iGEM 2019 — Team Tunghai_TAPG

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# Team:Tunghai TAPG/Notebook

TeamPage

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* [## **Home**

  (current)](https://2019.igem.org/Team%3ATunghai_TAPG)
* ## **Project**

  (current)

  [#### **Description**](https://2019.igem.org/Team%3ATunghai_TAPG/Description)
  [#### **AMP's**](https://2019.igem.org/Team%3ATunghai_TAPG/AMPs)
  [#### **Part**](https://2019.igem.org/Team%3ATunghai_TAPG/Parts)
  [#### **Result**](https://2019.igem.org/Team%3ATunghai_TAPG/Results%20)
  [#### **Notebook**](https://2019.igem.org/Team%3ATunghai_TAPG/Notebook)
  [#### **Protocol**](https://2019.igem.org/Team%3ATunghai_TAPG/Protocol)
  [#### **Demonstration**](https://2019.igem.org/Team%3ATunghai_TAPG/Demonstrate)
* ## **Human Practice**

  (current)

  [#### **Overview**](https://2019.igem.org/Team%3ATunghai_TAPG/Human_Practices/Overview)
  [#### **Integrated Human Practice**](https://2019.igem.org/Team%3ATunghai_TAPG/Human_Practices)
  [#### **Collaboration**](https://2019.igem.org/Team%3ATunghai_TAPG/Collaborations)
* [## **Dry Lab**

  (current)](https://2019.igem.org/Team%3ATunghai_TAPG/Model)
* ## **Team**

  (current)

  [#### **Team Members**](https://2019.igem.org/Team%3ATunghai_TAPG/Team)
  [#### **Attribution**](https://2019.igem.org/Team%3ATunghai_TAPG/Attributions)
* [## **Safety**

  (current)](https://2019.igem.org/Team%3ATunghai_TAPG/Safety)

![](https://static.igem.org/mediawiki/2019/e/ef/T--Tunghai_TAPG--notebook_cover.jpg)

| Name of medicament | Vendor | Serial number |
| --- | --- | --- |
| Acrilamide(29:1) | USB | AC0353 |
| Agar | BD | 214010 |
| Agarose | Invitrogen | 17852 |
| Ampicilin sodium salt | Sigma | A9518 |
| Beta-mercaptoethanol | Plus one | 17-1317-01 |
| Bisacrylamide | Sigma | 146072 |
| Calcium chloride | Sigma | 10043-52-4 |
| Comassie Brilliant Blue | Merck | 1.15444.00025 |
| DTT(Dithiothreitol) | Sigma | D632 |
| EDTA(Ethylenediaminetetraacetic acid) | USB | US15699 |
| Ethanol | Showa | 0502-3160 |
| Glycerol | Merck | 1.04092.1000 |
| Glyeine | USB | 123009 |
| Hydrogen chloride | TEDIA | 7647-01-0 |
| Imidazole | USB | 115860 |
| IPTG(Isopropyl Beta-D-1-Thiogalactopyranoside) | Calbiochem | B46679 |
| Kanamycin | Sigma | K0254 |
| PMSF(Phenylmethanesulfonyl floride) | Sigma | P7626 |
| Potassium chloride | Merck | 1.04936.1000 |
| Sodium Chloride | KANTO chemical | 37144-00 |
| Sodium dihtdrogen phosphate | Sigma | 1.06370.0050 |
| SDS(Sodium Dodecyl Sulfate) | USB | US75819 |
| Sodium hydrogen phosphate | SHOWA | 1932-7150 |
| Sodium hydroxide | Merck |  |
| TEMED(N,N,N’,N’,-Tetramethylenediamine) | Merck | 8.08742.0005 |
| Tris base | USB | US75825 |
| Tryptone | BD | 5027930 |
| Tween-20 | SHOWA |  |
| Yeast extract | Affymetrix | 4220471 |

| Biochemical reagent and kit name | Vendor | Serial number |
| --- | --- | --- |
| QIAprep Miniprep | QIAGEN | 27106 |
| Clean/Gel Extraction Kit | Biokit | Bio-C100 |
| 1Kb DNA Ladder | Biokit | Bio-1kb |
| 100bp DNA Ladder | Biokit | Bio-100 |
| 500bp DNA Ladder | Biokit | Bio-50 |
| 40mM dNTPs | Biokit | Bio-NTP-10 |
| Taq polymerase | Allbio | ABTGMBP02 |
| 6x Loading Dye | Biokit | Bio-LDY |
| Protein Marker | Ivitrogen | 02101-250 |
| SYBR safe DNA Gel Stain | Ivitrogen | S33120 |
| T4 DNA Ligase |  | M020T |
| BasI-HFv2 | NEB | R3733S |
| BasI-HF | NEB | R339L |
| BCA protein assay kit | Thermo | 23225 |

| Name of strain | Source of strain |
| --- | --- |
| Escherichia coil DH5α | Food Industry Research and Development Instiute |
| Escherichia coil BL21(DE3) | Lucigen |
| Escherichia coil C41(DE3) pLysS | Lucigen |

###### | | | | --- | --- | | **Date** | **Project fortnightly** | | 02/17-03/02 | * Wet Lab: Designed the vector(TAPG)* Dry Lab: The members were organized and everyone expressed their views on the project.* Human practice: Browsing the previous iGEM wiki, and brainstorming about our team project | | 03/03-03/16 | * Wet Lab: Designed the primers(eGFP、SUMO、6x His tag、intein)* Dry Lab: Because our product is based on antimicrobial peptide as the main component of hospital’s supply, we need to detail the relationship between the various pointers of the environment in the hospital and the growth of bacteria, such as the temperature and humidity in the influence on bacterial growth and the effect of peptide.* Human practice: Brainstorming possible outreach activities, including a science camp, high school lecture and volunteer etc. | | 03/17-03/30 | * Wet Lab: Primers have arrived .PCR Amplification and then check PCR product on agarose gel.* Dry Lab: For the project we were searching for paper to find out how to design model.* Human practice: Asking expert for the advise of our project. | | 03/31-04/13 | * Wet Lab: Ligase and overlap extension PCR.* Dry Lab: We thought that the growth status of bacteria had the effect on our final product, so we planned to set up the growth model of E.coli first.* Human practice: Meetup with different iGEM team and asking their experience about our current problem. | | 04/14-04/27 | * Wet Lab: Heat Shock Transformation of Chemically Component E.coli( DH5α). Used PCR to colony, afterward Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size* Dry Lab: We looked for the literature on the bacterial growth model and found that Gompertz model was recognized and recommended by many researchers.* Human practice: Conduct a Questionnaire ranging from under18 and above 65 about public cognition. | | 04/28-05/11 | * Wet Lab: DNA Sequencing (vector designed is Successful).* Dry Lab: We asked the experimental group for help to do the growth curve of E.coli in general environment, and then we continued to study the Gompertz model.* Human practice: Holding a camp for high school student, teaching them basic synthesis biology. | | 05/12-05/25 | * Wet Lab: Designed primers for expression of Antimicrobial peptide.* Dry Lab: We consolidated and analyzed the data from the experimental group, after that we set up our growth model based on the Gompertz model.* Human practice: Visit different alumni like department of chemical engineering. | | 05/26-06/08 | * Wet Lab: Designed primers for expression of Antimicrobial peptide.* Dry Lab: We made a preliminary model of the growth model, but we still searched papers in the hope that the model could be perfected.* Human practice: Target on tour project and ask help for more profession problem. | | 06/09-06/22 | * Wet Lab: Primers have arrived .PCR Amplification and then check PCR product on agarose gel. Ligase. Ligase product was subjected to DNA extraction using a Gene-Spin TM 1-4-3 DNA Purification Kit, and plasmids were used PCR. Then Run the gel to do initial analysis.* Dry Lab: We compared the improved model with the data from the experimental group.* Human practice: Hold an activity to kindergarden student and make them know how fun the science is. | | 06/23-07/06 | * Wet Lab: Heat Shock Transformation of Chemically Component C43 E. coli. Isolation of plasmid in E.coli(C43).* Dry Lab: We wanted or maximize product effect at the lowest cost. So we designed some experiments in order to obtain the best convention and the suitable effective time of peptide.* Human practice: Meet up with 2019 NCTU iGEM on video. | | 07/07-07/20 | * Wet Lab: Used PCR to colony, afterward Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size.* Dry Lab: We still search for information for suitable model references.* Human practice: Speech to student of other major. | | 07/21-08/03 | * Wet Lab: Protein Overexpression Protocol. And cell disruption.* Dry Lab: We asked the teachers who came from the department of bioscience and chemistry for the design and some related questions on the experiments.* Human practice: Meet up with 2019 mingdao iGEM onsite. | | 08/04-08/17 | * Wet Lab: Protein Overexpression Protocol. And cell disruption..* Dry Lab: We had an experimental discussion with the experimental group.* Human practice: Take part in 2019 Taiwan conference. | | 08/18-08/31 | * Wet Lab: protein purification* Dry Lab: We assisted the experimental group in the experiment.* Human practice: Collaborate with iGEM mingdao. | | 09/01-09/14 | * Wet Lab: Growth curve measurement (Antimicrobial peptide)* Dry Lab: We analyzed the data and tried to work out the equation.* Human practice: Edit iGEM wiki page | | 09/15-09/28 | * Wet Lab: MIC test.* Dry Lab: We applied the computer software matlab try to make the figure of model.* Human practice: Edit iGEM wiki page | | 09/29-10/12 | * Wet Lab: protein purification* Dry Lab: We asked the instructor whether the model right or not.* Human practice: Edit iGEM wiki page. | | 10/12-10/26 | * Wet Lab: Edit iGEM wiki page.* Dry Lab: We discussed our final models and submitted the data.* Human practice: Edit iGEM wiki page. |


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# Team:Tunghai TAPG/Experiments

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[HOME](https://2019.igem.org/Team%3ATunghai_TAPG)

TEAM

[Team Members](https://2019.igem.org/Team%3ATunghai_TAPG/Team)
[Collaborations](https://2019.igem.org/Team%3ATunghai_TAPG/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3ATunghai_TAPG/Description)
[Design](https://2019.igem.org/Team%3ATunghai_TAPG/Design)
[Experiments](https://2019.igem.org/Team%3ATunghai_TAPG/Experiments)
[Notebook](https://2019.igem.org/Team%3ATunghai_TAPG/Notebook)
[Contribution](https://2019.igem.org/Team%3ATunghai_TAPG/Contribution)
[Results](https://2019.igem.org/Team%3ATunghai_TAPG/Results)
[Demonstrate](https://2019.igem.org/Team%3ATunghai_TAPG/Demonstrate)
[Improve](https://2019.igem.org/Team%3ATunghai_TAPG/Improve)
[Attributions](https://2019.igem.org/Team%3ATunghai_TAPG/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3ATunghai_TAPG/Parts)
[Basic Parts](https://2019.igem.org/Team%3ATunghai_TAPG/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3ATunghai_TAPG/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3ATunghai_TAPG/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3ATunghai_TAPG/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3ATunghai_TAPG/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3ATunghai_TAPG/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3ATunghai_TAPG/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3ATunghai_TAPG/Hardware)
[Measurement](https://2019.igem.org/Team%3ATunghai_TAPG/Measurement)
[Model](https://2019.igem.org/Team%3ATunghai_TAPG/Model)
[Plant](https://2019.igem.org/Team%3ATunghai_TAPG/Plant)
[Software](https://2019.igem.org/Team%3ATunghai_TAPG/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Tunghai_TAPG)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:Tunghai TAPG/Results

TeamPage

![](https://static.igem.org/mediawiki/2019/b/b6/T--Tunghai_TAPG--LOG.jpg)

* [## **Home**

  (current)](https://2019.igem.org/Team%3ATunghai_TAPG/Model)
* ## **Project**

  (current)

  [#### **Description**](https://2019.igem.org/Team%3ATunghai_TAPG/Description)
  [#### **AMP's**](https://2019.igem.org/Team%3ATunghai_TAPG/AMPs)
  [#### **Part**](https://2019.igem.org/Team%3ATunghai_TAPG/Parts)
  [#### **Result**](https://2019.igem.org/Team%3ATunghai_TAPG/Results%20)
  [#### **Notebook**](https://2019.igem.org/Team%3ATunghai_TAPG/Notebook)
  [#### **Protocol**](https://2019.igem.org/Team%3ATunghai_TAPG/Protocol)
  [#### **Demonstration**](https://2019.igem.org/Team%3ATunghai_TAPG/Demonstrate)
* ## **Human Practice**

  (current)

  [#### **Overview**](https://2019.igem.org/Team%3ATunghai_TAPG/Human_Practices/Overview)
  [#### **Integrated Human Practice**](https://2019.igem.org/Team%3ATunghai_TAPG/Human_Practices)
  [#### **Collaboration**](https://2019.igem.org/Team%3ATunghai_TAPG/Collaborations)
* [## **Dry Lab**

  (current)](https://2019.igem.org/Team%3ATunghai_TAPG/Model)
* ## **Team**

  (current)

  [#### **Team Members**](https://2019.igem.org/Team%3ATunghai_TAPG/Team)
  [#### **Attribution**](https://2019.igem.org/Team%3ATunghai_TAPG/Attributions)
* [## **Safety**

  (current)](https://2019.igem.org/Team%3ATunghai_TAPG/Model)

![](https://static.igem.org/mediawiki/2019/0/01/T--Tunghai_TAPG--result_cover.jpg)

# *Overview*

### **JJ01** is our core subject, it is an antimicrobial peptide that has been verified that it has excellent antibacterial effect and lower hemolysis. We discovered there are three key proteins could help us to produce antimicrobial peptide effectively.

### Goal:

#### 1. Improved the engineering bacteria provided by iGEM by adding four proteins which could be optimized--- eGFP (enhanced green fluorescent protein), SUMO (small ubiquitin-like modifier), sec VMA intine and 6xHis tag. 2. 2. Using eGFP as an identifier to monitor the yield of the proteins.

### Results:

#### 1. Execute the production of antimicrobial peptide **JJ01**. 2. Verify the effect of antimicrobial peptide **JJ01**.

# *Results*

## Design the Vector

### Improved the engineered bacteria that provided by iGEM by adding four special proteins with different effects. Adding His 6x which can bind to metal divalent ions, and facilitate the purification of the target protein. The proteins with His tag can be purified by divalent metal ions affinity chromatography column under non-denaturing conditions. Then addition of SUMO is to avoid the possible toxicity of our target proteins to the bacterial host and increase its solubility as well. And using eGFP as a reporter to help us quickly screen for successful transformation of colonies and also allow us to quickly monitor the protein production. In order to obtain the pure target peptide, we have added the Intein. which is a self-cleavable polypeptide chain under specific conditions.

![](https://static.igem.org/mediawiki/2019/6/65/T--Tunghai_TAPG--_PCR_SUMO_eGFP.jpg)

### **Fig.1.** Successful PCR 1% Agarose gel electrophoresis.

### - Lane 1: 100 bp DNA Ladder - Lane 2: sample 1 (SUMO: 297 bp) - Lane 3: sample 2 (eGFP: 717 bp) - Lane 4: sample 3 (Intein: 1534 bp)

### **Figure 1.** Three DNA fragments were produced by PCR for the major parts in the recombinant plasmid followed by the agarose gel electrophoresis analysis. We could notice the size of each fragment is very close to our expectation. We have designed a vector that contains eGFP(enhanced green fluorescent protein), SUMO (small ubiquitin-like modifier), and sec VMA intein, then using PCR to amplify these fragments followed by agarose gel electrophoresis. **Fig.1** demonstrates the size of each PCR product is similar to that of our expected.

![](https://static.igem.org/mediawiki/2019/a/a9/T--Tunghai_TAPG--_Successful_PCR_0.7%25_Agarose_gel_electrophoresis.jpg)

### **Fig.2.** Successful PCR 0.7 % Agarose gel electrophoresis

### - Lane 1: 1kb DNA Ladder - Lane 2: sample1 (vector with Intein:4784 bp) - Lane 3: sample2 (vector without Intein:3250 bp)

### **Figure 2.**Two vectors were selected for PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis. The size of each fragment is very close to our expectation and their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan. Ligation with BsaⅠ-HFv2 enzyme and T7 DNA Ligase, then using PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis. The size of each fragment is very close to our expectation and their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan. Heat shock transformation into chemically component *E. coli* (DH5α) was performed and cells were streaked on a plate to obtain isolated colonies. The colonies were selected by PCR amplification followed by the agarose gel electrophoresis analysis. And their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan. Plasmid construction We have designed the target gene **(JJ01)** and contracted with commissioned Mission Biotech for **JJ01** DNA synthesis as well as sequencing later on. Because protein cleavage and purification are commonly not easy and enzymes for protein cleavage are quit expensive. we therefore constructed two plasmids, one of which contains sec VMA Intine for easy purification. We learned from the Journal of Engineering that the addition of Intein is not always conducive to soup up the expression of the recombinant protein due to the large molecular weight of Intein. This result is consistent with our experimental results, which inferred that the addition of Intein in the vector made the target protein to be at the precipitation. Later on, we will commission it to a Bio company for protein purification with HPLC.

![](https://static.igem.org/mediawiki/2019/6/6b/T--Tunghai_TAPG--_Successful_PCR_4%25_Agarose_gel_electrophoresis.jpg)

### **Fig.3.** Successful PCR 4% Agarose gel electrophoresis. - Lane 1:50 bp DNA Ladder - Lane 2-4: sample 1-3 (target: 33 bp)

### **Figure 3.** target gene **(JJ01)** were used PCR to carry on a small amount of expansion, afterward, Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size.

### **JJ01** antibacterial peptide was composited by 11 amino acids. We reverse transcript the JJ01 antibacterial peptide from amino acid according to genetic code. We then can design the primers for PCR to amplify the DNA fragment, followed by the agarose gel electrophoresis analysis.

![](https://static.igem.org/mediawiki/2019/7/70/T--Tunghai_TAPG--_Checkout_plasmid_4817_bp_1%25_Agarose_gel_electrophoresis.jpg)

### **Fig.4.** Checkout plasmid 4817 bp 0.7% Agarose gel electrophoresis.

### - Lane 1: sample 1 (plasmid without Extraction) - Lane 2: sample 2 (plasmid with Extraction) - Lane 3: 1 kb DNA Ladder

### **Figure 4.** Plasmids were used PCR to carry on a small amount of expansion, afterward, Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size and the content is low but, the relative purity is higher.

### The ligation reaction was carried out using the cleavage enzyme BsaI-HFv2 and ligase T7 DNA Ligation, and the ligated product was subjected to DNA extraction using a Gene-Spin TM 1-4-3 DNA Purification Kit, and plasmids were used PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis with the proper size. Although the intensity is weak, the purity is relatively higher.

![](https://static.igem.org/mediawiki/2019/e/ee/T--Tunghai_TAPG--Checkout_E.coli%28DH5%CE%B1with_TAPG-JJo1%29_4817_bp_1%25_Agarose_gel_electrophoresis.jpg)

### **Fig.5.** Checkout *E.coli* (**DH5α** with TAPG-**JJ01**) 4817 bp 0.7 % Agarose gel electrophoresis.

### - Lane 1: 1 kb DNA Ladder - Lane 2-6: DNA sample 1-5 - Lane 7-11: DNA sample 6-11

### **Figure 5.**Heat shock transformation of plasmid into of cComponent *E. coli* **(DH5α)** due to its stabilityle and easy forto transformation. properties andThe cells were streaked on a plate to obtain isolated colonies (determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed). Using used PCR to select the coloniesy., afterward Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size According to, gel analysis, the colonies 1, 3, 5 (plasmid with intein **DH5α**), and colonies 7, 10 (plasmid with intein **DH5α**) are roughly verifiedcorrect, so we choose colonies 1 and ,7 for further study. again coated, Their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan.

![](https://static.igem.org/mediawiki/2019/4/42/T--Tunghai_TAPG--DNA_sequence_%28JJ01%29.jpg)

### **Fig.6.** DNA sequence**(JJ01)**.

### **Figure 6.** DNA Sequencing of target gene **(JJ01)** to checkout DNA Sequence is correct.

### Through the DNA sequence result map, the DNA sequence was compared with the originally designed target gene **(JJ01)** sequence, and it was found that this sequence was consistent with the sequence we originally designed, and we can know that we have successfully linked the target gene **(JJ01)** into our plasmid (Pet6) and successfully transformed into the competent cell **(DH5α)**..

![](https://static.igem.org/mediawiki/2019/e/e8/T--Tunghai_TAPG--Dish_with_bacterial_%28E.coli%28C43_with_TAPG-JJ01%29%29_under_the_ultraviolet_light.jpg)

### **Fig.7.** Dish with bacterial (*E.coli* (C43 with PATG-**JJ01**)) under the ultraviolet light.

### **Figure 7.** Dish with bacterial (*E.coli* (C43 with PATG-**JJ01**)) under the ultraviolet light. And determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed into competent cells (C43).

### Using heat shock for second transformation, we chose to transformation into *E. coli* (C43) because it can efficiently enhance the expression of toxic proteins. The following figure shows the image of bacterial disk under ultraviolet light, which can preliminarily determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed into competent cells (C43) and containing the target gene as well.

![](https://static.igem.org/mediawiki/2019/0/03/T--Tunghai_TAPG--Checkout_protein_44kDa_%28plasmid_without_sec_VMA_intine%29.jpg)

### **Fig.8.** Checkout protein 44 kDa (plasmid without sec VMA intine).

### - Lane 1: Protein Maker (10-245 kDa) - Lane 2-3: protein samples collected from cell disruption (one-time centrifugation)

### **Figure 8.** SDS-PAGE.(protein without Intein) indicating that the correct position and there are more products in the soup.

![](https://static.igem.org/mediawiki/2019/c/cb/T--Tunghai_TAPG--_Checkout_protein_100_kDa_%28plasmid_with_sce_VMA_intein%29.jpg)

### **Fig.9.** Checkout protein 100 kDa (plasmid with sce VMA intein).

### - Lane 1-3: protein samples collected from cell disruption (two-time centrifugation) - Lane 4-6: protein samples collected from cell disruption (one-time centrifugation) - Lane 7: Protein Maker (10-245 kDa)

### **Figure 9.** SDS-PAGE. (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.

### After pProtein oOverexpression, the cells were harvested by centrifugation and disruptedion process was utilized by a Ultrasonic Cell Disruptor to obtain the cell extract containing for the recombinant protein. The protein expression was and also analyzed by SDS-PAGE. Fig.8. The checkout protein at 44 kDa (plasmid without sec VMA intine) indicatsing that the correct position and high expression of target protein, and also demonstrating the ration between the supernatant and the precipitation up to 90 percent.We added sec VMA intine protein in order to splice target protein also reduce the amino acid residue. According to Fig.9. checking out protein at 100 kDa (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.We added sec VMA intine protein in order to splice target protein also reduce the amino acid residue. According to **Fig.9.** checking out protein at 100 kDa (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.

![]( https://static.igem.org/mediawiki/2019/7/7b/T--Tunghai_TAPG--jj01_kill.png)

### **Fig.10.** We grew the bacteria on the Petri dishes. Left: with **JJ01**, right: without **JJ01**.

![](https://static.igem.org/mediawiki/2019/6/67/T--Tunghai_TAPG--_JJ01activity.jpg)

### **Fig.11.** Antimicrobial activity of **JJ01**.

### We tested the MIC90 (*E. coli*) of **JJ01** with the purified target peptide and confirmed that the **JJ01** has the same antibacterial effect as the patent. So we can believe that we have succeeded in doing the Genetic engineering experiments.

#### Reference

##### - 1. Wang SJ, Chen BY, Wang YJ, et al. Application of inteins in building protein affinity purification system. Chin J Biotech, **2016**.

##### - 2. Bailey A. Fong, W.-Y. W, David W. Wood, The potential role of self-cleaving Purification tags in commercial-scale processes. Trends in Biotechnology, **2010**.

##### - 3. Li , Y., Self-cleaving fusion tags for recombinant protein production Biotechnology Letters, **2011**.

##### - 4. DW, W., New trends and affinity tag designs for recombinant protein purification. Current Opinion in Structural Biology, **2014**.

##### - 5. Hassouneh W,C.T.,Chilkoti A, Elastin-like polypeptides as a purification tag for recombinant proteins. Current Protocols in Protein Science, **2010**.

##### - 6. Micklos DA, Freyer GA **(2003)** DNA Science: A First Course, 2nd ed. Cold Spring Harbor Laboratory Press, New York.