iGEM 2018 — Team CCU_Taiwan

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# Team:CCU Taiwan/Notebook

![](https://static.igem.org/mediawiki/2018/0/08/T--CCU_Taiwan--home_button.png)

* ![](https://static.igem.org/mediawiki/2018/2/24/T--CCU_Taiwan--aboutus.png)About Us
  [+ Team](https://2018.igem.org/Team%3ACCU_Taiwan/Team)
  [+ Attributions](https://2018.igem.org/Team%3ACCU_Taiwan/Attributions)
  [+ Medals](https://2018.igem.org/Team%3ACCU_Taiwan/Medal)
  [+ For Judges](https://2018.igem.org/Team%3ACCU_Taiwan/Judge)
  [+ Achievements](https://2018.igem.org/Team%3ACCU_Taiwan/Achievements)
* ![](https://static.igem.org/mediawiki/2018/6/6f/T--CCU_Taiwan--project.png)Project
  [+ Description](https://2018.igem.org/Team%3ACCU_Taiwan/Description)
  [+ Design](https://2018.igem.org/Team%3ACCU_Taiwan/Applied_Design)
  [+ Results](https://2018.igem.org/Team%3ACCU_Taiwan/Results)
  [+ Demonstration](https://2018.igem.org/Team%3ACCU_Taiwan/Demonstrate)
  [+ InterLab](https://2018.igem.org/Team%3ACCU_Taiwan/InterLab)
* ![](https://static.igem.org/mediawiki/2018/1/17/T--CCU_Taiwan--part.png)Parts
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Parts)
  [+ Basic Part](https://2018.igem.org/Team%3ACCU_Taiwan/Basic_Part)
  [+ Composite Part](https://2018.igem.org/Team%3ACCU_Taiwan/Composite_Part)
* ![](https://static.igem.org/mediawiki/2018/0/09/T--CCU_Taiwan--model.png)Modeling
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Model)
  [+ Binding Model](https://2018.igem.org/Team%3ACCU_Taiwan/Binding)
  [+ Polymer Model](https://2018.igem.org/Team%3ACCU_Taiwan/Polymer)
* ![](https://static.igem.org/mediawiki/2018/f/fc/T--CCU_Taiwan--Dry_lab.png)Product
  [+ Analysis](https://2018.igem.org/Team%3ACCU_Taiwan/Our_Plan)
  [+ Production Line](https://2018.igem.org/Team%3ACCU_Taiwan/Engineering)
  [+ Safety](https://2018.igem.org/Team%3ACCU_Taiwan/Safety)
* ![](https://static.igem.org/mediawiki/2018/9/96/T--CCU_Taiwan--humanpractice.png)HP
  [+ Human Practice](https://2018.igem.org/Team%3ACCU_Taiwan/Human_Practices)
  [+ Public Engagement](https://2018.igem.org/Team%3ACCU_Taiwan/Public_Engagement)
  [+ Entrepreneurship](https://2018.igem.org/Team%3ACCU_Taiwan/Entrepreneurship)
  [+ Engaging Experts](https://2018.igem.org/Team%3ACCU_Taiwan/engaging_experts)
  [+ Integrated HP](https://2018.igem.org/Team%3ACCU_Taiwan/Integrate)
* ![](https://static.igem.org/mediawiki/2018/c/c9/T--CCU_Taiwan--notebook.png)Notebook
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Notebook)
  [+ Collaborations](https://2018.igem.org/Team%3ACCU_Taiwan/Collaborations)
  [+ Protocols](https://2018.igem.org/Team%3ACCU_Taiwan/Protocols)
  [+ Experiments](https://2018.igem.org/Team%3ACCU_Taiwan/Experiments)
  [+ Materials](https://2018.igem.org/Team%3ACCU_Taiwan/Materials)

[Notebook](#ca1)

# NOTEBOOK

Notebook

February

    Molecular biology class
    *E. coli* genomic DNA preparation
    *E. coli* transformation

March

    Instrument operation

April

    Project brainstorming: Product Positioning, Monolignol proportion

May

    Culture selection: compare Yeast, *E. coli*, Acetobacter aceti
    Gene design

June

    Interlab experiment: Calibration 1, 2, 3

July

**7/3**
    Start Interlab experiment: cell measurement
  **7/5**
    Yeast Extract–Peptone–Dextrose (YPD) formulation
  **7/9**
    Yeast (X33) culture
  **7/13**
    Fundraising briefing session
  **7/16**
    Communicate with NCKU (Interlab & project)
  **7/18**
    Communicate with BIT (project)

August

**8/5~8/12**

    Cell growth, sampling, and assay
    Digest pGAPZ A(X3)/ pUCIDT\_Lac1/ pUCIDT\_Px16/ pUCIDT\_Px18 with AgeI, EcoRI
    Transformation pGAPZ A\_Lac1/ pGAPZ A\_Px16/ pGAPZ A\_Px18 (15µl) into 20µl ECOS™
    101 Competent Cells [DH5a]
    Ligation pGAPZ A\_Lac1/ pGAPZ A\_Px16/ pGAPZ A\_Px18
    Digest pUCIDT\_Lac1/ pUCIDT\_Px16/ pUCIDT\_Px18 with AgeI, EcoRI

**8/13~8/19**

    Miniprep pGAPZ A\_Lac1/ pGAPZ A\_Px16/ pGAPZ A\_Px18
    Digest pGAPZ A(X3)/ pUCIDT\_Lac1/ pUCIDT\_Px16/ pUCIDT\_Px18 with AgeI, EcoRI
    Digest pGAPZ A with SalI
    Incubate with DNA Polymerase I, Large (Klenow) Fragment remove SalI restriction site
    Ligation pGAPZ A-SalI(pGAPZ A-1)
    Transformation of pGAPZ A-SalI(pGAPZ A-1)
    Minipreparation of pGAPZ A-SalI(pGAPZ A-1)

**8/20~8/26**

  Gel extract
    Digestion – pGAPZ A-1+HIS4 cut with AgeI and EcoRI (x3)
    Ligation - pGAPZ A\_Lac1/ pGAPZ A\_Px16/ pGAPZ A\_Px18

**8/27~9/2**

    Digestion – pGAPZ A-1+HIS4 cut with AgeI and EcoRI (x3)
    Ligation – pGAPZ A-1+HIS4 cut with AgeI and EcoRI (x3)

    Transform
      1. pGAPZ A-1 + HIS4 + Px16
      2. pGAPZ A-1 + HIS4 + Px18
      3. pGAPZ A-1 + HIS4 + Lac1
    Digest pGAPZ A HIS4(X2) with AgeI, EcoRI
    pGAPZ A\_Px16, pGAPZ A\_Px18 , pGAPZ A\_Lac1 Traditional plasmid DNA extraction
    Yeast SMD1168 Transformation(pGAPZ A\_Px16, pGAPZ A\_Px18 , pGAPZ A\_Lac1)

September

**9/3~9/9**

    PCR—pGAPZ A-1 + HIS4 + Px16, Px18, Lac1
    miniprep – pGAPZ A-1 + HIS4 + Px16, Px18
    Transform – pGAPZ A-1 + HIS4 + Lac1
    Digestion – pGAPZ A-1 + HIS4+lac1

**9/12~9/14**

    Colony PCR gel electrophoresis check - HIS4+Px16, HIS4+Lac1
    Culture HIS4+Px16, HIS4+Lac1
    Colony PCR gel electrophoresis check - HIS4+Px16, HIS4+Lac1
    Yeast SMD1168 pGAPZ A\_Px16, pGAPZ A\_Px18, pGAPZ A\_Lac1 colony PCR
    Plasmid miniprep - HIS4+Lac1, HIS4+Px16

**9/24~9/27**

    Culture HIS4+Lac1 x3
    Prepare yeast competent cells (SMD1168)
    Traditional plasmid DNA extraction Day1
    Yeast SMD1168 pGAPZ A\_Px16, pGAPZ A\_Px18, pGAPZ A\_Lac1 colony PCR
    Yeast SMD1168 pGAPZ A\_Px16, pGAPZ A\_Px18, pGAPZ A\_Lac1 Western blot
    Prepare YPD plates
    Prepare the amino acids of SD plates
    Traditional plasmid DNA extraction Day2
    Plasmids contain HIS4 cut with SalI
    Yeast transformation - Px16, Px18, Lac1 (plasmids contain HIS4)

October

**10/8~10/11**

    Prepare yeast competent cells (SMD1168)
    Colony PCR (HIS4+Px16)
    Safety (30℃, 50℃, 70℃)
    Yeast transformation - Px16, Lac1 (plasmids contain HIS4)

    Yeast SMD1168 pGAPZ A\_Px16, pGAPZ A\_Px18, pGAPZ A\_Lac1 Western blot

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# Team:CCU Taiwan/Engineering

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* ![](https://static.igem.org/mediawiki/2018/2/24/T--CCU_Taiwan--aboutus.png)About Us
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  [+ Attributions](https://2018.igem.org/Team%3ACCU_Taiwan/Attributions)
  [+ Medals](https://2018.igem.org/Team%3ACCU_Taiwan/Medal)
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  [+ Achievements](https://2018.igem.org/Team%3ACCU_Taiwan/Achievements)
* ![](https://static.igem.org/mediawiki/2018/6/6f/T--CCU_Taiwan--project.png)Project
  [+ Description](https://2018.igem.org/Team%3ACCU_Taiwan/Description)
  [+ Design](https://2018.igem.org/Team%3ACCU_Taiwan/Applied_Design)
  [+ Results](https://2018.igem.org/Team%3ACCU_Taiwan/Results)
  [+ Demonstration](https://2018.igem.org/Team%3ACCU_Taiwan/Demonstrate)
  [+ InterLab](https://2018.igem.org/Team%3ACCU_Taiwan/InterLab)
* ![](https://static.igem.org/mediawiki/2018/1/17/T--CCU_Taiwan--part.png)Parts
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Parts)
  [+ Basic Part](https://2018.igem.org/Team%3ACCU_Taiwan/Basic_Part)
  [+ Composite Part](https://2018.igem.org/Team%3ACCU_Taiwan/Composite_Part)
* ![](https://static.igem.org/mediawiki/2018/0/09/T--CCU_Taiwan--model.png)Modeling
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Model)
  [+ Binding Model](https://2018.igem.org/Team%3ACCU_Taiwan/Binding)
  [+ Polymer Model](https://2018.igem.org/Team%3ACCU_Taiwan/Polymer)
* ![](https://static.igem.org/mediawiki/2018/f/fc/T--CCU_Taiwan--Dry_lab.png)Product
  [+ Analysis](https://2018.igem.org/Team%3ACCU_Taiwan/Our_Plan)
  [+ Production Line](https://2018.igem.org/Team%3ACCU_Taiwan/Engineering)
  [+ Safety](https://2018.igem.org/Team%3ACCU_Taiwan/Safety)
* ![](https://static.igem.org/mediawiki/2018/9/96/T--CCU_Taiwan--humanpractice.png)HP
  [+ Human Practice](https://2018.igem.org/Team%3ACCU_Taiwan/Human_Practices)
  [+ Public Engagement](https://2018.igem.org/Team%3ACCU_Taiwan/Public_Engagement)
  [+ Entrepreneurship](https://2018.igem.org/Team%3ACCU_Taiwan/Entrepreneurship)
  [+ Engaging Experts](https://2018.igem.org/Team%3ACCU_Taiwan/engaging_experts)
  [+ Integrated HP](https://2018.igem.org/Team%3ACCU_Taiwan/Integrate)
* ![](https://static.igem.org/mediawiki/2018/c/c9/T--CCU_Taiwan--notebook.png)Notebook
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Notebook)
  [+ Collaborations](https://2018.igem.org/Team%3ACCU_Taiwan/Collaborations)
  [+ Protocols](https://2018.igem.org/Team%3ACCU_Taiwan/Protocols)
  [+ Experiments](https://2018.igem.org/Team%3ACCU_Taiwan/Experiments)
  [+ Materials](https://2018.igem.org/Team%3ACCU_Taiwan/Materials)

[Sketch](#ca1)

[Devices](#ca2)

[Design diagram](#ca3)

# PRODUCTION LINE

When we come to production line, the most important things is that our product need to meet those modern lamination industries' needs. We designed the entire production line, and separate it into five major parts.

Sketch

![](https://static.igem.org/mediawiki/2018/b/b0/T--CCU_Taiwan--LAI_ProductionLine.jpg)

Figure1: Sketch of our designed production line.

Production Process : Fermenting→ Filtering→ Reacting→ Extraction→ Particle Manufacturing

Devices

**1. Fermentation tank:**
TFY-MS automatic mechanical stirring fermentation tank 50L
Working capacity: 65~75%
Product features: The fermentation tank adopts a mechanical stirring system, the stirrer speed can be adjusted continuously. The fermentation temperature, pH, dissolved oxygen and other parameters are automatically controlled, automatically recorded, stored, printed, and suitable for small test and pilot test of fermentation products.

![](https://static.igem.org/mediawiki/2018/3/32/T--CCU_Taiwan--LAI_production_fermentationtank.jpg)

Figure2: 3D model of the fermentation tank.

**2. Filtration device:**
Use membrane filtration to prevent yeast from leaving the system.

![](https://static.igem.org/mediawiki/2018/a/ad/T--CCU_Taiwan--LAI_production_filter.jpg)

Figure3: 3D model of the filtration device.

**3. Reaction tank**
Reacted the enzyme with the monolignol, and added other essential material (buffer and H2O2). The capabilities are acid and alkali control, stirring, temperature control and pressure control.
Type: T.T-M.I-2000
Full capacity: 2000 L
Output: 15-50 kg / Hr

![](https://static.igem.org/mediawiki/2018/e/e7/T--CCU_Taiwan--LAI_production_reactiontank.jpg)

Figure4: 3D model of the reaction tank.

**4. Low temperature concentration separation extraction system:**
The device was filtered by membrane and separated by different molecular weight to separate different substances. Using this equipment, LIGGREEN would be extracted and purified.
Microfiltration membrane: MF Membrane 0.1~0.3um
Membrane area: 5m2
Diluted liquid volume: approximately 100 L / Hr
Temperature: up to 50° C

![](https://static.igem.org/mediawiki/2018/7/70/T--CCU_Taiwan--LAI_productionline_extrction.jpg)

Figure5: 3D model of the low temperature concentration separation extraction system.

**5. Extrusion granulation (Twin screw)**
After heated material to the melting point, it was pumped (Liquid flowed out a tube and cut when it was cooled by air) to form particles, which made LIGREEN into a smaller particulates.
Type: TS 30
Output: 15-50 kg / Hr
Extruder: 7.5-18 kW

![](https://static.igem.org/mediawiki/2018/f/f5/T--CCU_Taiwan--LAI_production_pelletforming.jpg)

Figure6: 3D model of the Extrusion granulation, and we would get production at last.

Design diagram

Follow figures were complete design diagram of our production line

![](https://static.igem.org/mediawiki/2018/9/93/T--CCU_Taiwan--LAI_productionline.jpg)

Figure7: Design of our entire production line.

[![](https://static.igem.org/mediawiki/2018/0/07/T--CCU_Taiwan--CCUP1.png)](http://huitong.com.tw)
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# Team:CCU Taiwan/Experiments

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* ![](https://static.igem.org/mediawiki/2018/2/24/T--CCU_Taiwan--aboutus.png)About Us
  [+ Team](https://2018.igem.org/Team%3ACCU_Taiwan/Team)
  [+ Attributions](https://2018.igem.org/Team%3ACCU_Taiwan/Attributions)
  [+ Medals](https://2018.igem.org/Team%3ACCU_Taiwan/Medal)
  [+ For Judges](https://2018.igem.org/Team%3ACCU_Taiwan/Judge)
  [+ Achievements](https://2018.igem.org/Team%3ACCU_Taiwan/Achievements)
* ![](https://static.igem.org/mediawiki/2018/6/6f/T--CCU_Taiwan--project.png)Project
  [+ Description](https://2018.igem.org/Team%3ACCU_Taiwan/Description)
  [+ Design](https://2018.igem.org/Team%3ACCU_Taiwan/Applied_Design)
  [+ Results](https://2018.igem.org/Team%3ACCU_Taiwan/Results)
  [+ Demonstration](https://2018.igem.org/Team%3ACCU_Taiwan/Demonstrate)
  [+ InterLab](https://2018.igem.org/Team%3ACCU_Taiwan/InterLab)
* ![](https://static.igem.org/mediawiki/2018/1/17/T--CCU_Taiwan--part.png)Parts
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Parts)
  [+ Basic Part](https://2018.igem.org/Team%3ACCU_Taiwan/Basic_Part)
  [+ Composite Part](https://2018.igem.org/Team%3ACCU_Taiwan/Composite_Part)
* ![](https://static.igem.org/mediawiki/2018/0/09/T--CCU_Taiwan--model.png)Modeling
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Model)
  [+ Binding Model](https://2018.igem.org/Team%3ACCU_Taiwan/Binding)
  [+ Polymer Model](https://2018.igem.org/Team%3ACCU_Taiwan/Polymer)
* ![](https://static.igem.org/mediawiki/2018/f/fc/T--CCU_Taiwan--Dry_lab.png)Product
  [+ Analysis](https://2018.igem.org/Team%3ACCU_Taiwan/Our_Plan)
  [+ Production Line](https://2018.igem.org/Team%3ACCU_Taiwan/Engineering)
  [+ Safety](https://2018.igem.org/Team%3ACCU_Taiwan/Safety)
* ![](https://static.igem.org/mediawiki/2018/9/96/T--CCU_Taiwan--humanpractice.png)HP
  [+ Human Practice](https://2018.igem.org/Team%3ACCU_Taiwan/Human_Practices)
  [+ Public Engagement](https://2018.igem.org/Team%3ACCU_Taiwan/Public_Engagement)
  [+ Entrepreneurship](https://2018.igem.org/Team%3ACCU_Taiwan/Entrepreneurship)
  [+ Engaging Experts](https://2018.igem.org/Team%3ACCU_Taiwan/engaging_experts)
  [+ Integrated HP](https://2018.igem.org/Team%3ACCU_Taiwan/Integrate)
* ![](https://static.igem.org/mediawiki/2018/c/c9/T--CCU_Taiwan--notebook.png)Notebook
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Notebook)
  [+ Collaborations](https://2018.igem.org/Team%3ACCU_Taiwan/Collaborations)
  [+ Protocols](https://2018.igem.org/Team%3ACCU_Taiwan/Protocols)
  [+ Experiments](https://2018.igem.org/Team%3ACCU_Taiwan/Experiments)
  [+ Materials](https://2018.igem.org/Team%3ACCU_Taiwan/Materials)

[Notebooks](#ca1)

# EXPERIMENTS

Laboratory notebooks

Our project aims to produce new material— LIGGREEN. In our experiment, we select *P. pastoris* as our strain to secrete enzymes which helps the polymerization of coniferyl alcohol. Besides, we divide into two groups to conduct experiment— gene sequence with or without Zeocin™.

Here are the common steps of our experiment：
1. Clone our vector in *E.coli*
2. Minipreparate our vector and purified from *E.coli*
3. Transform the linear vector into *P. pastoris*
4. Select *P. pastoris* strains
 5. Conduct western blot to detect our target enzyme
6. Purified target enzyme.

[![](https://static.igem.org/mediawiki/2018/0/07/T--CCU_Taiwan--CCUP1.png)](http://huitong.com.tw)
[![](https://static.igem.org/mediawiki/2018/e/e3/T--CCU_Taiwan--CCUP2.png)](https://www.facebook.com/ccuigemteam/?ref=br_rs)
[![](https://static.igem.org/mediawiki/2018/8/89/T--CCU_Taiwan--CCUP3.png)](http://ewww.ccu.edu.tw/)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:CCU Taiwan/Results

![](https://static.igem.org/mediawiki/2018/0/08/T--CCU_Taiwan--home_button.png)

* ![](https://static.igem.org/mediawiki/2018/2/24/T--CCU_Taiwan--aboutus.png)About Us
  [+ Team](https://2018.igem.org/Team%3ACCU_Taiwan/Team)
  [+ Attributions](https://2018.igem.org/Team%3ACCU_Taiwan/Attributions)
  [+ Medals](https://2018.igem.org/Team%3ACCU_Taiwan/Medal)
  [+ For Judges](https://2018.igem.org/Team%3ACCU_Taiwan/Judge)
  [+ Achievements](https://2018.igem.org/Team%3ACCU_Taiwan/Achievements)
* ![](https://static.igem.org/mediawiki/2018/6/6f/T--CCU_Taiwan--project.png)Project
  [+ Description](https://2018.igem.org/Team%3ACCU_Taiwan/Description)
  [+ Design](https://2018.igem.org/Team%3ACCU_Taiwan/Applied_Design)
  [+ Results](https://2018.igem.org/Team%3ACCU_Taiwan/Results)
  [+ Demonstration](https://2018.igem.org/Team%3ACCU_Taiwan/Demonstrate)
  [+ InterLab](https://2018.igem.org/Team%3ACCU_Taiwan/InterLab)
* ![](https://static.igem.org/mediawiki/2018/1/17/T--CCU_Taiwan--part.png)Parts
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Parts)
  [+ Basic Part](https://2018.igem.org/Team%3ACCU_Taiwan/Basic_Part)
  [+ Composite Part](https://2018.igem.org/Team%3ACCU_Taiwan/Composite_Part)
* ![](https://static.igem.org/mediawiki/2018/0/09/T--CCU_Taiwan--model.png)Modeling
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Model)
  [+ Binding Model](https://2018.igem.org/Team%3ACCU_Taiwan/Binding)
  [+ Polymer Model](https://2018.igem.org/Team%3ACCU_Taiwan/Polymer)
* ![](https://static.igem.org/mediawiki/2018/f/fc/T--CCU_Taiwan--Dry_lab.png)Product
  [+ Analysis](https://2018.igem.org/Team%3ACCU_Taiwan/Our_Plan)
  [+ Production Line](https://2018.igem.org/Team%3ACCU_Taiwan/Engineering)
  [+ Safety](https://2018.igem.org/Team%3ACCU_Taiwan/Safety)
* ![](https://static.igem.org/mediawiki/2018/9/96/T--CCU_Taiwan--humanpractice.png)HP
  [+ Human Practice](https://2018.igem.org/Team%3ACCU_Taiwan/Human_Practices)
  [+ Public Engagement](https://2018.igem.org/Team%3ACCU_Taiwan/Public_Engagement)
  [+ Entrepreneurship](https://2018.igem.org/Team%3ACCU_Taiwan/Entrepreneurship)
  [+ Engaging Experts](https://2018.igem.org/Team%3ACCU_Taiwan/engaging_experts)
  [+ Integrated HP](https://2018.igem.org/Team%3ACCU_Taiwan/Integrate)
* ![](https://static.igem.org/mediawiki/2018/c/c9/T--CCU_Taiwan--notebook.png)Notebook
  [+ Overview](https://2018.igem.org/Team%3ACCU_Taiwan/Notebook)
  [+ Collaborations](https://2018.igem.org/Team%3ACCU_Taiwan/Collaborations)
  [+ Protocols](https://2018.igem.org/Team%3ACCU_Taiwan/Protocols)
  [+ Experiments](https://2018.igem.org/Team%3ACCU_Taiwan/Experiments)
  [+ Materials](https://2018.igem.org/Team%3ACCU_Taiwan/Materials)

[Project Achievements](#ca1)

# RESULTS

Project Achievements

1. Prove our vectors successfully constructed
2. Prove three enzymes successfully produced
3. Prove enzymes are functional

Prove our vectors successfully constructed

  In LIGGREEN synthesis, monolignols react mainly with two enzymes, peroxidase and laccase. We decided to synthesize these enzymes through synthetic biology. In our experiments, we use *P. pastoris* as our host. Our goal is to transform the insert into *P. pastoris*. We did westernblot to prove the yeast transform was successful and the enzymes were produced.

![](https://static.igem.org/mediawiki/2018/5/57/T--CCU_Taiwan--JOE_results_figure1.jpg)

Figure 1：Plasmid digest( EcoRI, AgeI) after miniprep (pGAPZ A-SalI\_His4, pGAPZ A-SalI)

![](https://static.igem.org/mediawiki/2018/0/0e/T--CCU_Taiwan--JOE_results_figure2.jpg)

Figure 2：Plasmid digest( EcoRI, AgeI) after miniprep (Px16, Px18, Lac1)

![]( https://static.igem.org/mediawiki/2018/6/6b/T--CCU_Taiwan--JOE_results_figure3.jpg)

Figure 3：PCR result of E.coli colony (pGAPZ A\_Px16, pGAPZ A\_Px18, pGAPZ A\_Lac1)

  According to gel electrophoresis, we have confirm that our cloning is successful.

Prove three enzymes successfully produced

  We use western blot to analyze our protein expression. To detect Lac1, Px16, Px18, we select mouse anti-FLAG tag, mouse anti-HA tag, mouse anti-V5 tag, respectively as our primary antibody. As our secondary antibody, we use goat anti-mouse HRP. We to detect secondary antibody, we apply standard enhanced chemilumescent (ECL) substrates for detecting horseradish peroxidase (HRP) enzyme activity.

Western blot：

![](https://static.igem.org/mediawiki/2018/8/87/T--CCU_Taiwan--western_blot_%28lac1_protein_extraction_from_pellet%29.jpeg)

Figure 4：Western blot (lac1 protein extraction from pellet) of high copy strain, middle copy strain, high copy strain at 96hr

![](https://static.igem.org/mediawiki/2018/9/90/T--CCU_Taiwan--western_blot_%28prx18_protein_extraction_from_pellet%29.png)

Figure 5：Western blot (px18 protein extraction from pellet) of high copy strain

![](https://static.igem.org/mediawiki/2018/e/e5/T--CCU_Taiwan--western_blot_%28px18_soup%29.png)

Figure 6：Western blot (px18 protein extraction from soup) of high copy strain

![](https://static.igem.org/mediawiki/2018/6/65/T--CCU_Taiwan--western_blot_%28px16_protein_extraction_from_pellet%29.png)

Figure 7：Western blot (px16 protein extraction from pellet) of low copy strain

Prove enzymes are functional

  Coniferyl alcohol will react with our enzymes, Px16, Px18 and Lac1. We want to prove that coniferyl alcohol will polymerize into an oligomer. We use UV-visible for measurements. Through UV-visible, we can prove the difference in absorbance wavelength between monomer and oligomer.

UV-visible :

![](https://static.igem.org/mediawiki/2018/4/48/T--CCU_Taiwan--JOE_results_4-5.jpg)

Figure 8：the difference between monomer and monomer + Lac1

![](https://static.igem.org/mediawiki/2018/f/fb/T--CCU_Taiwan--JOE_results_4-6.jpg)

Figure 9：the difference between monomer and monomer + Px16 and Px18

![](https://static.igem.org/mediawiki/2018/a/a9/T--CCU_Taiwan--JOE_results_4-7.jpg)

Figure 10：the difference between monomer and monomer + Lac1 + Px16 and Px18

  According to UV-visible, when the wavelength reaches some specific positions, the oligomer will shift to the left more than the monomer. Therefore, we found that only the peroxidase case did not produce spectral absorption. We have shown that our enzymes can polymerize the monomer into the oligomer.

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