iGEM 2022 — Team shsid

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{% block title %}Notebook{% endblock %}
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# Notebook

## 7/9

Team ice-breaking

Project Introduction

Project discussion

![](https://static.igem.wiki/teams/4280/wiki/notebook/t-shsid-notebook-01.png)

## 7/10

Laboratory Safety Training

Preparation of LB medium

PCR amplify parts of pUC57 and DexA70

![](https://static.igem.wiki/teams/4280/wiki/notebook/t-shsid-notebook-02.jpg)

Gel extraction

## 7/11

Homologous recombination

Transformation of plasmid into DN5α

Sending for sequencing

Coat plate for selection of plasmid

![](https://static.igem.wiki/teams/4280/wiki/notebook/t-shsid-notebook-03.png)

## 7/12

Plasmid Extraction

PCR verification of plasmid

Culture bacteria colony

Take team casual photo

![](https://static.igem.wiki/teams/4280/wiki/notebook/t-shsid-notebook-04.jpg)

## 7/13

Use CaCl2 solution for the preparation of sensitized cells

Repeat PCR

Preparation of LB medium

## 7/14

Determine the experimental scheme

Transform DexA and Cly R to *E. coli* Nissle 1917

Store samples of 96 well plate

![](https://static.igem.wiki/teams/4280/wiki/notebook/t-shsid-notebook-05.jpg)

## 7/15

Determination of redusing sugar using DNS method

SDS PAGE electrophoresis

Examine ability of film suppression and film removal

Repeat Transformation

![](https://static.igem.wiki/teams/4280/wiki/notebook/t-shsid-notebook-06.png)

## 7/16

Prepare His Buffer for the purification of protein

Repeat the storage of samples in 96 well plate

Repeat the DNS method

Repeat SDS PAGE electrophoresis

![](https://static.igem.wiki/teams/4280/wiki/notebook/t-shsid-notebook-07.png)

## 7/17-18

Preparation of Brain Heart Infusion Broth (BHI) medium

Culture *Streptococcus mutans*

## 7/19-20

Inhibition zone experiment of *Streptococcus mutans*

![](https://static.igem.wiki/teams/4280/wiki/notebook/t-shsid-notebook-08.png)

## 7/21

Collect and analyze experimental data and write wiki materials

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{% block title %}Engineering{% endblock %}
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# Engineering

## Introduction

Caries is a bacterial infection that occurs in dental tissues. *Streptococcus mutans* is one of the genus
Streptococcus with the largest proportion in the natural flora of the oral cavity, and it is also the main
pathogenic bacteria of caries. *Streptococcus mutans* adheres to teeth layer by layer, forming a
so-called
"biofilm" that is highly resistant to the environment and resistant to antimicrobial treatments. In this
study, we designed a genetically engineered bacterium that produces glucanase (DexA)and a phage lyase (ClyR).
The former can effectively prevent the formation of biofilm and dissolve the formed biofilm, and the latter
can specifically kill *Streptococcus mutans*. The genetically engineered bacteria prepared in this
project have
great potential in preventing and treating dental caries.

![](https://static.igem.wiki/teams/4280/wiki/engineering/t-shsid-engineering-01.jpg)

Figure 1. The principle of genetically engineered bacteria kills *Streptococcus mutans*.

## Design

We inserted the DexA70 and ClyR gene sequences into pUC57 and pBAD expression vectors, respectively. The
expression of DexA70 is regulated by the constitutive strong promoter J23101. And the expression of clyR is
regulated with an arabinose-inducible promoter araBAD. After the two plasmids were transferred into the same
host E. coli, they have the dual functions of destroying the biofilm formed by denatured Streptococcus and
being bactericidal, thus providing a more effective solution for the treatment of dental caries.

![](https://static.igem.wiki/teams/4280/wiki/engineering/t-shsid-engineering-02.jpg)

Figure 2. The map of plasmid.
A . puc57- OmpA-smDexA plasmid
B. pBAD-Myc-OmpA-ClyR plasimd

## Build

### 1. PCR amplification

For the construction, we firstly amplified the DexA fragment and pUC57 vector by PCR assay. As indicated in
Figure 3, DexA and pUC57 strands were correctly replicated. Then, ligase the DexA fragment and pUC57 carrier
to obtain the recombinant plasmid puc57- OmpA-smDexA.

![](https://static.igem.wiki/teams/4280/wiki/engineering/t-shsid-engineering-03.jpg)

Figure 3. Identification of DexA (1899 bp) and pUC57 (2126 bp) DNA strands by agarose gel electrophoresis.

### 2. Enzymatic digestion verification

To identify the recombinant plasmid, we used double enzyme digestion to the puc57- OmpA-smDexA plasmid. PCR
was used to verify the monoclonal colony of the strain. As indicated in Figure 4, lines 1 and 2 were
positive clones.

![](https://static.igem.wiki/teams/4280/wiki/engineering/t-shsid-engineering-04.jpg)

Figure 4. Identification of puc57- OmpA-smDexA by agarose gel electrophoresis.

### 3. Sequencing to identify recombinant plasmids

Gene sequencing is used to double verification. As indicated in Figure 5, the returned sequencing comparison
results showed that there were no mutations in the ORF region, and the plasmid was successfully constructed.
Another plasmid pBAD-Myc-OmpA-ClyR is to be given away by the 2021 iGEM team Shanghai\_United\_HS. So far, we
have successfully obtained the two recombinant plasmids, which can be used to express glucanases and phage
lyases proteins.

![](https://static.igem.wiki/teams/4280/wiki/engineering/t-shsid-engineering-05.jpg)

Figure 5. Sequencing of puc57- OmpA-smDexA

## Test

### 1. SDS-PAGE analysis of the target protein.

In order to obtain the two proteins smDexA and ClyR, we transferred the recombinant plasmids into *E.coli
BL21(DE3)*, expanded the culture in the LB medium, and added IPTG to induce protein expression when the
OD600
reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of
cells to release the intracellular proteins. Then we used SDS-PAGE to test the expression of smDexA and
ClyR. As shown in Figure 6, the proteins were expressed successfully. At this point, we got the two protein
solutions we wanted.

![](https://static.igem.wiki/teams/4280/wiki/engineering/t-shsid-engineering-06.jpg)

Figure 6. SDS-PAGE assay

Glue diagram of DexA and ClyR proteins. MS is medium supernatant, P is precipitation, and S is supernatant.
The molecular weight of DexA protein is 74.33 kDa. The molecular weight of ClyR protein was 30.49 kDa.

### 2. Activity assay

To confirm the ability of the DexA and ClyR proteins to inhibit bacterial growth, we used *Streptococcus
mutans* as bacteria for inhibition zone method experiments. As indicated in Figure 7, a transparent
circle
can be seen around the Oxford cup treated with DexA protein, indicating that the DexA protein can destroy or
inhibit the biofilm produced by *Streptococcus mutans*.

![](https://static.igem.wiki/teams/4280/wiki/engineering/t-shsid-engineering-07.jpg)

Figure 7. Strain functional test
(A) The white circle of ClyR, dexA-6, ClyR + DexA-6, ClyR + DexA-1 and DexA-1 covers the entire plate,
adding up to 6.8 cm.
(B) The white circle of ClyR is 4.2 cm; The white circle of DexA-6 is 1.4 cm; The white circle of ClyR +
DexA-6 is 1.3 cm;
(C)The white circle of ClyR + DexA-1 is 1.6 cm; The white circle of DexA-1 is 1.0 cm.

In conclusion, the results of the inhibition zone experiment showed that the protein expressed by the
plasmid we constructed could successfully inhibit the growth of *Streptococcus mutans*, which was in
line with
the expectations of the project.

## Learn

We have already collected the figures from our experiments. Combinding DexA and ClyR has the effect of
controlling *Streptococcus mutans* proliferation. By the way, when we compared these data with the
positive
control group, we surprisingly find that the effect of the DexA+ClyR is better than that.

Because of the effect of the two protein, we believe that if we can use the product in the future and promote
it in the market, it will become a great power to against *Streptococcus mutans*, so as to achieve the
purpose
of anti-plaque, protect human's teeth, and prevent dental caries.

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
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# Experiments

{% include 'experiments/section-1.html' %}
{% include 'experiments/section-2.html' %}
{% include 'experiments/section-3.html' %}

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
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# Results

## 1. Construction of the plasmids

We inserted the DexA70 and ClyR gene sequences into pUC57 and pBAD expression vectors, respectively. The
expression of DexA70 is regulated by the constitutive strong promoter J23101. And the expression of clyR is
regulated with an arabinose-inducible promoter araBAD. After the two plasmids were transferred into the same
host *E. coli*, they have the dual functions of destroying the biofilm formed by denatured Streptococcus
and
being bactericidal, thus providing a more effective solution for the treatment of dental caries.

![](https://static.igem.wiki/teams/4280/wiki/results/t-shsid-results-01.jpg)

Figure 1. The map of plasmid.
A . puc57- OmpA-smDexA plasmid
B. pBAD-Myc-OmpA-ClyR plasimd

### 1.1 PCR amplification

For the construction, we firstly amplified the DexA fragment and pUC57 vector by PCR assay. As indicated in
Figure 2, DexA and pUC57 strands were correctly replicated. Then, ligase the DexA fragment and pUC57 carrier
to obtain the recombinant plasmid puc57- OmpA-smDexA.

![](https://static.igem.wiki/teams/4280/wiki/results/t-shsid-results-02.jpg)

Figure 2. Identification of DexA (1899 bp) and pUC57 (2126 bp) DNA strands by agarose gel electrophoresis.

### 1.2 Enzymatic digestion verification

To identify the recombinant plasmid, we used double enzyme digestion to the puc57- OmpA-smDexA plasmid. PCR
was used to verify the monoclonal colony of the strain. As indicated in Figure 3, lines 1 and 2 were
positive
clones.

![](https://static.igem.wiki/teams/4280/wiki/results/t-shsid-results-03.jpg)

Figure 3. Identification of puc57- OmpA-smDexA by agarose gel electrophoresis.

### 1.3 Sequencing to identify recombinant plasmids

Gene sequencing is used to double verification. As indicated in Figure 4, the returned sequencing comparison
results showed that there were no mutations in the ORF region, and the plasmid was successfully constructed.
Another plasmid pBAD-Myc-OmpA-ClyR is to be given away by the 2021 iGEM team Shanghai\_United\_HS. So far, we
have successfully obtained the two recombinant plasmids, which can be used to express glucanases and phage
lyases proteins.

![](https://static.igem.wiki/teams/4280/wiki/results/t-shsid-results-04.jpg)

Figure 4. Sequencing of puc57- OmpA-smDexA

## 2. SDS-PAGE analysis of the target protein.

In order to obtain the two proteins smDexA and ClyR, we transferred the recombinant plasmids into *E.coli
BL21(DE3)*, expanded the culture in the LB medium, and added IPTG to induce protein expression when the
OD600
reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of
cells to release the intracellular proteins. Then we used SDS-PAGE to test the expression of smDexA and ClyR.
As shown in Figure 5, the proteins were expressed successfully. At this point, we got the two protein
solutions we wanted.

![](https://static.igem.wiki/teams/4280/wiki/results/t-shsid-results-05.jpg)

Figure 5. SDS-PAGE assay
Glue diagram of DexA and ClyR proteins. MS is medium supernatant, P is precipitation, and S is supernatant.
The molecular weight of DexA protein is 74.33 kDa. The molecular weight of ClyR protein was 30.49 kDa.

## 3. Activity assay

To confirm the ability of the DexA and ClyR proteins to inhibit bacterial growth, we used *Streptococcus
mutans*
as bacteria for inhibition zone method experiments. As indicated in Figure 6, a transparent circle can be seen
around the Oxford cup treated with DexA protein, indicating that the DexA protein can destroy or inhibit the
biofilm produced by *Streptococcus mutans*.

![](https://static.igem.wiki/teams/4280/wiki/results/t-shsid-results-06.jpg)

Figure 6. Strain functional test
(A) The white circle of ClyR, dexA-6, ClyR + DexA-6, ClyR + DexA-1 and DexA-1 covers the entire plate,
adding up to 6.8 cm.
(B) The white circle of ClyR is 4.2 cm; The white circle of DexA-6 is 1.4 cm; The white circle of ClyR +
DexA-6 is 1.3 cm;
(C)The white circle of ClyR + DexA-1 is 1.6 cm; The white circle of DexA-1 is 1.0 cm.

In conclusion, the results of the inhibition zone experiment showed that the protein expressed by the plasmid
we constructed could successfully inhibit the growth of *Streptococcus mutans*, which was in line with
the
expectations of the project.

{% endblock %}