iGEM 2021 — Team United_Shanghai_HS

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# Team:United Shanghai HS/Notebook

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# Team:CAU China/Team Member

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# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:United Shanghai HS/Engineering

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# United\_Shanghai\_HS

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# Engineering

# 1.Obtaining the fusion gene sequence

We found in the literature that Wu Bian’s team from the Institute of
Microbiology of the Chinese Academy of Sciences optimized the enzymes required by the Osaka
bacteria to degrade PET plastics, and obtained a redesigned enzyme with significantly
enhanced stability, which made the Osaka bacteria more efficient in degrading PET films. It
was improved by 300 times, and we found its optimized ISF6\_4831 protein sequence. According
to the relevant data of NCBI, we found the protein sequence of Osaka bacteria ISF6\_0224; the
protein sequence of GFP fluorescent protein. We fused the gene sequences of ISF6\_4831,
ISF6\_0224, and GFP fluorescent protein, and added linker (GGGGS/GGGGSGGGGS) between the two
sequences. The linker protein has a high degree of freedom and is not expected to affect the
protein function expression.

Through literature review, ethylene glycol can affect the conformation of the glycine
riboswitch and enhance transcription. The structure of ethylene glycol is very similar to
that of glycine. Although it is not a ligand for the glycine riboswitch, it will affect the
riboswitch and enhance transcription [1]. Therefore, this ribose switch can be used to
switch the positive feedback path at the right time. mCherry can be used as a smooth
expression of the positive feedback pathway.

The transcription of the sacB promoter is not strictly regulated by sucrose, and can be
transcribed without an inducer, but the intensity is 100 times lower than that with sucrose
[2].

We have found in the literature that the signal peptide SPlamB has the highest exocrine
efficiency. We deleted the signal peptide sequence before the ISF6\_4831 (expressing PETase)
and ISF6\_0224 (expressing MHETase) gene fragments, and added the signal peptide SPLamB
before the fusion sequence. Gene sequence to improve exocrine expression of protein.

All in all, the pathway we hope is (as shown in Figure 2): When transferred into the
transformed plasmid, Bacillus subtilis can degrade a certain amount of ethylene glycol due
to the loose regulation of the sacB promoter. With the release of ethylene glycol, the
terminator contained in the riboswitch will be hidden, and the fusion protein of MHETase and
PETase will be expressed more. At this time, mCherry is also expressed as a reporter gene.
At this time, sucrose amplification effect needs to be added, and more ethylene glycol will
increase the transcription of PETase-MHETase fusion protein. It presents a positive feedback
effect, and the front end of each fusion protein will have the signal peptide SPLamB,
thereby secreting more fusion proteins to the outside of the cell for degradation.

![](https://static.igem.org/mediawiki/2021/4/49/T--United_Shanghai_HS--team_eng1.jpg)
![](https://static.igem.org/mediawiki/2021/f/fa/T--United_Shanghai_HS--team_eng2.jpg)
![](https://static.igem.org/mediawiki/2021/9/9b/T--United_Shanghai_HS--team_eng3.jpg)
Figure 1 Fusion expression of PET
degrading enzyme
![](https://static.igem.org/mediawiki/2021/8/80/T--United_Shanghai_HS--team_eng4.jpg)

[1] Hamachi K, Hayashi H, Shimamura M, Yamaji Y,
Kaneko A, Fujisawa
A, Umehara T, Tamura K. Glycols modulate terminator stem stability and ligand-dependency of
a glycine riboswitch. Biosystems. 2013 Aug; 113:59-65.

[2] Zukowski M M, Miller L. Hyperproduction of an
intracellular heterologous protein in a sacUh mutant of Bacillus subtilis[J]. Gene, 1986,46:
247-255.

# 2.Plasmid construction

1) Plasmid construction and degradation efficiency detection of pET28a fusion protein
First of all, you need to know the degradation efficiency of the PETase-MHETase fusion
protein and the acquisition of each fragment. Therefore, first use the commonly used pET28a
plasmid as the intermediate plasmid, and connect the fusion protein including the
PETase-MHETase-GFP fragment and PETase-MHETase-mCherry to pET28a on the plasmid.

①　Codon optimization
Codon optimization of PETase-MHETase makes the final expression in Bacillus subtilis more
efficient. Use the website http://www.kazusa.or.jp/codon/ to analyze the codon optimization
problem. And send the optimized gene fragments to the gene company for synthesis.

②　Obtaining the plasmid of fusion expression of pET28a
The linearized plasmid was obtained by double digestion with Nde Ⅰ and BamH Ⅰ, and the
synthesized fusion fragment was introduced into the restriction sites of Nde Ⅰ and BamH Ⅰ by
means of PCR, and pET28a and PETase-MHETase- GFP and PETase-MHETase-mCherry are connected.
Thus, the recombinant plasmid of pET28a was obtained.

③　Detection of degradation efficiency and detection of fusion protein secretion
The recombinant plasmid of pET28a was transferred into BL21(DE3) large intestine expression
strain, and T7 universal primers were used to test and verify the correctness of the
fragment size, and then sequenced. After the verification is correct, the detection of the
degradation efficiency of the protein and the detection of the secretion of the fusion
protein are performed.

Use 500 mL of LB medium for secondary culture of BL21(DE3) large intestine, and shake it to
OD600 of 0.6-0.8 at secondary level. At this time, add IPTG for 16 hours of induction.
Purify the protein, and save the supernatant and precipitate, use SDS-PAGE method to detect
protein expression and secretion.

In addition, you can also use a microplate reader to detect GFP or mCherry, separate the
cultured cells and supernatant, and observe the presence of GFP or mCherry in the
supernatant.
The degradation of PET film can be assessed by weight loss, carbon dioxide generation
through PET catabolism, and changes in the surface morphology and increment of the film
determined by scanning electron microscopy (SEM) [3].

And the purified fusion protein is incubated with the PET film for a certain period of time,
then the incubated PET film is removed from the protein solution, the film is soaked in 1%
SDS, and gently rotated for 5 minutes. Repeat the 1% SDS soaking film and rotate gently for
5 minutes. Put it in distilled water and rotate gently for 5 minutes. Repeat to soak the
film with 1% SDS, rotate it gently for 5 minutes, and do this twice. Put it in ethanol. Air
dry at room temperature for 1 hour (vacuum drying is best). Weigh the film and calculate the
weight loss.

[3]Yoshida S, Hiraga K, Taniguchi I, Oda K.
Ideonella sakaiensis, PETase, and MHETase: From
identification of microbial PET degradation to enzyme characterization. Methods Enzymol.
2021;648:187-205.

2)Plasmid construction of pWB980 fusion protein
and detection of
degradation efficiency

(1) Obtaining each fragment

PETase-MHETase-GFP (includes SP LamB signal peptide) was amplified from the previous
recombinant pET28a plasmid using PCR.

PETase-MHETase-mCherry (which already contains SP LamB signal peptide) was amplified from the
previous recombinant pET28a plasmid using PCR.

The core sequence of the known Bacillus subtilis SacB gene was retrieved from GenBank, and
the sacB promoter sequence was found. Specific primers were designed. Using Bacillus
subtilis as a template, the promoter was amplified by PCR.

The sequences of the laps promoter and the glycine riboswitch were obtained in the
literature, and the sequences were sent to the gene company for synthesis.

(2) Fragment connection

First carry out the first double digestion and ligation, use EcoR Ⅰ and BamH Ⅰ double
digestion of pWB980 plasmid, recombine with sacB promoter and PETase-MHETase-GFP (which
already contains SP LamB signal peptide) (the process will add RBS and retain EcoR) Ⅰ and
BamH Ⅰ restriction sites).

Then perform the second digestion. At this time, the digestion is BamH Ⅰ single digestion.
The synthesized laps promoter and glycine riboswitch are combined with the
PETase-MHETase-mCherry (which already contains SP LamB signal peptide) amplified by PCR. Cut
the connection. Finally, the recombinant plasmid backbone shown in the figure below is
obtained (as shown in Figure 3).

![](https://static.igem.org/mediawiki/2021/3/3d/T--United_Shanghai_HS--team_eng5.jpg)

(3) Fusion protein expression detection

The obtained recombinant plasmid was introduced into Bacillus Subtilis 168 for expression,
the intensity and presence of GFP fluorescence were detected to determine the expression of
the fusion protein, and the expression was detected by Western blot using mCherry and GFP
antibodies.

(4) Fusion protein expression detection

Incubate the expressed Bacillus subtilis with the PET film for a certain period of time.
During this period, add sucrose to increase the expression and degradation efficiency of the
fusion protein, and then remove the incubated PET film from the protein solution and use 1%
SDS Soak the film and rotate gently for 5 minutes. Repeat the 1% SDS soaking film and rotate
gently for 5 minutes. Put it in distilled water and rotate gently for 5 minutes. Repeat to
soak the film with 1% SDS, rotate it gently for 5 minutes, and do this twice. Put it in
ethanol. Air dry at room temperature for 1 hour (vacuum drying is best). Weigh the film and
calculate the weight loss. Once effective, the degradation system can be increased, such as
collecting plastic waste in a simulated degradation reactor for further degradation tests.

![](https://static.igem.org/mediawiki/2021/0/02/T--United_Shanghai_HS--team_simg6.jpg)
![](https://static.igem.org/mediawiki/2021/c/cd/T--United_Shanghai_HS--team_simg4.jpg)
![](https://static.igem.org/mediawiki/2021/7/7d/T--United_Shanghai_HS--team_simg3.jpg)

#### Contact us:woxihuan36@foxmail.com

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# Team:United Shanghai HS/Experiments

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# United\_Shanghai\_HS

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![](https://static.igem.org/mediawiki/2021/6/60/T--United_Shanghai_HS--team_simg2.jpg)
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![](https://static.igem.org/mediawiki/2021/c/cd/T--United_Shanghai_HS--team_simg4.jpg)

# Experiments

1. Plasmid construction

1) Plasmid construction and degradation efficiency
detection of pET28a fusion protein

First of all, you need to know the degradation efficiency
of the PETase-MHETase fusion protein and the acquisition of each fragment. Therefore, first use
the
commonly used pET28a plasmid as the intermediate plasmid, and connect the fusion protein
including the
PETase-MHETase-GFP fragment and PETase-MHETase-mCherry to pET28a on
the
plasmid.

①　Codon optimization

Codon optimization of PETase-MHETase makes the final
expression in Bacillus subtilis more efficient. Use the website http://www.kazusa.or.jp/codon/
to
analyze the codon optimization problem. And send the optimized gene fragments to the gene
company for
synthesis.

②　Obtaining the plasmid of fusion expression of
pET28a

The linearized plasmid was obtained by double digestion
with Nde Ⅰ and BamH Ⅰ, and the synthesized fusion fragment was introduced into the restriction
sites of
Nde Ⅰ and BamH Ⅰ by means of PCR, and pET28a and PETase-MHETase- GFP and PETase-MHETase-mCherry
are
connected. Thus, the recombinant plasmid of pET28a was obtained.

③　Detection of degradation efficiency and detection of
fusion protein secretion

The recombinant plasmid of pET28a was transferred into
BL21(DE3) large intestine expression strain, and T7 universal primers were used to test and
verify the
correctness of the fragment size, and then sequenced. After the verification is correct, the
detection
of the degradation efficiency of the protein and the detection of the secretion of the fusion
protein
are performed.

Use 500 mL of LB medium for secondary culture of BL21(DE3)
large intestine, and shake it to OD600 of 0.6-0.8 at secondary level.
At this
time, add IPTG for
16 hours of induction. Purify the protein, and save the supernatant and precipitate, use
SDS-PAGE method
to detect protein expression and secretion.

In addition, you can also use a microplate reader to
detect GFP or mCherry, separate the cultured cells and supernatant, and observe the presence of
GFP or
mCherry in the supernatant.

The degradation of PET film can be assessed by weight
loss, carbon dioxide generation through PET catabolism, and changes in the surface morphology
and
increment of the film determined by scanning electron microscopy (SEM)[1]
.

And the purified fusion protein is incubated with the PET
film for a certain period of time, then the incubated PET film is removed from the protein
solution, the
film is soaked in 1% SDS, and gently rotated for 5 minutes. Repeat the 1% SDS soaking film and
rotate
gently for 5 minutes. Put it in distilled water and rotate gently for 5 minutes. Repeat to soak
the film
with 1% SDS, rotate it gently for 5 minutes, and do this twice. Put it in ethanol. Air dry at
room
temperature for 1 hour (vacuum drying is best). Weigh the film and calculate the weight
loss.

[1] Yoshida S, Hiraga K,
Taniguchi
I, Oda K. Ideonella sakaiensis, PETase, and MHETase: From identification of microbial PET
degradation to
enzyme characterization. Methods Enzymol. 2021;648:187-205.

2) Plasmid construction of pWB980 fusion protein and
detection of degradation efficiency

(1) Obtaining each fragment

PETase-MHETase-GFP (includes SP LamB signal peptide) was
amplified from the previous recombinant pET28a plasmid using PCR.

PETase-MHETase-mCherry (which already contains SP LamB
signal peptide) was amplified from the previous recombinant pET28a plasmid using PCR.

The core sequence of the known Bacillus subtilis SacB gene
was retrieved from GenBank, and the sacB promoter sequence was found. Specific primers were
designed.
Using Bacillus subtilis as a template, the promoter was amplified by PCR.

The sequences of the laps promoter and the glycine
riboswitch were obtained in the literature, and the sequences were sent to the gene company for
synthesis.

(2) Fragment connection

First carry out the first double digestion and ligation,
use EcoR Ⅰ and BamH Ⅰ double digestion of pWB980 plasmid, recombine with sacB promoter and
PETase-MHETase-GFP (which already contains SP LamB signal peptide) (the process will add RBS and
retain
EcoR) Ⅰ and BamH Ⅰ restriction sites).

Then perform the second digestion. At this time, the
digestion is BamH Ⅰ single digestion. The synthesized laps promoter and glycine riboswitch are
combined
with the PETase-MHETase-mCherry (which already contains SP LamB signal peptide) amplified by
PCR. Cut
the connection. Finally, the recombinant plasmid backbone shown in the figure below is obtained
(as
shown in Figure 3).

![](https://static.igem.org/mediawiki/2021/d/d4/T--United_Shanghai_HS--team_exp1.png)

![](https://static.igem.org/mediawiki/2021/0/02/T--United_Shanghai_HS--team_simg6.jpg)
![](https://static.igem.org/mediawiki/2021/c/cd/T--United_Shanghai_HS--team_simg4.jpg)

#### Contact us:woxihuan36@foxmail.com

![](https://static.igem.org/mediawiki/2021/2/2f/T--United_Shanghai_HS--qiuying.jpg)

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# Team:United Shanghai HS/Results

Document

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# United\_Shanghai\_HS

* [Team](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Team_Members)
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* [Project](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Description)
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  + [Proof
    Of Concept](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Proof_Of_Concept)
  + [Results](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Results)
* [Parts](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Parts_Overview)
  + [Parts
    Overview](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Parts_Overview)
  + [Contribution](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Contribution)
  + [Engnieering
    Success](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Engineering)
  + [Improvement](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Improvement)
  + [New
    Composite Part](https://2021.igem.org/Team%3AUnited_Shanghai_HS/New_Composite_Part)
* [Lab](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Experiments)
  + [Experiments](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Experiments)
* [Model](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Model)
* [HP](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Human_Practices)
  + [Human
    Practices](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Human_Practices)
  + [Education](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Communication)
  + [Integrated
    Human
    Practices](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Integrated_Human_Practices)
* [Safety](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Lab_Safety)
  + [Lab
    Safety](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Lab_Safety)
  + [Implementation](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Implementation)
* [Judge](https://2021.igem.org/Team%3AUnited_Shanghai_HS/Judge)

Search

The experiment was delayed due to COVID-19, so there was no experiment. But we have carried out experimental
design, and related reagents and primers have been ordered. We will start the experiment when the epidemic slows
down.

![](https://static.igem.org/mediawiki/2021/c/c0/T--United_Shanghai_HS--team_covid1.png)

#### Contact us:woxihuan36@foxmail.com

![](https://static.igem.org/mediawiki/2021/2/2f/T--United_Shanghai_HS--qiuying.jpg)