iGEM 2020 — Team NWU-CHINA-A

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

# Team:NWU-CHINA-A/Notebook

Project

* [Description](https://2020.igem.org/Team%3ANWU-CHINA-A/Description)
* [Design](https://2020.igem.org/Team%3ANWU-CHINA-A/Design)
* [Implementation](https://2020.igem.org/Team%3ANWU-CHINA-A/Implementation)
* [Engineering](https://2020.igem.org/Team%3ANWU-CHINA-A/Engineering)
* [Model](https://2020.igem.org/Team%3ANWU-CHINA-A/Model)
* [Plastic
  Pollution](https://2020.igem.org/Team%3ANWU-CHINA-A/Plastic_Pollution)
* [Proof of
  Concept](https://2020.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3351)

Parts

* [Parts](https://2020.igem.org/Team%3ANWU-CHINA-A/Parts)
* [Contribution](https://2020.igem.org/Team%3ANWU-CHINA-A/Contribution)

Team

* [Team
  Members](https://2020.igem.org/Team%3ANWU-CHINA-A/Team_Members)
* [Attributions](https://2020.igem.org/Team%3ANWU-CHINA-A/Attributions)

Experiment

* [Notebook](https://2020.igem.org/Team%3ANWU-CHINA-A/Notebook)
* [Safety](https://2020.igem.org/Team%3ANWU-CHINA-A/Safety)
* [Protocols](https://2020.igem.org/Team%3ANWU-CHINA-A/Protocols)

HP

* [Integrated HP](https://2020.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
* [Education](https://2020.igem.org/Team%3ANWU-CHINA-A/Education)
* [Collaborations](https://2020.igem.org/Team%3ANWU-CHINA-A/Collaborations)
* [Partnership](https://2020.igem.org/Team%3ANWU-CHINA-A/Partnership)

Project

* Design
* Protocols
* Description
* Safety

Model

* Overview
* XXX Model
* XXX Model
* XXX Model

Parts

* Overview
* Basic
* Composite
* XXXXXX

Team

* Team member
* Attributions
* Collaborations
* Note

HP

* Overview
* Intergrated HP
* Public
* Notes

![](https://static.igem.org/mediawiki/2020/7/76/T--NWU-CHINA-A--school_logo.png)
![](https://static.igem.org/mediawiki/2020/3/35/T--NWU-CHINA-A--life-science-college-icon.png)
![](https://static.igem.org/mediawiki/2020/6/66/T--NWU-CHINA-A--snapgenelogodark.png)
![](https://static.igem.org/mediawiki/2020/7/77/T--NWU-CHINA-A--GenScript-icon.jpg)

Contact us:
Email:igem\_nwu\_a@outlook.com

Copyright 2020 NWU-CHINA-A

Notebook

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

# Team:NWU-CHINA-A/Engineering

Project

* [Description](https://2020.igem.org/Team%3ANWU-CHINA-A/Description)
* [Design](https://2020.igem.org/Team%3ANWU-CHINA-A/Design)
* [Implementation](https://2020.igem.org/Team%3ANWU-CHINA-A/Implementation)
* [Engineering](https://2020.igem.org/Team%3ANWU-CHINA-A/Engineering)
* [Model](https://2020.igem.org/Team%3ANWU-CHINA-A/Model)
* [Plastic
  Pollution](https://2020.igem.org/Team%3ANWU-CHINA-A/Plastic_Pollution)
* [Proof of
  Concept](https://2020.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
* [Judging Form](https://igem.org/2020_Judging_Form?id=3351)

Parts

* [Parts](https://2020.igem.org/Team%3ANWU-CHINA-A/Parts)
* [Contribution](https://2020.igem.org/Team%3ANWU-CHINA-A/Contribution)

Team

* [Team
  Members](https://2020.igem.org/Team%3ANWU-CHINA-A/Team_Members)
* [Attributions](https://2020.igem.org/Team%3ANWU-CHINA-A/Attributions)

Experiment

* [Notebook](https://2020.igem.org/Team%3ANWU-CHINA-A/Notebook)
* [Safety](https://2020.igem.org/Team%3ANWU-CHINA-A/Safety)
* [Protocols](https://2020.igem.org/Team%3ANWU-CHINA-A/Protocols)

HP

* [Integrated HP](https://2020.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
* [Education](https://2020.igem.org/Team%3ANWU-CHINA-A/Education)
* [Collaborations](https://2020.igem.org/Team%3ANWU-CHINA-A/Collaborations)
* [Partnership](https://2020.igem.org/Team%3ANWU-CHINA-A/Partnership)

Project

* Design
* Protocols
* Description
* Safety

Model

* Overview
* XXX Model
* XXX Model
* XXX Model

Parts

* Overview
* Basic
* Composite
* XXXXXX

Team

* Team member
* Attributions
* Collaborations
* Note

HP

* Overview
* Intergrated HP
* Public
* Notes

![](https://static.igem.org/mediawiki/2020/7/76/T--NWU-CHINA-A--school_logo.png)
![](https://static.igem.org/mediawiki/2020/3/35/T--NWU-CHINA-A--life-science-college-icon.png)
![](https://static.igem.org/mediawiki/2020/6/66/T--NWU-CHINA-A--snapgenelogodark.png)
![](https://static.igem.org/mediawiki/2020/7/77/T--NWU-CHINA-A--GenScript-icon.jpg)

Contact us:
Email:igem\_nwu\_a@outlook.com

Copyright 2020 NWU-CHINA-A

Engineering

# Research

PhaP contains a hydrophobic granule binding domain and a cytosol-facing hydrophilic domain. Complete
biodegradable nature of PhaP. PhaP-tagged proteins could interact with various types of hydrophobic
surfaces. A (Gly4Ser2)2 flexible linker is inserted between PhaP and HD5d5 to facilitate attaching of
HD5d5 on surface and displaying sufficient flexibility of this peptide.

The noncationic and nonhydrophobic residues of HD5 were replaced with electropositive Arg to increase the
antibacterial activity of HD5 derivative that contains a Cys2-4 bond, obtaining another derivative
termed HD5d5. The in vitro antibacterial assay and irradiation-wound-infection animal experiment both
showed that HD5d5 was much more effective than HD5 at eliminating MDRAb.

![](https://static.igem.org/mediawiki/2020/2/2b/T--NWU-CHINA-A--engineering-img-1.png)

# Imagine

The fusion protein PhaP-HD5d5 was expressed in E. coli strain Transetta (DE3).Because PhaP-tagged
proteins could interact with various types of hydrophobic surfaces. PhaP-HD5d5 will anchor to
hydrophobic polymer surface efficiently to make band-aids with antibacterial functions.

# Design/build

1. HD5d5: Mining Gene Information from the Literature.

2. The HD5d5 fragment was subcloned into the pWDX plasmid using the HindⅢ and SpeⅠ sites to construct the
pWDX-HD5d5 plasmid.

![](https://static.igem.org/mediawiki/2020/a/a2/T--NWU-CHINA-A--engineering-img-2.png)

3. The plasmids were then transformed into E. coli BL21(DE3), and transformant clones were screened.

4. E. coli BL21 (DE3) was used as the protein expression host.

5. Extraction and Purification of Proteins (PhaP-HD5d5) from E. coli BL21 (DE3).

6. The in vitro antibacterial activities of PhaP-HD5d5 was measured against S. aureus using the filter
paper disc diffusion method

7. PhaP-HD5d5 will be adhered to the surface of PHA.

8. The bactericidal activity of PhaP, PhaP-HD5d5, and their coated PHA films will be tested against
selected bacteria

# Method

① Positive colonies (E. coli BL21 (DE3) pWDX(+)::PhaP-HD5d5) from September 12, 2020 were transferred to
test tubes with 2 mL LB+amp and incubated at 37°C, 200 rpm overnight. One 250 mL Erlenmeyer flask with
100 mL LB and 100µL amp/kan was inoculated with 1 mL of one culture above in a 250 mL flask, which both
were incubated at 37°C, 200 rpm. OD measurement after three hours showed an OD(600) = 0.6. E. coli BL21
cultures were induced with 100 µL IPTG for incubated at 37°C, 200 rpm overnight.

The bacterial growth curves at OD600 were recorded at 30-min intervals for 15 hours.

![](https://static.igem.org/mediawiki/2020/0/0e/T--NWU-CHINA-A--engineering-img-3.png)

After induction for 85~6 hours, the sludge was collected by centrifugation and analyzed by SDS-PAGE. The
results showed that the antibacterial peptides fused with PhaP were all expressed, among which PhaP-HD5
and PhaP-HD5d5 expressed the most.

![](https://static.igem.org/mediawiki/2020/2/27/T--NWU-CHINA-A--engineering-img-4.png)

② The incubated medium was transferred to 50 mL centrifuge tube and the supernatant collected after
centrifuging (6791×g) for 3 min at 4°C. After removal of the supernatant by centrifugation (6791×g, 15
min), it was collected precipitate.

③ the bacteria were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval
time: 1s) on ice for 2 min. (A volume of 20 ml of the Bacterial lysates was added to each baterial
precipitat, Bacterial lysates 2.0: 20mM/L tris-HCl 150mM/L NaCl)

④ PhaP-HD5d5 was not observed in the supernatant analyzed by SDS-PAGE.

# Research and design cycle

Through analysis, the reason we thought was the Bacterial lysates or the ultrasonic power/total time,so
we change the Bacterial lysates and ultrasonic total time.

* The bacterias were crushed with an ultrasonic (ultrasonic power: 400 W, crushing time: 1 s, interval
  time: 1s) on ice for 15 min. (A volume of 20 ml of the Bacterial lysates was added to each baterial
  precipitat, Bacterial lysates 2.0: 20mM/L tris-HCl 150mM/L NaCl). But PhaP-HD5d5 also was not
  observed
  in the supernatant analyzed by SDS-PAGE.
* This time,we increased the power of ultrasound and the time to crush the bacteria,which were
  crushed with an ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice
  for 30 min.it is pity that we couldn’t observed the PhaP-HD5d5 in in the supernatant analyzed by
  SDS-PAGE.
* So we increased the total time of ultrasound to 45 min, the bacterias were crushed with an
  ultrasonic (ultrasonic power: 500 W, crushing time: 3 s, interval time: 5s) on ice for 45 min.The
  sonicated bacterial lysate was centrifuged 6791 × g for 15 min at 4°C. Equivalent amounts of
  supernatant and precipitate were subjected to SDS-PAGE analysis. The supernatant, containing target
  proteins(PhaP-HD5d5)

![](https://static.igem.org/mediawiki/2020/2/21/T--NWU-CHINA-A--engineering-img-5.png)

# Bacteriostatic experiment

It was noticed that PhaP-HD5d5 showed the antibacterial effects when examined on S. aureus strain RN4220.

![](https://static.igem.org/mediawiki/2020/7/79/T--NWU-CHINA-A--engineering-img-6.png)

In order to verify the adhesion between PHA and PhaP-AMPs ,we did the following experiments:

First ,we used electrospinning technology to make PHA films. Secondly the films was immersed in the
supernatant after bacterial sonication for 2 hours. Thirdly, we washed the films gently with lysis
buffer, then put them into a centrifuge tube and added SDS-PAGE buffer, heat at 90℃ until the thin
membrane softens.

In order to make the adhesion between PhaP-AMPs and PHA better, we used nano-level PHA for verification.
Added PHA and supernatant in a ratio of 1:20, stirred two hours by the magnetic stirrer, then
centrifuged at 8000rpm for 2min, rinsed twice with lysate, and added SDS-PAGE buffer.

We took the liquid from the above product for SDS-PAGE, as we can see, PhaP-HD5d5 combines the most
products with PHA，whether it is electrospinning or nanoparticles.

![](
                    https://static.igem.org/mediawiki/2020/4/4e/T--NWU-CHINA-A--engineering-img-7.png)

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

# Team:NWU-CHINA-A/Experiments

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position: relative;
width: 100%;
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background-image: url(![T--NWU-CHINA-A--description_banner.png](https://static.igem.org/mediawiki/2020/b/b0/T--NWU-CHINA-A--description_banner.png)

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position: absolute;
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z-index: 9999;
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.text-bg {
position: absolute;
font-size: 1.12rem !important;
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p {
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position: fixed;
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box-shadow: 0 .5rem 1rem rgba(0, 0, 0, .15) !important;
border-radius: 7px;
}

.indicator-title {
display: block;
font-size: 0.875rem;
font-weight: 330;
margin: 0;
padding: 10px 0 10px 16px;
cursor: pointer;
user-select: none;
}

.indicator-title-current {
font-size: 1.2rem;
font-weight: 600;
cursor: pointer;
user-select: none;
box-shadow: 0 .3rem .5rem rgba(0, 0, 0, .15) !important;
}

.indicator-banner-title {
margin-bottom: 1.6rem;
padding-left: 5px;
font-size: 1.4rem;
font-weight: 800;
user-select: none;
}

.text-img {
width: 100%;
margin: 50px auto !important;
align-items: center;
}

.text-table {
margin: 50px auto !important;
width: 75%;
}

/\*时间线部分\*/
.time-line-sector {
display: block !important;
margin: 80px 0 80px 0 !important;
min-height: 200px !important;
}

.time-line-sector h2 {
margin-left: -20px !important;
margin-bottom: 50px !important;
}

.time-sub-sector {
display: block !important;
padding: 0 50px 50px 50px !important;
min-height: 150px !important;
border-left: 1px solid #000 !important;
}

.time-sub-sector p {
padding-left: 20px !important;
margin-top: 18px !important;
margin-bottom: 18px !important;
}

.time-label {
display: block !important;
font-size: 0.8rem !important;
color: #fff !important;
font-weight: 700 !important;
padding: 0 5px !important;
width: 40px !important;
text-align: center;
background-color: rgb(25, 212, 150) !important;
border-radius: 5px !important;
}

.time-label-wrapper {
position: relative !important;
margin-bottom: 18px;
}

.time-label-wrapper::before {
content: "" !important;
position: absolute !important;
top: -18px !important;
left: -76px !important;
height: 50px !important;
width: 50px !important;
background-color: #e3f3fd !important;
background-image: url(<https://static.igem.org/mediawiki/2020/a/ad/T--NWU-CHINA-A--partnership_icon.svg>) !important;
background-position: center !important;
background-size: 70% 70% !important;
background-repeat: no-repeat !important;
}

/\*照片显隐部分\*/
.img-switch-btn {
display: block;
width: 60px;
height: 60px;
margin: 50px auto;
background-image: url(<https://static.igem.org/mediawiki/2020/d/d4/T--NWU-CHINA-A--HP-img-switch-btn.svg>);
background-position: center;
background-repeat: no-repeat;
background-size: 60%;
background-color: rgb(25, 212, 150);
border-radius: 30px;
}

.img-switch-btn:hover {
box-shadow: rgba(36, 168, 244, 0.5) 0px 4px 10px;
background-color: rgb(103, 235, 179);
}

.HP-img-box {
display: none;
flex-direction: row;
justify-content: space-evenly;
user-select: none;
}

.HP-img {
display: block;
margin: 0 10px;
max-height: 230px;
}

.clearspace {
height: 300px;
width: 100%;
}

# 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.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### 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)

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

# Team:NWU-CHINA-A/Results

.text-banner {
position: relative;
width: 100%;
height: 652px;
padding-bottom: 50px;
background-image: url(![T--NWU-CHINA-A--description_banner.png](https://static.igem.org/mediawiki/2020/b/b0/T--NWU-CHINA-A--description_banner.png)

.banner-title {
position: absolute;
top: 50%;
left: 50%;
width: 600px;
height: 4.5rem;
margin: -3rem 0 0 -300px;
text-align: center;
font-family: "a Atmospheric";
font-size: 4.5rem;
color: rgb(56, 50, 45);
}

.text-wrapper {
display: block;
margin: 0;
padding: 80px 0 100px 0;
background-image: url(![T--NWU-CHINA-A--description_bg.gif](https://static.igem.org/mediawiki/2020/4/41/T--NWU-CHINA-A--description_bg.gif));
z-index: -1;
}

.text-sector {
font-family: OpenSans;
}

.text-subsection {
position: relative;
margin: 0px 6% 80px 24%;
padding: 50px 80px 80px 80px;
font-size: 1.12rem !important;
box-shadow: 0 .3rem .5rem rgba(0, 0, 0, .15) !important;
z-index: 9999;
}

.text-bg {
position: absolute;
font-size: 1.12rem !important;
/\*box-shadow: 0 .3rem .5rem rgba(0,0,0,.15)!important;\*/
/\*background-color: #8fd0f7;\*/
background-color: #fff;
opacity: 0.75;
z-index: 1000;
}

.text-sector,
p {
font-size: 1.125rem !important;
font-weight: 530 !important;
}

.sector-title {
font-size: 2rem;
font-weight: 1000;
}

.title-indicator {
position: fixed;
top: 96px;
left: 4%;
width: 180px;
margin: 200px auto auto 0%;
padding: 15px 20px 15px 20px !important;
background-color: #fff;
font-family: OpenSans;
box-shadow: 0 .5rem 1rem rgba(0, 0, 0, .15) !important;
border-radius: 7px;
}

.indicator-title {
display: block;
font-size: 0.875rem;
font-weight: 330;
margin: 0;
padding: 10px 0 10px 16px;
cursor: pointer;
user-select: none;
}

.indicator-title-current {
font-size: 1.2rem;
font-weight: 600;
cursor: pointer;
user-select: none;
box-shadow: 0 .3rem .5rem rgba(0, 0, 0, .15) !important;
}

.indicator-banner-title {
margin-bottom: 1.6rem;
padding-left: 5px;
font-size: 1.4rem;
font-weight: 800;
user-select: none;
}

.text-img {
width: 100%;
margin: 50px auto !important;
align-items: center;
}

.text-table {
margin: 50px auto !important;
width: 75%;
}

/\*时间线部分\*/
.time-line-sector {
display: block !important;
margin: 80px 0 80px 0 !important;
min-height: 200px !important;
}

.time-line-sector h2 {
margin-left: -20px !important;
margin-bottom: 50px !important;
}

.time-sub-sector {
display: block !important;
padding: 0 50px 50px 50px !important;
min-height: 150px !important;
border-left: 1px solid #000 !important;
}

.time-sub-sector p {
padding-left: 20px !important;
margin-top: 18px !important;
margin-bottom: 18px !important;
}

.time-label {
display: block !important;
font-size: 0.8rem !important;
color: #fff !important;
font-weight: 700 !important;
padding: 0 5px !important;
width: 40px !important;
text-align: center;
background-color: rgb(25, 212, 150) !important;
border-radius: 5px !important;
}

.time-label-wrapper {
position: relative !important;
margin-bottom: 18px;
}

.time-label-wrapper::before {
content: "" !important;
position: absolute !important;
top: -18px !important;
left: -76px !important;
height: 50px !important;
width: 50px !important;
background-color: #e3f3fd !important;
background-image: url(<https://static.igem.org/mediawiki/2020/a/ad/T--NWU-CHINA-A--partnership_icon.svg>) !important;
background-position: center !important;
background-size: 70% 70% !important;
background-repeat: no-repeat !important;
}

/\*照片显隐部分\*/
.img-switch-btn {
display: block;
width: 60px;
height: 60px;
margin: 50px auto;
background-image: url(<https://static.igem.org/mediawiki/2020/d/d4/T--NWU-CHINA-A--HP-img-switch-btn.svg>);
background-position: center;
background-repeat: no-repeat;
background-size: 60%;
background-color: rgb(25, 212, 150);
border-radius: 30px;
}

.img-switch-btn:hover {
box-shadow: rgba(36, 168, 244, 0.5) 0px 4px 10px;
background-color: rgb(103, 235, 179);
}

.HP-img-box {
display: none;
flex-direction: row;
justify-content: space-evenly;
user-select: none;
}

.HP-img {
display: block;
margin: 0 10px;
max-height: 230px;
}

.clearspace {
height: 300px;
width: 100%;
}

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)