iGEM 2021 — Team The_Webb_Schools

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

# Team:The Webb Schools/Notebook

The\_Webb\_Schools

* [Home](https://2021.igem.org/Team%3AThe_Webb_Schools)
* Project

  + [Description](https://2021.igem.org/Team%3AThe_Webb_Schools/Description)
  + [Design](https://2021.igem.org/Team%3AThe_Webb_Schools/Design)
  + [Experiments](https://2021.igem.org/Team%3AThe_Webb_Schools/Experiments)
  + [Results](https://2021.igem.org/Team%3AThe_Webb_Schools/Results)
  + [Proof Of Concept](https://2021.igem.org/Team%3AThe_Webb_Schools/Proof_Of_Concept)
  + Notebook
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* Parts

  + [Parts Collection](https://2021.igem.org/Team%3AThe_Webb_Schools/Parts)
  + [Engineering](https://2021.igem.org/Team%3AThe_Webb_Schools/Engineering)
* Human Practices

  + [Integrated Human Practice](https://2021.igem.org/Team%3AThe_Webb_Schools/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AThe_Webb_Schools/Communication)
  + [Fundraising](https://2021.igem.org/Team%3AThe_Webb_Schools/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AThe_Webb_Schools/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AThe_Webb_Schools/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AThe_Webb_Schools/Model)
* Team

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  + [Collaborations](https://2021.igem.org/Team%3AThe_Webb_Schools/Collaborations)

Project Notebook

We would love to talk with you! Reach us at:

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Email - LightHouse.gw@outlook.com

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

# Team:The Webb Schools/Engineering

The\_Webb\_Schools

* [Home](https://2021.igem.org/Team%3AThe_Webb_Schools)
* Project

  + [Description](https://2021.igem.org/Team%3AThe_Webb_Schools/Description)
  + [Design](https://2021.igem.org/Team%3AThe_Webb_Schools/Design)
  + [Experiments](https://2021.igem.org/Team%3AThe_Webb_Schools/Experiments)
  + [Results](https://2021.igem.org/Team%3AThe_Webb_Schools/Results)
  + [Proof Of Concept](https://2021.igem.org/Team%3AThe_Webb_Schools/Proof_Of_Concept)
  + [Notebook](https://2021.igem.org/Team%3AThe_Webb_Schools/Notebook)
  + [Safety](https://2021.igem.org/Team%3AThe_Webb_Schools/Safety)
* Parts

  + [Parts Collection](https://2021.igem.org/Team%3AThe_Webb_Schools/Parts)
  + Engineering
* Human Practices

  + [Integrated Human Practice](https://2021.igem.org/Team%3AThe_Webb_Schools/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AThe_Webb_Schools/Communication)
  + [Fundraising](https://2021.igem.org/Team%3AThe_Webb_Schools/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AThe_Webb_Schools/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AThe_Webb_Schools/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AThe_Webb_Schools/Model)
* Team

  + [Team
    Members](https://2021.igem.org/Team%3AThe_Webb_Schools/Members)
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  + [Collaborations](https://2021.igem.org/Team%3AThe_Webb_Schools/Collaborations)

Engineering

Introduction

In modern society, more and more people suffer from mild or severe depression. It
has been reported that malondialdehyde (MDA) level in the plasma of depressed patients is significantly
increased. After receiving conventional antidepressant treatment, the patient's MDA level decreased to the
same as that of healthy people. Therefore, researchers believe that oxidative stress may play an important
role in the occurrence and development of depression, and the activity of antidepressant therapy may be
mediated by improving oxidative stress/antioxidant function. We attempt to express an antioxidant enzyme
system (peroxidase gene efeB) in the cell or on the surface of the engineered probiotic bacteria to inhibit
the production of malondialdehyde (MDA) by human cells and prevent cell oxidation from causing health damage
to the body. So as to prevent or alleviate the condition of depression.

Design

The oxidation rate of peroxisome increases in proportion to the increase of oxygen
tension. Especially in the case of high oxygen concentrations, the oxidation reaction of peroxisomes
dominates. This characteristic allows peroxisomes to protect cells from the toxic effects of high
concentrations of oxygen. efeB reduces the oxidative stress in the cell by competing with mitochondria for
oxygen, and reduces the MDA produced during the oxidation of lipids.

![](https://static.igem.org/mediawiki/2021/8/8e/T--The_Webb_Schools--engineering01.jpg)

Build

Four recombinant plasmids were constructed (plasmid A：pUC57-Pro-efeB; plasmid
B：pUC57-amilGFP; plasmid C：pUC57-Pro-efeB-amilGFP) (Figure 1). Plasmid A is used to produce recombinant efeB
in probiotic bacteria. Plasmids B and C are used to confirm function of the composite part and expression of
efeB, respectively. All the constructs are confirmed by DNA sequencing.

![](https://static.igem.org/mediawiki/2021/f/f8/T--The_Webb_Schools--engineering02.jpg)
Figure 1. Schematic map of plasmids.

Test

Bacteria A (contains plasmid A, pUC57-efeB), and the bacteria D (contains plasmid
D, pUC57 empty vector). Bacteria A, which produces efeB enzyme, can survive in high concentration of
hydrogen peroxide (Figure 2).

![](https://static.igem.org/mediawiki/2021/6/69/T--The_Webb_Schools--engineering03.jpg)
Figure 2. Histogram of relative biomass of bacteria A and B against different concentrations of
H2O2.

Bacteria A and C kept the MDA concentration dramatically lower than those of
Bacteria D (transformed with pUC57 vector) under the hydrogen peroxide concentration of 0mM, 2mM and 2.5mM.
Bacteria A and C with the efeB gene all effectively reduced the MDA concentration to the level lower than
Bacteria D (Figure 3).

![](https://static.igem.org/mediawiki/2021/0/0b/T--The_Webb_Schools--engineering04.jpg)
Figure 3. Histogram of MDA in the systems with different H2O2 concentration.

Learn

This shows that bacteria with the efeB gene is able to survive when the level of
ROS increases. As the concentration of hydrogen peroxide increases to 4mM, the relative biomass of both
bacteria A and bacteria D are zero. This shows that when the level of ROS is too high, bacteria are unable
to survive regardless of the presence of the efeB gene. Bacteria with the efeB gene effectively reduced the
MDA concentration to the lower level. Our genetically engineered bacteria are promising in preventing or
alleviating the condition of depression.

We would love to talk with you! Reach us at:

---

Instagram - LightHouse.gw

Email - LightHouse.gw@outlook.com

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

# Team:The Webb Schools/Experiments

The\_Webb\_Schools

* [Home](https://2021.igem.org/Team%3AThe_Webb_Schools)
* Project

  + [Description](https://2021.igem.org/Team%3AThe_Webb_Schools/Description)
  + [Design](https://2021.igem.org/Team%3AThe_Webb_Schools/Design)
  + Experiments
  + [Results](https://2021.igem.org/Team%3AThe_Webb_Schools/Results)
  + [Proof Of Concept](https://2021.igem.org/Team%3AThe_Webb_Schools/Proof_Of_Concept)
  + [Notebook](https://2021.igem.org/Team%3AThe_Webb_Schools/Notebook)
  + [Safety](https://2021.igem.org/Team%3AThe_Webb_Schools/Safety)
* Parts

  + [Parts Collection](https://2021.igem.org/Team%3AThe_Webb_Schools/Parts)
  + [Engineering](https://2021.igem.org/Team%3AThe_Webb_Schools/Engineering)
* Human Practices

  + [Integrated Human Practice](https://2021.igem.org/Team%3AThe_Webb_Schools/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AThe_Webb_Schools/Communication)
  + [Fundraising](https://2021.igem.org/Team%3AThe_Webb_Schools/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AThe_Webb_Schools/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AThe_Webb_Schools/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AThe_Webb_Schools/Model)
* Team

  + [Team
    Members](https://2021.igem.org/Team%3AThe_Webb_Schools/Members)
  + [Attributions](https://2021.igem.org/Team%3AThe_Webb_Schools/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AThe_Webb_Schools/Collaborations)

Experiments

Plasmid Construction

Transformation, Culture and Preservation

Function Test

Medium Preparation

We would love to talk with you! Reach us at:

---

Instagram - LightHouse.gw

Email - LightHouse.gw@outlook.com

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

# Team:The Webb Schools/Results

The\_Webb\_Schools

* [Home](https://2021.igem.org/Team%3AThe_Webb_Schools)
* Project

  + [Description](https://2021.igem.org/Team%3AThe_Webb_Schools/Description)
  + [Design](https://2021.igem.org/Team%3AThe_Webb_Schools/Design)
  + [Experiments](https://2021.igem.org/Team%3AThe_Webb_Schools/Experiments)
  + Results
  + [Proof Of Concept](https://2021.igem.org/Team%3AThe_Webb_Schools/Proof_Of_Concept)
  + [Notebook](https://2021.igem.org/Team%3AThe_Webb_Schools/Notebook)
  + [Safety](https://2021.igem.org/Team%3AThe_Webb_Schools/Safety)
* Parts

  + [Parts Collection](https://2021.igem.org/Team%3AThe_Webb_Schools/Parts)
  + [Engineering](https://2021.igem.org/Team%3AThe_Webb_Schools/Engineering)
* Human Practices

  + [Integrated Human Practice](https://2021.igem.org/Team%3AThe_Webb_Schools/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AThe_Webb_Schools/Communication)
  + [Fundraising](https://2021.igem.org/Team%3AThe_Webb_Schools/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AThe_Webb_Schools/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AThe_Webb_Schools/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AThe_Webb_Schools/Model)
* Team

  + [Team
    Members](https://2021.igem.org/Team%3AThe_Webb_Schools/Members)
  + [Attributions](https://2021.igem.org/Team%3AThe_Webb_Schools/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AThe_Webb_Schools/Collaborations)

Results

Plasmid Construction Result

![](https://static.igem.org/mediawiki/2021/e/ee/T--The_Webb_Schools--design01.jpg)

![](https://static.igem.org/mediawiki/2021/c/cc/T--The_Webb_Schools--result02.jpg)
Figure 2. Sequencing result of the plasmid C from the sequencing company

The sequencing result (Figure 2) shows that the gene efeB
and amilGFP are successfully inserted namely the plasmid C was constructed successfully which was used for
the subsequent function tests same as the other 3 plasmids.

Function Test Result

**1. Bacteria Growth**

![](https://static.igem.org/mediawiki/2021/5/5e/T--The_Webb_Schools--result03.jpg)
Table 1. relative biomass percents of bacteria A and B with different concentrations of H2O2

![](https://static.igem.org/mediawiki/2021/2/2e/T--The_Webb_Schools--result04.jpg)
Figure 3. Histogram of relative biomass of bacteria A and B against different concentrations of
H2O2

Bacteria A contains plasmid A, pUC57-efeB, and the bacteria
D contains plasmid D, pUC57. When the concentration of hydrogen peroxide is 0 mM, the relative biomass of
bacteria A and bacteria C are the same. As the concentration of hydrogen peroxide increases to 1mM, the
relative biomass of bacteria A increases while the relative biomass of bacteria C remains the same, which
shows that without efeB, bacteria is unable to grow when ROS is present. As the concentration of hydrogen
peroxide increases to 2mM, the relative biomass of bacteria A decreases by half while the relative biomass
of bacteria D decreases to 0mM. This shows that bacteria with the efeB gene is able to survive when the
level of ROS increases. As the concentration of hydrogen peroxide increases to 4mM, the relative biomass of
both bacteria A and bacteria D are zero. This shows that when the level of ROS is too high, bacteria are
unable to survive regardless of the presence of the efeB gene.

![](https://static.igem.org/mediawiki/2021/8/86/T--The_Webb_Schools--result05.jpg)
Figure 4. Line chart of the OD600 of bacteria A, C and D against hours in 2.5mM H2O2

The bacteria A contains plasmid A, pUC57-efeB. The bacteria
C contains plasmid C, pUC57-efeB-amilGFP. The bacteria D contains plasmid D, pUC57. The bacteria are placed
in 2.5mM hydrogen peroxide and their growth is measured for the first 4 hours. Bacteria D, without the efeB
gene, has lower growth than bacteria A and bacteria C. This shows that efeB gene helps bacteria to grow when
ROS is present.

**2. MDA and Fluorescence Measuring**

![](https://static.igem.org/mediawiki/2021/6/60/T--The_Webb_Schools--result06.jpg)
Figure 5. Histogram of MDA in the systems with different H2O2 concentration of bacteria A, C and
D

Bacteria A, C, D were placed in hydrogen peroxide solution
for 21 hours and the concentration of MDA was measured. Bacteria A and C kept the MDA concentration
dramatically lower than those of Bacteria D under the hydrogen peroxide concentration of 0mM, 2mM and 2.5mM.
There is a certain amount of MDA concentration within DH5α when the H2O2 concentration is 0mM. Starting from
the original DH5α MDA concentration to the higher MDA concentration imposed by the 2mM and 2.5mM H2O2
concentration, Bacteria A and C with the efeB gene all effectively reduced the MDA concentration to the
level lower than Bacteria D.

![](https://static.igem.org/mediawiki/2021/f/f6/T--The_Webb_Schools--result07.jpg)
Figure 6. Histogram of fluorescence intensity of bacteria C system with different H2O2 concentration

In bacteria C, the expression of GFP is controlled by the
expression of efeB. The more intense the fluorescence is, the better efeB will express. H2O2 climbed from 0
to 2.5 mM, and the intensity of fluorescence climbed higher as well. The tendency of the intensity shows
that the expression of efeB increases with the H2O2 concentration. Expression of efeB disturbs the
production of MDA.

![](https://static.igem.org/mediawiki/2021/5/51/T--The_Webb_Schools--result08.jpg)
Figure 7. Line chart of MDA in bacteria C system with 2.5mM H2O2 against hours

Bacteria C was placed in the 2.5nM hydrogen peroxide
solution, and the level of MDA was measured under different hours. During the first 4 hours, the
concentration of MDA increased up to 20 mmol/mg. The concentration of MDA decreased to 6 mmol/mg for the
next four hours. The measured levels of MDA demonstrate that the efeB gene can curb the increase of MDA and
keep it in a relative low level.

In order to deeper analyze the factors which may affect the
ability of bacteria C and bacteria A to inhibit MDA production, more function tests would be designed and
conducted in the future.

We would love to talk with you! Reach us at:

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Email - LightHouse.gw@outlook.com