iGEM 2021 — Team Shanghai_United_HS

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# Team:Shanghai United HS/Notebook

Shanghai\_United\_HS

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Project Notebook

Contact Info.

---

Email: 3389014902@qq.com; ruiruihuang@gfs.org

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# Team:Shanghai United HS/Engineering

Shanghai\_United\_HS

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Engineering

Introduction

Dental caries is a common disease. It not only directly affects human oral health,
but also often causes adverse symptoms in other parts of the body. Global disease statistics in 2016 show
that the incidence of dental caries in the population is ranking second among common diseases. Studies have
shown that the formation of dental plaque is the result of the joint action of a variety of bacteria,
including Streptococcus mutans, Lactobacillus, Actinomycetes, etc. Phage lyase ClyR (combined from different
bacteriophage lytic enzymes) has a broad bactericidal spectrum, especially the only one reported to be
extremely strong against Streptococcus mutans and Streptococcus mulberry. The enzyme is promising to kill
these two kinds of streptococci are the main cause of dental caries.

Design

This project designed a genetically engineered probiotic (Escherichia coli strain
Nissle 1917 (EcN)), which can be induced and expressed of lysate enzyme ClyR upon exposed to monosaccharides
(arabinose) which released from food arabinoxylan in the oral (Figure 1).

![](https://static.igem.org/mediawiki/2021/b/bf/T--Shanghai_United_HS--Engineering01.jpg)

Build

Genetically engineered probiotics bacteria which produce and secret ClyR are
constructed. Construct puc57-kan-mini-J23101-OmpA-arab-TT secretes arabinosidase to hydrolyze
polysaccharides into monosaccharides; construct B pBAD-Myc-HisA-OmpA-ClyR-6His-TT is induced by arabinose
and produce the ClyR enzyme. Construct C pBAD-Myc-HisA-OmpA-amilGFP-TT is used to test the function of the
system. All the constructs were confirmed by DNA sequencing.

![](https://static.igem.org/mediawiki/2021/2/20/T--Shanghai_United_HS--Engineering02.jpg)
Figure 2. Schemtic maps of plasmids A, B, and C..

Test

**(1) AmilGFP expression in cells transformed with plasmid C**

In the cells transformed with plasmid C, the fluoroscopic data of cells increased
with the addition of arabinose. Meanwhile, the fluoroscopic data become larger with the prolongation of
incubation time.

![](https://static.igem.org/mediawiki/2021/2/23/T--Shanghai_United_HS--Engineering03.jpg)
Figure 3. The fluoroscopic data of cells transformed with plasmid C with the addition of
arabinose.

**(2) AmilGFP expression in cells transformed with plasmids A and C**

In the cells transformed with constructs A and C, the fluoroscopic data of cells
increased with the addition of araboxylan. Meanwhile, the fluoroscopic data become larger with the
prolongation of incubation time (Figure 4).

![](https://static.igem.org/mediawiki/2021/2/28/T--Shanghai_United_HS--Engineering04.jpg)
Figure 4. The fluoroscopic data of cells transformed with plasmids A and C.

**(3) ClyR expression**

Plasmid A and plasmid B were co-transformed to E. coli Nissle 1917 by
electroporation. The araboxylan with final concentration 0, 0.3%, 0.6%, 1.0%, 1.5% and 2.0% (w/v) was added
into the culture to induce ClyR expression. For the comparison, E. coli Nissle 1917 with plasmid B was
cultured by the same way and the expression of ClyR was induced by the addition of arabinose with final
concentration of 0 μM, 10 μM,30 μM,0.1 mM, 0.2 mM,0.5 mM and 2 mM.

The protein concentration was monitored at 595 nm using Multiscan Spectrum
(BioTek). Read the data for three times, recorded the average of the A595 data (Table 1). The A595 of the
culture supernatant of E. coli with plasmid A and B suggested the possible expression of ClyR (Table 2). The
A595 data at “0 %” was referred as blank, and the protein concentration could be calculated referred to the
standard formula.

Table 2. The protein concentration of the culture supernatant of E. coli with
plasmids A and B.

![](https://static.igem.org/mediawiki/2021/9/9b/T--Shanghai_United_HS--Engineering05.jpg)

As can be seen from figure 5, in the transformed bacteria with plasmid A and B, the
concentration of the reducing sugar increased with the elongation of the incubation time. During the
expression process, bacteria lysis occurred with the elongation of the incubation time. But the ClyR band
could obviously be obtained as is shown in figure 5.

![](https://static.igem.org/mediawiki/2021/4/4a/T--Shanghai_United_HS--Engineering06.jpg)
Figure 5. The SDS-PAGE of supernatant.

**(4) In vitro activity assay of ClyR**

E. coli Nissle 1917 transferred with plasmid A and B were culture with
Gram-positive bacteria Lactobacillus casei subsp. casei (ATCC334). The value of OD600 was monitored using
Multiscan Spectrum (BioTek). The OD600 of the L. casei subsp. casei cell suspension gradually reduced with
the addition of supernatant (Figure 6).

![](https://static.igem.org/mediawiki/2021/3/38/T--Shanghai_United_HS--Engineering07.jpg)
Figure 6. The in vitro assay of ClyR.

Learn

AmilGFP was successfully expressed, suggesting the feasibility and possibility of
the inducible secretory expression of ClyR. We have successfully obtained the ClyR protein in the culture
supernatant of cells carrying plasmids with araB and ClyR genes. The concentration of ClyR was low, so more
efforts are still needed in future. It is worth to note that the expression of ClyR protein may cause the
host to suicide, which can be supported by the obvious cell lysis and the presence of flocculent
precipitate.

Contact Info.

---

Email: 3389014902@qq.com; ruiruihuang@gfs.org

Search “DrPHAGE” in Wechat to find more about us!

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# Team:Shanghai United HS/Experiments

Shanghai\_United\_HS

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Experiments

Media Preparation

![](https://static.igem.org/mediawiki/2021/e/e2/T--Shanghai_United_HS--newimg1.jpg)

Molecular Cloning

![](https://static.igem.org/mediawiki/2021/b/b4/T--Shanghai_United_HS--newimg2.jpg)

PCR and Electrophoresis

![](https://static.igem.org/mediawiki/2021/0/04/T--Shanghai_United_HS--newimg3.jpg)

Transformation

Expression

![](https://static.igem.org/mediawiki/2021/1/19/T--Shanghai_United_HS--newimg5.jpg)

Vitro Activity Assay

Contact Info.

---

Email: 3389014902@qq.com; ruiruihuang@gfs.org

Search “DrPHAGE” in Wechat to find more about us!

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

# Team:Shanghai United HS/Results

Shanghai\_United\_HS

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Results

Group 1

**Experimental bacteria：***E. coli* Nissle 1917

**Experimental plasmid:**plasmid A (puc57-kan-mini-J23101-OmpA-araB-TT).

**PROCEDURE**

**1) Electroporation**
Plasmid A (~ 100 ng) was transformed to E. coli
Nissle 1917 by electroporation.
**2) AraB activity assay**
In the engineering cells
simultaneously containing plasmid A and plasmid B, the expression of ClyR in plasmid B is to be induced by
arabinose, which can be supplied by AraB encoded by plasmid A via the hydrolysis of araboxylan. So, for the
sake of ClyR expression, plasmid A must express active AraB. We tried to monitor the concentration of the
reducing sugar arabinose and thus access the activity of AraB.
The concentration of reducing sugar was
determined by the DNS kit and the A540 were recorded accordingly.

**RESULT**

Table 1. The A540 data of the culture supernatant of E. coli with plasmid A and B.
![](https://static.igem.org/mediawiki/2021/6/63/T--Shanghai_United_HS--results01.jpg)

**CONCLUSION**

All the data at “0 h” with different concentration of araboxylan was referred as
blank, and the concentration of the reducing sugar could be calculated.
As can be seen from figure 1,
in the transformed bacteria with plasmid A, the concentration of the reducing sugar increased with the
elongation of the incubation time. The plasmid A can work normally and possess hydrolyzing arabinoxylan
activity, which supply the arabinose for the expression of ClyR.

Group 3

**Experimental bacteria：***E. coli* Nissle 1917

**Experimental plasmid:**

plasmid A (puc57-kan-mini-J23101-OmpA-araB-TT).

plasmid C (pBAD-Myc-HisA-OmpA-amilGFP).

**PROCEDURE**

**1) Electroporation**
Plasmid A and plasmid C were co-transformed to E.
coli Nissle 1917 by electroporation.
**2) AmilGFP expression**
Based on the data of Group 1,
the cells containing plasmid A could hydrolyze araboxylan to arabinose. To test the produced arabinose could
then induce the expression of genes under the control of pBAD. We choose green fluorescent protein AmilGFP
as a reporter protein. In the cells simultaneously containing plasmid A and plasmid C, if the active AraB
from plasmid A work effectively, the AmilGFP would be monitored.
**3) AmilGFP assessment**
The
existence of AmilGFP was monitored by the fluoroscopic examination using SpectraMax i3x..Read the
fluoroscopic data of the supernatant, and recorded the average data. After this timepoint, 100 ul of the
culture was sampled and marked as “6 h”, “8 h”, “10 h”, “12 h” and “13 h”, respectively, and the
fluoroscopic data were recorded accordingly.

**RESULT**

**1) AmilGFP expression in cells transformed with plasmid A**

![](https://static.igem.org/mediawiki/2021/4/4b/T--Shanghai_United_HS--results03.png)
Figure 2. The fluoroscopic data of cells transformed with plasmid A.

In the cells transformed with only
plasmid A, the
fluoroscopic data of cells with the addition of different concentration of araboxylan showed no difference
with that of cells without the addition of araboxylan. This data suggested no AmilGFP was produced in cells
with or without addition of araboxylan.

**2) AmilGFP expression in cells transformed with plasmids A and C**

![](https://static.igem.org/mediawiki/2021/1/15/T--Shanghai_United_HS--results04.png)
Figure 3. The fluoroscopic data of cells transformed with plasmids A and C.

In the cells transformed with plasmids A and C, the
fluoroscopic data of cells increased with the addition of araboxylan. Meanwhile, the fluoroscopic data
become larger with the prolongation of incubation time.

**3) AmilGFP expression in cells transformed with plasmid C**

![](https://static.igem.org/mediawiki/2021/1/1d/T--Shanghai_United_HS--results05.jpg)
Figure 4. The fluoroscopic data of cells transformed with plasmid C with the addition of
arabinose.

In the cells transformed with plasmid C, the fluoroscopic
data of cells increased with the addition of arabinose. Meanwhile, the fluoroscopic data become larger with
the prolongation of incubation time.

**CONCLUSION**

AmilGFP was successfully expressed, suggesting the
feasibility and possibility of the inducible secretory expression of ClyR.

Group 2

**Experimental bacteria：***E. coli* Nissle 1917

**Experimental plasmid:**

plasmid A (puc57-kan-mini-J23101-OmpA-araB-TT)

plasmid B (pBAD-Myc-HisA-OmpA-ClyR-6His-TT)

**PROCEDURE**

**1) Electroporation**
Plasmid A and plasmid B were co-transformed to E.
coli Nissle 1917 by electroporation.
**2) ClyR expression**
The araboxylan with final
concentration 0, 0.3%, 0.6%, 1.0%, 1.5% and 2.0% (w/v) was added into the culture to induce ClyR expression.

For the comparison, E. coli Nissle 1917 with plasmid B was cultured by the same way and the expression
of ClyR was induced by the addition of arabinose with final concentration of 0 μM, 10 μM,30 μM,0.1 mM, 0.2
mM,0.5 mM and 2 mM.
**3) The exported protein concentration assay**
The protein concentration
was monitored at 595 nm using Multiscan Spectrum (BioTek). Read the data for three times, recorded the
average of the A595 data..
**4) SDS-PAGE analysis of ClyR**
Gels were scanned with the
ImageQuant™ LAS 4000 mini (GE Healthcare).

**RESULT**

Table 2. The protein concentration of the culture supernatant of E. coli with plasmids A and B.
![](https://static.igem.org/mediawiki/2021/9/9b/T--Shanghai_United_HS--results06.jpg)
![](https://static.igem.org/mediawiki/2021/3/3b/T--Shanghai_United_HS--results07.jpg)
Figure 5. The SDS-PAGE of supernatant.

The A595 of the culture supernatant of E. coli with plasmid
A and B suggested the possible expression of ClyR (Table 2). The A595 data at “0 %” was referred as blank,
and the protein concentration could be calculated referred to the standard formula. As can be seen from
figure 5, in the transformed bacteria with plasmid A and B, the concentration of the reducing sugar
increased with the elongation of the incubation time.

During the expression process, bacteria lysis occurred with
the elongation of the incubation time. But the ClyR band could obviously be obtained as is shown in figure
5.

**CONCLUSION**

In the engineering cells containing plasmid A and plasmid B, the expression of ClyR
was successful.

In vitro activity assay of ClyR

**Experimental bacteria：***E. coli* Nissle 1917 transferred with plasmid A
and B, Lactobacillus casei subsp. casei (ATCC334)

**PROCEDURE**

1) The culture of Lactobacillus casei subsp. casei (ATCC334)
2) In vitro
assay
The value of OD600 was monitored using Multiscan Spectrum (BioTek).

**RESULT**

![](https://static.igem.org/mediawiki/2021/d/de/T--Shanghai_United_HS--results08.jpg)
Figure 6. The in vitro assay of ClyR.

The OD600 of the Lactobacillus casei subsp.
casei cell suspension gradually reduced with the addition of supernatant (figure 6).

**CONCLUSION**

We have successfully obtained the ClyR protein in the
culture supernatant of cells carrying plasmids with araB and clyR genes. The concentration of ClyR was low,
so more efforts are still needed in future. It is worth to note that the expression of ClyR protein may
cause the host to suicide, which can be supported by the obvious cell lysis and the presence of flocculent
precipitate.

Contact Info.

---

Email: 3389014902@qq.com; ruiruihuang@gfs.org

Search “DrPHAGE” in Wechat to find more about us!