iGEM 2024 — Team basis-china

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{% block title %}Notebook{% endblock %}
{% block header\_title %}Notebook{% endblock %}
{% block header\_desc %}
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{% block page\_aside %}
- [##### Overview](#Overview)
- [##### Experiment Notes](#Experiment Notes)
- [##### Our Foolish Mistakes 🙄💅](#Our Foolish Mistakes)
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## **Overview**

After recruiting team members from across 8 BASIS campuses all across China,
our team was officially formed in December, 2023. Due to our geographical
dispersion during the school year, we held online meetings once a week to
brainstorm our project. Starting from February, our team members started
meeting offline to make progress on our experiments and dry lab. Throughout May,
June, and October, we held offline training camps for all of our team members.
As we made progress with our experiments, we faced various challenges, some
technical and some just foolish (see below), which caused our progress to
jump back and forth. Ultimately, after months of rigorous experimentation and
dedication, our wet lab experiments came to an end in October.

## **Experiment Notes**

## **Our Foolish Mistakes 🙄💅**

Overcoming various challenges was a crucial part of our learning process.
Although some were rather foolish, these trips themselves have
turned our own errors into the building blocks of our growth.

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block header\_title %}Engineering Success{% endblock %}
{% block header\_desc %} {% endblock %}
{% block page\_aside %}
- [##### Overview](#Overview)
- [##### Cycle 1: PETase from iGEM Distribution Kit](#Cycle 1: PETase from iGEM Distribution Kit)
- [##### Cycle 2: AI Generated Mutants](#Cycle 2: AI Generated Mutants)
- [##### Cycle 3: Signal Peptide](#Cycle 3: Signal Peptide)
- [##### Cycle 4: E. coli to Cyanobacteria](#Cycle 4: E. coli to Cyanobacteria)
- [##### Cycle 5.1: Co-culture System](#Cycle 5.1: Co-culture System)
- [##### Cycle 5.2: Bioreactors](#Cycle 5.2: Bioreactors)
{% endblock %}
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## **Overview**

We consistently adhere to the Design-Build-Test-Learn Cycle methodology to advance our project step by step.
This year's project can be broken down into five distinct cycles. In the first cycle, we successfully
constructed and tested the PETase synthesis circuit in an *E. coli* chassis. In the second cycle, in order
to improve the PETase efficiency, we use AI tools to generate new PETase mutants and tested in *E. coli*.
In the third cycle, to maintain continued production and functioning of PETase, we designed a signal peptide to allow for efficient
extracellular excretion of PETase. In the fourth cycle, we further introduced the PETase into cyanobacteria.
Finally, in the fifth cycle, we designed and tailored bioreactors to support the optimized growth and
productivity of the engineered cyanobacteria.

![](https://static.igem.wiki/teams/5236/engineering-success/img-4426.jpeg)

Figure 1. Engineering cycle diagram of our project

## **Cycle 1: PETase from iGEM Distribution Kit**

### Design

Our goal is to identify enzymes capable of degrading PET at ambient temperatures. Initially, we utilized the
PETase available in the iGEM distribution kit and tested their efficiency in PET degradation. Additionally,
we took this chance to establish a standardized testing process for PET degradation: measuring the weight
loss of *E. coli*-treated PET particles and a testing method outlined by a previous iGEM team.

### Build

From the iGEM distribution kit, we identified five PETase available for use: BBa\_K2910000, BBa\_K2910003,
BBa\_J428065, BBa\_J428067 and BBa\_K3039002. After obtaining these parts, we assembled them into plasmids
using the pET28a vector and transformed then into *E. coli* BL21(DE3).

![](https://static.igem.wiki/teams/5236/engineering-success/1.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/2.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/3.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/4.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/5.jpg)

Figure 2. Snapgene diagrams of the five plasmids constructed

### Test

We verified successful plasmid construction through gel electrophoresis.

![](https://static.igem.wiki/teams/5236/engineering-success/6.jpg)

Figure 3. Gel electrophoresis results

After transforming the plasmid into *E. coli*, we tested for its efficiency by
measuring the weight loss of a 1g PET plastic film following a two-week incubation in our
*E. coli* culture. However, we found out that there were no significant differences
prior and post the *E. coli* treatment.

![](https://static.igem.wiki/teams/5236/engineering-success/7.jpg)

Figure 4. Graph of initial and final weight.

For additional testing, we adopted the method used by the team iGEM19\_Toronto:
*p*-nitrophenyl butryte degradation assay. This assay measures the activity level of PETase
through monitoring light absorption at 415 nm. PETase depolymerizes *p*-nitrophenyl butyrate
producing the monomers butyric acid and *p*-nitrophenolate, which absorb light at a 415nm
wavelength. Hence, greater 415nm light absorption correlates with higher enzyme activity.
(see protocol)

![](https://static.igem.wiki/teams/5236/engineering-success/8.jpg)

Figure 5. Diagram of *p*-nitrophenyl butyrate degradation assay

We obtained positive results when testing with samples extracted from our modified *E. coli*
culture. The relative enzyme efficiency (A415/protein concentration) that we are looking at takes into consideration both the efficiency of the enzyme itself and the PETase synthesis rate of the chassis, since our end goal is to implement the engineered organism in a self-sufficient PET degrading system as a whole.

![](https://static.igem.wiki/teams/5236/yh/relative-activity.jpg)

Figure 6. Result of *p*-nitrophenyl butyrate degradation assay.

### Learn

Our gel results and *p*-nitrophenyl butryte assay demonstrated that we successfully constructed
plasmids that encode PETase in *E. coli* capable of cleaving the ester bonds in PET
polymers. Additionally, we confirmed the *p*-nitrophenyl butryte assay to be an effective method
for measuring PETase activity. However, we were not able to detect any changes in the weight of
PET plastic after incubating it with our modified *E. coli*. We hypothesized that this was
due to two factors: (1) the enzymes were not very effective at degrading PET microplastics, and
(2) PET plastics have varying degrees of crystallinity depending on their synthesis process and
physical aging.

## **Cycle 2: AI Generated Mutants**

### Design

Following the completion of Cycle 1, we aimed to further enhance the efficiency and
effectiveness of PET degradation by PETase through identifying variants of the PETase
enzyme. In a discussion with Professor Qi, we were inspired by his insights on the PETase
mutant — LCC-A2, which lead us to explore the potential of improving degradation
efficiencies through mutations. Building on to this idea, in addition to reviewing existing
literature to identify PETase variants, we also employed AI tools we trained to generate
mutants that could possibly result in enzymes that surpass the performance of wild-type
PETase.

### Build

After conducting research, we identified a PETase variant — BhrPETase. The sequence of
BhrPETase was identified by the Shingo group in a metagenomic study on uncultured
thermophiles, and was deposited into the NCBI database by the group in 2018 and
annotated as a PET hydrolase [[1]](https://www.jstage.jst.go.jp/article/jsme2/33/1/33_ME17165/_article).
Together with the previous PETase obtained from the iGEM distribution kit, we created a
series of mutants for both IsPETase and BhrPETase using an AI-assisted protein design
tool (see model). These sequences were expressed in *E. coli* using the pET28a
vector.

![](https://static.igem.wiki/teams/5236/engineering-success/screenshot-2024-10-01-at-20-09-55.png)

Figure 7. Table of mutated version of IsPETase/BhrPETase

### Test

We successfully verified the construction of plasmids containing the mutated
sequences using gel electrophoresis.

![](https://static.igem.wiki/teams/5236/part-images/marker-colony-pcr.png)

Figure 8. Gel electrophoresis Results

The *p*-nitrophenyl butryte degradation assay, on the other hand, demonstrated that the
mutated variants of PETase outperformed wild-type PETase.

![](https://static.igem.wiki/teams/5236/yh/ispet-relative-activity.jpg)
![](https://static.igem.wiki/teams/5236/yh/bhrpet-relative-activity.jpg)

Figure 9. Result of of *p*-nitrophenyl butyrate degradation assay

### Learn

From the results we obtained, we determined that certain mutated variants
designed by our AI tool are more effective in degrading PET. Yet, we realized a
few drawbacks. Firstly, although the *p*-nitrophenyl butryte degradation assay is
a viable method for detecting the hydrolysis of ester bonds, it does not
specifically measure the actual degradation of PET. Secondly, lysing the
*E. coli* cells was required when testing for the activity of the enzymes.
However, considering that we desire to develop a self-sustaining PET degrading
system, breaking down the cells to release the enzyme is an unfeasible method.

## **Cycle 3: Signal Peptide**

### Design

With the drawbacks in mind, we consulted Dr. Wang in search for
solutions. He suggested the use of signal peptides for the secretion of
the enzymes. Following his advice, we inserted an additional sequence
into our plasmid design that encoded a signal peptide, which allows the
enzyme to exit without causing any disturbance to the cell.

### Build

ChatGPT provided us with several preliminary signal peptide
sequences.

![](https://static.igem.wiki/teams/5236/engineering-success/14.jpg)

Figure 10. Answer from ChatGPT

After reading more papers and doing more research, we decided to use
pelB as our signal peptide; when fused to the front end of a protein,
this signal peptide allows the protein to be transported across the two
membranes of *E. coli*. Plasmids were reconstructed with this
sequence inserted before the enzyme sequence.

![](https://static.igem.wiki/teams/5236/engineering-success/15.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/16.jpg)

Figure 11. Diagram of new plasmid including the signal peptide sequences

### Test

The effectiveness of the signal peptides was measured using the
*p*-nitrophenyl butryte degradation assay. We extracted cultures from
both *E. coli* containing the newly designed plasmid and *E.
coli* containing the original plasmid. After centrifuging the
samples, we retained the supernatant for the assay. Theoretically,
enzymes with the additional signal peptide will be able to leave the
cell and be present in the supernatant, thus result in better enzyme
activity. Final results indicated that signal peptides allowed for
the secretion of the enzymes.

![](https://static.igem.wiki/teams/5236/engineering-success/17.png)

Figure 12. Result of of *p*-nitrophenyl butyrate degradation assay

Subsequently, to verify the enzymes secreted were in fact the PETase
enzymes, we performed SDS-PAGE.

![](https://static.igem.wiki/teams/5236/engineering-success/18.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/19.jpg)

Figure 13. SDS-PAGE results

In hopes of detecting PETase activity through a more intuitive
approach, we utilized a scanning electron microscope (SEM) to
directly observe the degradation of PET. Different plastic samples
were placed in the culture medium of the engineered *E. Coli*.
After two weeks, the samples were observed under an SEM for any
alterations in the surface of the plastic by technicians at Shenzhen
University. The results demonstrated that plastic with low level of
crystallinity were degraded under the exposure to PETase synthesized
by our engineered *E. coli*. Further, the fact that plastic
with high crystallinity did not show any significant changes
addresses our hypothesis in Cycle 1: PET degradation is affected by
the crystallinity of the plastic, which varies depending on its
manufacturing process.

![](https://static.igem.wiki/teams/5236/engineering-success/20.png)
![](https://static.igem.wiki/teams/5236/engineering-success/21.png)
![](https://static.igem.wiki/teams/5236/engineering-success/22.png)

Figure 14. Results from SEM

### Learn

Upon analyzing our results, we determined that the signal
peptide pelB successfully enables the secretion of the actual
PETase synthesized within then engineered *E. coli* cells.
Using a SEM we were also able to detect the degradation of low
crystallinity plastics by the secreted enzymes. Still, the
results are not sufficient; the alteration of the plastic
surfaces might also be due to physical damages and not chemical
degradation. Additionally, we realized another problem
concerning the use of *E. coli* as our chassis. To ensure
the *E. coli* cells are still living, we fed them with
growth medium. However, this contradicts with our vision of a
fully self-sustaining PET degradation system.

## **Cycle 4: *E. coli* to Cyanobacteria**

### Design

Following the progress in cycle 3 and considering the
problems identified, we consulted more experts and
decided to use a new chassis — cyanobacteria
*Synechococcus elongatus* PCC 7942. Hence, we
reconstructed our plasmids using a transfer vector that
transport the target gene into cyanobacteria to be
incorporated into its genome.

### Build

The plasmids were reconstructed using a transfer
vector, with the signal peptide and PETase sequence
inserted between promoter PpsbA2 and terminator
Bba\_B0015.

![](https://static.igem.wiki/teams/5236/engineering-success/23.png)

Figure 15. Snapgene diagram of plasmid for
cyanobacteria

### Test

The reconstructed plasmids were transformed into
cyanobacteria and coated on BG11 plates
containing the antibiotic spectacular ionomycin.
Single colonies appeared after two weeks of
incubation.

![](https://static.igem.wiki/teams/5236/engineering-success/24.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/25.jpg)

Figure 16. Cyanobacteria culture plate

The single colonies were cultured and
samples were extracted for the *p*-nitrophenyl
butryte degradation assay. Results
demonstrated that the engineered
cyanobacteria can synthesize active PETases.

![](https://static.igem.wiki/teams/5236/engineering-success/26.jpg)

Figure 17. Result of of *p*-nitrophenyl
butyrate degradation assay

To address the limitations existing in our
previous methods for measuring the
effectiveness of PETase in degrading PET, we
performed high performance liquid
chromatography to detect the amount of PET
monomers — tetrephtalic acid (TPA) — present
after treatment of PET with engineered
*E. coli* and cyanobacteria. Samples
were prepared and sent to technicians at
Shenzhen Univeristy, whom performed HPLC.
The results, were positive for both.

![](https://static.igem.wiki/teams/5236/engineering-success/27.png)

Figure 18. HPCL result

### Learn

We concluded that cyanobacteria is a
suitable chassis for PETase
synthesis. We also obtained positive
results from the alternative PET
degradation testing method, HPLC,
directly indicating that the PETase
synthesized are capable of breaking
down the polymer PET into its
monomers. Yet, our end goal is to
achieve complete degradation of PET
into harmless, naturally occurring
compounds. Moreover, our current
engineered organisms lack a clear
application scenario.

## **Cycle 5.1: Co-culture System**

### Design

Upon doing more research, we
learned that further degradation
of terephthalic acid (TPA) into
carbon dioxide and water can be
achieved by the bacteria
*Rhodococcus jostii*. This
led us to consider co-culturing
cyanobacteria with
*Rhodococcus* to attain
further degradation of PET.

### Build

To culture the two different
organism together, their
respective growth mediums,
R2A for rhodobacteria and
BG-11 for cyanobacteria,
were mixed together in
different ratios: 1:1, 1:2,
and 1:3 (R2A: BG-11).

### Test

During the two week
incubation, pictures
were taken weekly to
observe the growth of
the rhodobacteria and
cyanobacteria. Results
demonstrate the the
organisms performed the
best at 1:3 R2A to BG-11
ratio.

![](https://static.igem.wiki/teams/5236/engineering-success/28.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/29.jpg)

Figure 19. Cyanobacteria
and Rhodococcus
co-culture results

### Learn

We determined that
cyanobacteria can be
co-cultured with
*Rhodococcus
jostii*.

## **Cycle 5.2: Bioreactors**

### Design

After
successfully
designing a PET
degrading
biological
system, we aim
to give it a
real life
application
through
implementing our
co-culture
system in
bioreactors.
Involving discussions with our
design group,
and after
interviews with
experts Dr. Fu
and Dr. Zhou, we
created several
bioreactor
design.

![](https://static.igem.wiki/teams/5236/engineering-success/30.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/31.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/2024-10-02-01-50-27.png)
![](https://static.igem.wiki/teams/5236/engineering-success/2024-10-02-01-35-16.png)

Figure 20.
Bioreactor
designs

### Build

Out of
the
multiple
designs,
a few
were
constructed.

![](https://static.igem.wiki/teams/5236/engineering-success/before-building.jpeg)
![](https://static.igem.wiki/teams/5236/engineering-success/after-building.jpeg)
![](https://static.igem.wiki/teams/5236/engineering-success/36.jpg)
![](https://static.igem.wiki/teams/5236/engineering-success/wechatimg225.jpg)

Figure
21.
Hardware

### Test

Out
of
the
ones
constructed,
some
demonstrated
to
be
viable,
such
as
the
life-based
version.

![](https://static.igem.wiki/teams/5236/engineering-success/37.jpg)

Figure
22.
Life-based
hardware

### Learn

Although
some
of
our
bioreactor
designs
are
feasible,
there
still
exists
some
limitations.
Further
improvement
still
needs
to
be
made
in
the
future.
(see
hardware
for
more
details)

{%
endblock
%}

{% extends "layout.html" %}
{% block title %} Experiments {% endblock %}
{% block header\_title %} Experiments {% endblock %}
{% block header\_desc %} {% endblock %}
{% block page\_aside %}
- [##### Experiments Overview](#Experiments Overview)
- [##### Experiments Process](#Experiments Process)
- [##### Protocols](#Protocols)
{% endblock %}
{% block page\_content %}

## **Experiments Overview**

Our experiment aims to engineer a chassis to degrade micro-plastics in an efficient
and accessible manner in the natural environment. We utilized PCR, recombination
and transformation to construct plasmid for *E.coli* to express the PETase.
The mutated PETase and signal peptide sequence were inserted during this process
for enhancing efficiency and out-membrane PETase expression. Then, we changed our
chassis to Cyanobacteria due to its self-sufficient characteristic. Last, the
modified Cyanobacteria was co-cultivating with *Rhodococcus* to further
break down the monomer initially broken down by PETase.

## **Experiments Process**

**1. Initial Construction:**

1. Plasmid Extraction: extract the plasmid with our wanted segments (PETase)
   from iGEM distribution kit
2. PCR of Targeted Segment: acquire and amplify the segments that we want
   from the plasmid
3. PCR Product Purification: get rid of unwanted DNA, like the original plasmid,
   and other unwanted substances from PCR product
4. Assembly: assemble PETase segment with constructed shuttle vector
   backbone
5. Transformation into DH5α: transform the constructed plasmid into
   competent DH5α *E.coli*cells
6. Colony PCR: to see whether the plasmid is transformed into *E.coli* or not
7. Sequencing: send samples to sequencing company and get the exact
   sequence of constructed plasmid to check whether construction was
   correct

**2. Mutation construction:**

1. Normal PCR: acquire PCR product with mutated PETase
2. Assembly: assemble PCR product and signal peptide gene with linearized
   pET28a plasmid
3. Transformation into DH5α and Further Transformation into BL21(DE3):
   transform the constructed plasmid into competent DH5α *E.coli* cells;
   further transform plasmid into BL21(DE3) for protein expression

**3. *E.coli* Expression**

1. Protein Induction: cultivate the positive single colonies of BL21(DE3), add
   IPTG to induce the start of transcription and the expression of PETase
2. SDS-PAGE: check the size of the proteins that were expressed before and
   after induction
3. Enzyme Assay:
   1. *p*-nitrophenyl butyrate Degradation Assay: detect PETase’s activity to
      check the degradation efficiency
   2. Scanning Electron Microscope: to observe the degrading level of
      microplastic

**4. *Synechococcus elongatus* PCC 7942, Co-culture, and Final Justification**

1. Natural Transformation into *Synechococcus elongatus* PCC 7942: transform
   the plasmid constructed from BL21(DE3) into Syn 7942
2. Streaking Cultivation: culture positive monocultures in preparation for
   colony PCR
3. Colony PCR: to check whether the plasmid has been successfully
   transformed into *Syn* 7942
   or not
4. Enzyme Assay:
   1. P-nitrophenyl butyrate Degradation Assay: detect PETase’s activity in
      *Syn* 7942
      to check the degradation efficiency
   2. High Performance Liquid Chromatography (HPLC) check of *E.
      coli*product: to get a chromatogram (chemical composition) showing
      the products that the proteins have produced, and justify that the
      product consists of TPA- Co-cultivation: cultivate *Rhodococcus jostii* and modified *Syn* 7942
     together
     to allow for further breakdown of TPA

![](https://static.igem.wiki/teams/5236/steph-s-folder/601727861468-pic.jpg)

## **Protocols**

* [0001 Recombination](https://static.igem.wiki/teams/5236/protocol/assembly-seamless-cloning-kit-aka-recombination.pdf)
* [0002 BCA Protein Assay](https://static.igem.wiki/teams/5236/protocol/bca-protein-assay-in-96-well-microplate.pdf)
* [0003 Biosafety in 24-well Microplate](https://static.igem.wiki/teams/5236/steph-s-folder/biosafety-in-24-well-microplate-2.pdf)
* [0004 Co-culture of Cyanobacteria and Rhodobacteria](https://static.igem.wiki/teams/5236/steph-s-folder/co-culture-of-cyanobacteria-and-rhodococcus-jostii-2.pdf)
* [0005 Colony PCR](https://static.igem.wiki/teams/5236/protocol/colony-pcr-only-for-testing.pdf)
* [0006 Cyanobacteria Colony PCR](https://static.igem.wiki/teams/5236/protocol/cyanobacteria-colony-pcr.pdf)
* [0007 Cyanobacteria Transformation](https://static.igem.wiki/teams/5236/steph-s-folder/cyanobacteria-transformation-2.pdf)
* [0008 *E.coli* Competent Cell Transformation](https://static.igem.wiki/teams/5236/protocol/e-coli-competent-cell-transformation.pdf)
* [0009 Enzyme Kinase Measuring](https://static.igem.wiki/teams/5236/steph-s-folder/enzyme-kinase-measuring-2.pdf)
* [0010 Gel Electrophoresis](https://static.igem.wiki/teams/5236/steph-s-folder/gel-electrophoresis-2.pdf)
* [0011 High-Performance Liquid Chromatography](https://static.igem.wiki/teams/5236/protocol/high-performance-liquid-chromatography.pdf)
* [0012 Normal PCR](https://static.igem.wiki/teams/5236/protocol/normal-pcr-used-for-obtaining-dna-fragments.pdf)
* [0013 Plasmid Extraction](https://static.igem.wiki/teams/5236/protocol/plasmid-extraction.pdf)
* [0014 SDS-PAGE](https://static.igem.wiki/teams/5236/protocol/sds-page-sodium-dodecyl-sulfate-polyacrylamide-gel-electrop.pdf)
* [0015 Streaking Cultivation](https://static.igem.wiki/teams/5236/protocol/streaking-cultivation.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block header\_title %}Results{% endblock %}
{% block header\_desc %} {% endblock %}
{% block page\_aside %}
- [##### Overview](#Overview)
- [##### Validation of PETase activity from the iGEM distribution kit](#Validation of PETase activity from the iGEM distribution kit)
- [##### Plasmid and Mutant Construction](#Plasmid and Mutant Construction)
- [##### Signal Peptide Construction](#Signal Peptide Construction)
- [##### Construction and verification in Cyanobacteria](#Construction and verification in Cyanobacteria)
- [##### Co-culture System](#Co-culture System)
{% endblock %}
{% block page\_content %}

## **Overview**

In this section, we will present all the key results that have been critical in advancing our project. These
results include plasmid and mutant construction, enzyme efficiency assay in *E.Coli* and cyanobacteria, signal
peptide construction, PETase expression, transformation into *Syn*. PCC 7942 and co-culture of *Rhodococcus jostii*
and cyanobacteria.

## **Validation of PETase activity from the iGEM distribution kit**

We identified several PETases from the iGEM distribution kit, which are the first naturally evolved PETase from
*Ideonella sakaiensis* and their mutants. To estimate PETase enzyme activity, we followed 2019 Toronto iGEM team’s
method of measuring esterase degradation using *p*-Nitrophenol butyrate (from Part:BBa K2910000 - parts.igem.org).
The enzymatic activity of these IsPETases were measured and estimated accordingly.

![](https://static.igem.wiki/teams/5236/engineering-success/6.jpg)

Figure 1. Gel electrophoresis result of the mutants BBa\_K3039002, BBa\_J428067, BBa\_K2910003, BBa\_K2910000, and
BBa\_J428065 (WT).

![](https://static.igem.wiki/teams/5236/yh/relative-activity.jpg)

Figure 2. the enzymatic activities of WT IsPETase (BBa\_J428065) compared to its mutants BBa\_K3039002,
BBa\_J428067, BBa\_K2910003, BBa\_K2910000, which showed that three out of four mutants had higher enzymatic
activity than the WT.

The relative enzyme efficiency (A415/protein concentration) that we are looking at takes into consideration both the efficiency of the enzyme itself and the PETase synthesis rate of the chassis, since our end goal is to implement the engineered organism in a self-sufficient PET degrading system as a whole.

## **Plasmid and Mutant Construction**

After reviewing several papers predicting PETase protein efficiency and activity, we identified mutation sites in
IsPETase that could potentially enhance activity. We also found a PETase with higher predicted activity than
wild type IsPETase — BhrPETase. We utilized an AI-assisted protein design tool based on methods described in
literature. Using the vector pET28a, we constructed plasmids containing our PETase sequences. The sequence is
inserted between the T7 promoter and T7 terminator.

![](https://static.igem.wiki/teams/5236/result/img-3204.jpeg)
![](https://static.igem.wiki/teams/5236/result/img-3205.jpeg)

Figure 3. Diagram of plasmids.

We tested whether our plasmid constructions were successful using gel electrophoresis and sequencing.

![](https://static.igem.wiki/teams/5236/part-images/marker-colony-pcr.png)

Figure 4. Gel electrophoresis results of the mutations of IsPETase and BhrPETase. The electrophoresis of
IsPETase
M128L failed.

![](https://static.igem.wiki/teams/5236/result/2024-10-01-19-50-08.png)

Figure 5. The table shows the sequencing of our mutation. 11 out of 14 were successful. IsPETase K95F and
BhrPETase M57L failed in sequencing.

### Enzyme Efficiency assay in *E.coli*

![](https://static.igem.wiki/teams/5236/yh/ispet-relative-activity.jpg)

Figure 6. The graph depicts the enzymatic activity of all the mutation of IsPETase,(including the ones
we generated using AI and those found in the iGEM distribution kit) compared to that of WT IsPETase. The
results demonstrate that our mutated variant S12E/R224Q/N233K has a higher enzymatic activity than the
highest found in the iGEM distribution kit (BBa\_K2910000).

![](https://static.igem.wiki/teams/5236/yh/bhrpet-relative-activity.jpg)

Figure 7. The image shows the enzyme activities of BhrPETase WT and IsPETase WT compared to the
mutations N191S, M57L, W229F, N205G. The mutation N205G has a slight higher enzyme activity than BhrPETase WT
at 30 min, and has certain potential to significance surpass the efficiency of BhrPETase if given more time, but the
enzymatic activities of W229F, M57L, N191S are less than IsPETase WT which indicates they have a lower
efficiency.

## **Signal Peptide**

We used ultrasonic to lyse the *E. coli* cells to release the PETase so that they can decompose plastic found
outside of the cell. However, not only does the chassis die, the enzymes also lose their bioactivity when
released this way. Hence, signal peptides are needed to allow the enzyme exit the double *E. coli’s* double
membrane without disturbing the cell. We inserted the signal peptide pelB sequence before the enzyme sequence.
The newly constructed plasmids were verified through sequencing.

![](https://static.igem.wiki/teams/5236/result-4/m154l-sequence-cycle-3.png)
![](https://static.igem.wiki/teams/5236/result-4/m10l-sequence-cycle-3.png)
![](https://static.igem.wiki/teams/5236/result-4/w159hf229y-sequence-cycle-3.png)
![](https://static.igem.wiki/teams/5236/result/h104w-sequence-cycle3.png)
![](https://static.igem.wiki/teams/5236/result-4/s121e-r224q-n233k-sequence-cycle3.png)
![](https://static.igem.wiki/teams/5236/result-4/k95f-sequnce-cycle-3.png)

Figure 8. The image shows the successful sequencing of IsPETase S12E/R224Q/N233K and BhrPETase N205G with pelB
signal peptide.

## PETase expression assay in *E.coli*

To verify IsPETase S12E/R224Q/N233K and BhrPETase N205G expression in *E.coli*, we performed SDS-PAGE to
check the protein expression of wild type IsPETase (~30 kDa) after inducing with IPTG. (see protocol)

![](https://static.igem.wiki/teams/5236/result-4/3.png)

Figure 9. the image shows the results from SDS-PAGE. Cell Culture: represents the protein obtained from
cells. Cell Culture Medium: represents the proteins obtained from the supernatent after centrifuge.

The results demonstrate the successful expression of our enzymes in E.coli and its secretion into the
extracellular matrix.

## Scanning Electron Microscope

We used a Scanning electron microscope to observe whether the plastic was degraded by PETase (IsPETase
S12E/R224Q/N233K and BhrPETase N205G).

![](https://static.igem.wiki/teams/5236/result-3/wechate7df91a80ee1763ddede1660fdfea8f1.jpg)
![]( https://static.igem.wiki/teams/5236/result-3/1.png)
![](https://static.igem.wiki/teams/5236/result-3/wechat18b6f655be723cecd8f5bfa50b2d53cc.jpg)

Figure 10. SEM photograph of surface changes of PET microplastics. The results show that the engineered
bacteria
can degrade the neck of plastic bottles with low crystallinity, but cannot degrade plastic films with a
possible
high level of crystallinity.
(a) Neck of plastic bottles (crystallinity≈1.2%) treated with *E.coli* harbored
with
BhrPETase-N205G for 12 days, control is treated with E.coli pET28a,
(b) neck of plastic
bottles(crystallinity≈1.2%) treated with E.coli harbored with IsPETase-S121E/R224Q/N233K for 12 days,
control is
treated with E.coli pET28a,
 (c)Plastic film (crystallinity unknown) treated with IsPETase.

## **Construction and verification in cyanobacteria**

### Natural transformation of *Synechococcus elongatus* PCC 7942

For expression of IsPETase S12E/R224Q/N233K and BhrPETase N205G in cyanobacteria, we reconstructed plasmids
using a transfer vector. The plasmids were transformed into *Synechococcus elongatus* PCC 7942. Successful
transformations were selected using the antibiotic spectinomycin.

![](https://static.igem.wiki/teams/5236/result/img-3208.jpeg)

Figure 11. Petri dishes containing transformed cyanobacteria

![](https://static.igem.wiki/teams/5236/result/img-3211.jpeg)
![](https://static.igem.wiki/teams/5236/result/img-3212.jpeg)

Figure 12. The two petri dishes are transformed IsPETase –S121E/R224Q/N233K and BhrPETase-N205G. 3 single
colonies were identified for each.

### Enzyme efficiency assay

We applied the *p*-Nitrophenyl Butryte degradation assay to test enzyme efficiency of those expressed in
cyanobacteria. (see protocol)

![](https://static.igem.wiki/teams/5236/result-4/result-of-cyan.webp)

Figure 13. This graph depicts the enzyme efficiency of those expressed in cyanobacteria. Although the
overall enzyme activities are low, BhrPETase-N205G’s activity is relatively higher than the other
sample.

We think these results are possibly due to (1) signal peptides do not perform well in cyanobacteria and (2)
cyanobacteria has a relatively slower growth than *E.coli*; therefore, we need to grant it more time to grow.

### Analysis of Degradation Product

HPLC is a method to directly detect the presence of specific compound in a chemical mixture. During the
degradation
of PET by PETase, TPA is created as a byproduct; hence, the presence of TPA in the final product indicates
degradation occurred. (see protocol)

![](https://static.igem.wiki/teams/5236/result/chromatography-cycle-4.png)
![](https://static.igem.wiki/teams/5236/result/img-3209.jpeg)

Figure 14. The results show that TPA is present after a 2 week incubation of PET with the engineered
bacteria.

## **Co-culture System**

### *Rhodococcus jostii* and *cyanobacteria*co-culture

PETase is capable of converting PET into TPA; however, TPA can still potentially harm the environment.
Therefore, to further break down the monomers produced by PETase, we co-cultivate *Rhodococcus jostii* and
cyanobacteria. *Rhodococcus* breaks dow TPA into H2O and CO2, which do not cause harm to the environment.

### Growth Medium Optimization

Cyanobacteria has a slower growth rate compared to that of *Rhodococcus*, which is relatively fast. Therefore,
the ratio of the cyanobacteria growth medium, BG11, and that of *Rhodococcus*, R2A, need to be optimized in
order to achieve a balanced growth of both organisms. Growth of the organisms was observed in growth mediums
with a 1:3, 1:2, and 1:1 BG11 to R2A ratio. Through observing growth in both liquid and solid media, we
determine that the cyanobacteria and rhodobacteria co-culture achieves optimal growth in a 1: 3 ratio growth
medium.

![](https://static.igem.wiki/teams/5236/result-4/20240929183705.jpg)

Figure 15. The image shows the solutions of cyanobacteria and medium which different ratio, 1: 3, 1:2,
1:1.

![](https://static.igem.wiki/teams/5236/result/img-3210.jpeg)

Figure 16. The image shows the growth of cyanobacteria and *Rhodococcus* on cultural medium plate,
ratio 1: 3.

{% endblock %}