementation](https://2021.igem.org/Team%3AUTokyo/Implementation)
  * [Education Communication](https://2021.igem.org/Team%3AUTokyo/Communication)
- Team
  * [Members](https://2021.igem.org/Team%3AUTokyo/Team)
  * [Attributions](https://2021.igem.org/Team%3AUTokyo/Attributions)
  * [Collaborations](https://2021.igem.org/Team%3AUTokyo/Collaborations)
  * [Partnership](https://2021.igem.org/Team%3AUTokyo/Partnership)
- [Awards](https://2021.igem.org/Team%3AUTokyo/Awards)

- Project
  * [Description](https://2021.igem.org/Team%3AUTokyo/Description)
  * [Contribution](https://2021.igem.org/Team%3AUTokyo/Contribution)
  * [Engineering](https://2021.igem.org/Team%3AUTokyo/Engineering)
  * [Safety](https://2021.igem.org/Team%3AUTokyo/Safety)
  * [Notebook](https://2021.igem.org/Team%3AUTokyo/Notebook)
  * [Excellence in another area](https://2021.igem.org/Team%3AUTokyo/Excellence_Area)
- Wet Lab
  * [Results](https://2021.igem.org/Team%3AUTokyo/Results)
  * [Experiments](https://2021.igem.org/Team%3AUTokyo/Experiments)
  * [Parts](https://2021.igem.org/Team%3AUTokyo/Parts)
- Dry Lab
  * [Model](https://2021.igem.org/Team%3AUTokyo/Model)
  * [Hardware](https://2021.igem.org/Team%3AUTokyo/Hardware)
  * [Software](https://2021.igem.org/Team%3AUTokyo/Software)
- Human Practices
  * [Integrated Human Practices](https://2021.igem.org/Team%3AUTokyo/Human_Practices)
  * [Implementation](https://2021.igem.org/Team%3AUTokyo/Implementation)
  * [Education Communication](https://2021.igem.org/Team%3AUTokyo/Communication)
- Team
  * [Members](https://2021.igem.org/Team%3AUTokyo/Team)
  * [Attributions](https://2021.igem.org/Team%3AUTokyo/Attributions)
  * [Collaborations](https://2021.igem.org/Team%3AUTokyo/Collaborations)
  * [Partnership](https://2021.igem.org/Team%3AUTokyo/Partnership)
- [Awards](https://2021.igem.org/Team%3AUTokyo/Awards)

![](https://static.igem.org/mediawiki/2021/c/c4/T--UTokyo--theme--img--yeast-eyecatch.png)

# Results

##### Index

1. [1. Oxygen Sensing](#Oxygen-Sensing)
   1. [1.1. Overview](#Overview)
   2. [1.2. System Design](#System-Design)
   3. [1.3. Parts Construction](#Parts-Construction)
   4. [1.4. Oxygen Assay](#Oxygen-Assay)
   5. [1.5. Discussion](#Discussion)
2. [2. Defensin Secretion](#Defensin-Secretion)
   1. [2.1. Overview](#Overview-1)
   2. [2.2. System Design](#System-Design-1)
   3. [2.3. Parts Construction](#Parts-Construction-1)
   4. [2.4. Effect on the Yeast](#Effect-on-the-Yeast)
   5. [2.5. Antibacterial Assay](#Antibacterial-Assay)
3. [3. Pathogen Detection](#Pathogen-Detection)
   1. [3.1. Overview](#Overview-2)
   2. [3.2. System Design](#System-Design-2)
   3. [3.3. Plasmid Construction](#Plasmid-Construction)

## Oxygen Sensing

### Overview

#### Goal

To construct an oxygen sensing system using oxygen-responsive promoters pAnb1 and pRox1 from *Saccharomyces cerevisiae* that can be applied to wound monitoring.

#### Result

Differences between aerobic and anaerobic conditions were indicated by fluorescence protein using *S. cerevisiae* with the constructed oxygen detection system. However, oxygen-dependent change of fluorescence was also observed in the constitutive promoter(pCyc:BBa\_I766555).

#### Future Direction

It is necessary to re-examine whether the change of fluorescence is due to promoters or a decrease in metabolic activity in the cells by setting up proper controls, like ADH1 promoter. And also, the threshold of the sensor system has to be quantified by using an oxygen concentration meter. Furthermore, creating a system that can be applied to coating materials is expected for practical use. This can be possible by incorporating chromoproteins or other reporter genes visible to the naked eye downstream of the oxygen promoter.

### System Design

We aimed to construct a sensor system by hacking the oxygen response pathway in *S. cerevisiae*, and the first step was to determine which promoters to use. Upstream regions of ANB1 and ROX1 are promoters which have responsiveness to changes in oxygen concentration, and their expression is activated under aerobic conditions in plasmid conditions [[1]](#fn:1)[[2]](#fn:2)[[3]](#fn:3). In this system(see the [Description](https://2021.igem.org/Team%3AUTokyo/Description)), pAnb1 and pRox1 were used, and fluorescent protein mCherry is attached downstream of pAnb1 and pRox1, and the CYC1 terminator was placed downstream of mCherry. This system reports the change of oxygen concentration by expressing fluorescent protein.
The information of each construct is shown inTable1. A sequence in pROX1 was modified to conform to BioBrick specifications.

**Table 1** Promoter information

| Name | Length | Sequences |
| --- | --- | --- |
| Oxy sensor pAnb1 | 2286bp | ANB1 promoter - mCherry - CYC1terminator |
| Oxy sensor pRox1 | 1565bp | ROX1 promoter - mCherry - CYC1terminator |

### Parts Construction

The oxygen sensing system plasmid was constructed based on the design above. First, a plasmid in which the CYC1 promoter-mCherry-CYC1 terminator was incorporated into the MCS of the BBa\_K1680016 backbone a yeast-E. coli shuttle vector was prepared. The following linear backbone DNA was extracted from the plasmid by PCR. The target plasmid was obtained by combining the sequences of pROX1 (synthesized by IDT) and pANB1 (cloned from *S. cerevisiae* BY4741 strain) with the linear backbone by the SLiCE method, respectively. By transferring this plasmid into *S. cerevisiae* BY4741 strain, we constructed the yeast strains which have the oxygen sensing system.

### Oxygen Assay

Using the yeast strains established above, we observed the change of fluorescence under aerobic and anaerobic conditions.The pAnb1, pRox1, and mCherry strains (constitutively expressing mCherry) were cultured under anaerobic and aerobic conditions for one day each, and observed by a microscope (40x NA0.95).

**Table 2** Experimental setup

| Condition | Experimental setup |
| --- | --- |
| Aerobic condition | Incubate overnight with shaking in a normal tube for yeast incubation. |
| Anaerobic condition | Incubate overnight with shaking in a 5ml syringe filled with medium. |

![](https://static.igem.org/mediawiki/2021/f/f3/T--UTokyo--figures--results_oxygen_fig1.png)

**Fig. 1** Experimental setup of anaerobic (left) and aerobic condition(right).

Below(Fig. 2) is a microscopic image obtained from the above experiment. As can be seen from this image, the expression of mCherry is repressed in both pROX1 and pANB1 strains under anaerobic conditions.

![](https://static.igem.org/mediawiki/2021/8/83/T--UTokyo--figures--results_oxygen_fig2.png)

**Fig. 2** Change of fluorescence by pAnb1, pRox1, and Constitutive(pCyc) promoter under aerobic and anaerobic conditions. (40x NA0.95, BF:50ms, Fluorescence:600ms)

In addition, segmentation of cells was (segmented by cellpose [[4]](#fn:4) from Phase image) performed on the captured images to quantify the distribution of fluorescence states for each cell (Fig. 3). Intense fluorescence (over 1000) was observed only in aerobic conditions in any strains but in control and pRox1 strains change of fluorescence intensity were weak.
![](https://static.igem.org/mediawiki/2021/2/2a/T--UTokyo--figures--results_oxygen_fig3.png)
**Fig. 3** (A)Promoter activity of pAnb1, pRox1 and constitutive(pCyc) measured by means of Fluorescence of each cell. (B)Fluorescence distribution of cells in pAnb1 strain. (C)Fluorescence distribution of cells in pRox1 strain.(D)Fluorescence distribution of cells in Constitutive(pCyc) strain.

### Discussion

As can be seen from the experimental results, both pAnb1 and pRox1 showed a promoter activity increase with the change of oxygen concentration. However, the increase in activity was relatively small in pRox1, and also the change in activity was observed in the constitutive promoter(pCyc:BBa\_I766555). For the explanation of the results, two possibility can be thought:
**Possibility 1: The decrease in expression level is caused by a decrease in cellular metabolic activity under anaerobic conditions.**
**Possibility 2: The pCyc used as a constitutive promoter may be oxygen-responsive. (Some paper reported that pCyc have oxygen-dependent expression change [[5]](#fn:5))**
As a future prospect, we need to set up an experiment to re-evaluate these possibilities. And also, if possibility 1 actually makes a significant difference, we can build a system that directly uses this nature of yeast cells.

## Defensin Secretion

### Overview

#### Goal

To construct a system that prevents infection of wounds and keeps them clean by making *Saccharomyces cerevisiae* secrete HBD3 (human beta-defensin-3), an antimicrobial protein.

#### Result

A yeast strain that secretes HBD3 was developed, and its effect on the yeast itself was evaluated. Antimicrobial assays were also performed by using its culture supernatant. A yeast growth assay showed that HBD3 secretion affected the growth rate of yeast but did not affect the final cell density. This antimicrobial assay qualitatively suggested that HBD3 had antibacterial activity against *E. coli*. However, the results lacked certainty and required additional experiments.

#### Future Direction

We are going to qualify the secretion of HBD3 and reconsider which promoter to use. We will also test whether it shows antimicrobial activity when yeast is incorporated into a cell fiber.

### System Design

Among the various human antimicrobial proteins, HBD3 is known for its effectiveness against both Gram-negative and Gram-positive bacteria, independent of the environmental salt concentration [[6]](#fn:6). Therefore, we chose HBD3 for this project.

### Parts Construction

Based on the design described above, the plasmid was constructed by assembling a linear backbone and the sequence containing HBD3 (CYC1 promoter-HBD3-ADH1 terminator) using SLiCE. The backbone was linearized at both ends of MCS of BBa\_K1680016, and the fragment including HBD3 was synthesized in IDT. The desired yeast strain was established by transforming the plasmid into yeast.
The specific design was to incorporate the sequence CYC1 promoter-HBD3-ADH1 terminator into a plasmid and make it work in yeast.

### Effect on the Yeast

We constructed an HBD3-secreting yeast strain following the procedure described above and evaluated its growth characteristics. It was reported that HBD3 has antimicrobial activity against yeast [[6]](#fn:6), and secretion of HBD3 may affect the growth of yeast. Therefore, the growth rate between the HBD3-secreting strain and the control strain was compared in our project (n=3).
The results of the OD600 measurement of HBD3-secreting yeast were shown in Figure 4. The control strain was the pRS316 transformed one (BBa\_K1680016 compatible vector without insert). As shown in Figure 4, HBD3 dramatically affected yeast growth during the exponential phase. This property was observed particularly at 330 minutes after the inoculation. However, since the final concentration was almost the same, HBD3 secretion was considered to have little effect on the number of yeast in the end. Thus, this strain is expected to be sufficient for practical implementation.

![](https://static.igem.org/mediawiki/2021/2/26/T--UTokyo--figures--results_defensin_fig4.png)

**Fig. 4** The growth curve of the HBD3-secreting strain and the control (pRS316).

### Antibacterial Assay

Dressings were soaked with supernatant from the culture of HBD3-secretion strain and the control strain, and then they were placed on the plates coated with *E. coli*. The plates were incubated at 37℃ for 48 hours. As shown in Figure 5, a halo formed around the dressing soaked with HBD3-secretion strain culture supernatant, which was not observed with the control. The results indicated that HBD3 had antibacterial activity against *E. coli*.
However, this experiment was conducted with n=1, and the results were not reproducible. It is necessary to experiment with more samples to measure the antibacterial activity of HBD3 more accurately. (Antimicrobial assay of our yeast supernatant was also conducted by team Kyoto, and it brought similar results. Please check [here](https://2021.igem.org/Team%3AUTokyo/Notebook#Data-from-team-Kyoto).)

![](https://static.igem.org/mediawiki/2021/c/c7/T--UTokyo--figures--results_defensin_fig5.png)

**Fig. 5** The antibacterial activity of the HBD3-secreting strain. The red arrow indicates the halo.

## Pathogen Detection

### Overview

#### Goal

To construct an infection detection system by detecting quorum sensing signals of *Pseudomonas aeruginosa* using *S. cerevisiae*.

#### Result

We planned to construct the desired plasmids by assembling six DNA fragments. We successfully created DNA fragments consisting of three segments and two segments, but we did not have enough time to complete the whole construction of plasmids.

#### Future Direction

After completing the construction of the plasmids and transferring them into yeast, we are going to examine the threshold of infection detection ability by testing the response to various concentrations of AHL. In addition, by changing or mutating the promoters used, we will adjust the threshold.

### System Design

The aim is to construct a system to detect quorum sensing signals by modifying the quorum sensing pathway of prokaryotes and incorporating it into *S. cerevisiae*, but the first problem was to decide which path to use. We chose LasR and QscR proteins, which detect 3OC12-HSL [[7]](#fn:7)[[8]](#fn:8), an AHL that plays a central role in quorum sensing in *P. aerugionosa*. We also decided to use the LuxR protein pathway since the Tsinghua University iGEM team 2013 had demonstrated the detection of a quorum sensing signal of *P. aerugionosa* with LuxR [[9]](#fn:9). In incorporating the prokaryotic system into eukaryotes, we designed the plasmids based on the Tsinghua University 2013 project, as described in the [Description](https://2021.igem.org/Team%3AUTokyo/Description) page (Fig. 6).

![](https://static.igem.org/mediawiki/2021/c/c1/T--UTokyo--figures--results_pathogen_fig6.png)

**Fig. 6** The design of the plasmid.

### Plasmid Construction

#### Design

The DNA fragments shown in Table 3 were prepared by ordering them from IDT.

Table3. The details of the prepared fragments.

| Fragment name | Included regions or parts |
| --- | --- |
| fragment1 | pTEF & Flag |
| fragment2 | LuxR/LasR/QscR, pLux/pLas/pQsc, ADH1 terminator, & cyc 100 mini promoter |
| monoVP16 | VP16 |
| backbone | mCherry, CYC1 terminator, & pRS316 |

The specific plasmid construction plan is described below (Fig. 7).
Step1: To splice fragment1 and mono VP16
Step2: To splice two pieces of monoVP16
Step3: To splice fragment1+monoVP16 and monoVP16+monoVP16
Step4: To splice fragment2 and the fragment constructed in Step3
Step5: To splice backbone and the fragment constructed in Step4 by SLiCE

![](https://static.igem.org/mediawiki/2021/9/9b/T--UTokyo--figures--results_pathogen_fig7.png)

**Fig. 7** The initial plasmid construction plan.

#### Results

Step1 was successful, but Step2 did not go well, and smear bands were obtained after PCR (Fig. 8).

![](https://static.igem.org/mediawiki/2021/1/17/T--UTokyo--figures--results_pathogen_fig8.jpg)

**Fig. 8** The results of PCR of Step1 (right lane) and Step2 (left lane).

#### Redesign

Based on the results, the order of the plasmid assembly was changed: the construction was kept without tandem repeats at the initial steps because the tandem repeats could cause various by-products during PCR. The redesigned construction procedure was as follows (Fig. 9).
Step1: To splice fragment1 and monoVP16
Step2: To splice monoVP16 and fragment2
Step3: To splice backbone and the fragment constructed in Step1
Step4: To splice the fragments constructed in Step2 and Step3
Step5: To splice monoVP16 and the fragment constructed in Step4 by SLiCE

![](https://static.igem.org/mediawiki/2021/5/54/T--UTokyo--figures--results_pathogen_fig9.png)

**Fig. 9** The redesigned plasmid construction method.

#### Results

Step1, Step2, and Step3 were successful, but Step4 did not work (Fig. 10).

![](https://static.igem.org/mediawiki/2021/d/d8/T--UTokyo--figures--results_pathogen_fig10.png)

**Fig. 10** The results of PCR of Step2 (A), Step3 (B), and Step4 (C). For Step4, since Lux, Las, and Qsc (from right to left) were used for fragment2, there were three lanes.

We also tried to assemble the products of Step2, Step3, and monoVP16 by SLiCE, but no desired *E. coli* strain was obtained (Fig. 11).

![](https://static.igem.org/mediawiki/2021/2/22/T--UTokyo--figures--results_pathogen_fig11.jpg)

**Fig. 11** The results of PCR on *E. coli* colonies which could have the desired plasmid.

---

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