iGEM 2024 — Team hust-ueve-upsaclay

# Notebook

The Records of Our Experiment Process

➤

* [Notebook summary: *Escherichia coli (E. coli)* part](#topic)
* [Notebook summary: *Caenorhabditis elegans (C. elegans)* part](#background)

## Notebook summary: *Escherichia coli (E. coli)* part

### Daily Routine

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/notebook1-1.png)

### The scanned photos of our notebook of *E. coli* are listed below:

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/syn1.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/syn2.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/syn3.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/syn4.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/syn5.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/syn6.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/syn7.jpg)

## Notebook summary: *Caenorhabditis elegans (C. elegans)* part

### Daily Routine

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/nb2.png)

### The scanned photos of our notebook of *C. elegans* are listed below:

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/1.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/2.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/3.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/4.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/5.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/6.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/7.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/8.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/9.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/10.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/11.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/12.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/13.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/14.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/15.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/16.jpg)

![Expert Interview](https://static.igem.wiki/teams/5233/notebook/17.jpg)
![Expert Interview](https://static.igem.wiki/teams/5233/notebook/18.jpg)

# Engineering

Progress and improvement of our project

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* [Engineering on *E. coli*](#Introduction)
* [Engineering on *C. elegans*](#EngineeringOnCelegans)

## Engineering on *Escherichia coli (E. coli)*

### Introduction

We hope to construct two kinds of modified *E. coli* BL21 strains, enzyme secretor and TPA collector. Enzyme secretor bacteria express and secrete working enzymes that can efficiently degrade PET to TPA and EG for the degradation of polyethylene terephthalate (PET) in domestic waste water. Another modified *E. coli* expressing TPA transporter and TPA specific promoter induced fluorescent protein eGFP to achieve the preliminary enrichment and detection of the degradation product TPA in *E. coli*.

### Period 1: Intracellular expression of turboPETase

1.1 Objective: The turboPETase sequence was integrated into the pBAD plasmid vector, which could be precisely induced by arabinose, and 6xHis-tag was added to purify and detect the expressed turboPETase in subsequent experiments.

![Expert Interview](https://static.igem.wiki/teams/5233/engineering/dshc-1.png)

Fig. 1. pBAD- ParaBAD - TurboPETase - 6His

1.2 Practice: The plasmid was transformed into *E. coli* BL21 strain, and turboPETase was expressed in large amount under arabinose induction. The purified enzyme was isolated and purified to explore the activity and degradation ability of the enzyme under various conditions, in order to find the most suitable reaction conditions, and evaluate the degradation efficiency under natural conditions.

1.3 Conclusion: A series of enzyme activity tests were carried out using purified turboPETase. Our experimental results showed that the most suitable condition for the enzyme was 70 degrees Celsius and pH=8. There was no significant difference in the degradation or accumulation of substrate BHET and intermediate MHET at different temperatures, while the degradation or accumulation of product TPA was significantly different with different temperatures.

(1) Although the engineered strain expressed a large number of functional enzymes normally, the procedures of bacteria lysis, cytoplasm separation and purification were very complicated, which were not convenient to provide functional enzymes to environmental samples at a lower cost. Therefore, we hope to secrete turboPETase by adding signal peptides.

(2) By analyzing the changes of each component, it can be inferred that although the existing turboPETase has a strong degradation effect on the substrate, a large amount of intermediate product MHET has been accumulated in the reaction, which is not conducive to the reaction. We hope to add MHETase to degradate MHET and facilitate the degradation.

### Period 2: Construction of the PETase secretion plasmid

1.1 Objective: To construct a plasmid to achieve the secretion of our enzymes and to explore the signal peptide element with the best secretion effect on turboPETase, pET28a plasmid was used as the backbone of PV, and the T7 promoter with efficient expression ability was used to expand the expression efficiency of enzymes.

1.2 Design plasmid structure: According to literature review, we use four sec-denpending signal peptides (LamB, PelB, OmpA, MalE) for secretion, and add an enhancer element B1 (MERACVAV) that can greatly enhance the function of the signal peptide to improve the secretion efficiency.

According to the above, the structure of T7 promoter-B1 Enhancer-signal peptide-PETase plasmid was used.

![Expert Interview](https://static.igem.wiki/teams/5233/engineering/dshc-2.png)

Fig. 2. pET28a - PT7 - Lac operator - RBS- Enhancer- SP - TurboPETase

1.3 Practice: We used homologous recombination method to construct turboPETase secretory functional plasmids mentioned above. In the process of designing primers according to different signal peptides, we hope to achieve simple and low-cost access to other fixed components for four or more signal peptides, so we use the reusable "Plug" concept -- connecting homology arms in the design.

1.3.1 Enhancer elements (24bp) were introduced into the 3 'end of the PV backbone by PCR amplification primer design. The 5 '-end sequence (20bp) of the PV backbone was introduced into the 3' -end primer of turboPETase as a homology arm, and the pET28a PV backbone was preliminarily connected by the above design. The "-(Plug-5 ') Turbopetase-PV-enhancer (Plug-3 ')-" linear linker was formed.
20bp at the Enhancer3 'end was selected as Plug-3', and 20bp at the turboPETase 5'end was selected as Plug-5'. Through the design of primers, the interface sequences Plug-3 'and Plug-5' were introduced at both ends of the four signal peptide sequences to realize the homologous connection of different signal peptides and the linear linker of the previous step to form a circular plasmid.

![Expert Interview](https://static.igem.wiki/teams/5233/engineering/dshc-3.png)

Fig. 3. Homologous recombination of alternative elements

1.3.2 Due to the short fragments of the four signal peptides, in order to achieve the lowest cost and save the time of component synthesis, we can obtain short fragment elements by PCR by designing specific primers with length of 59bp or less.

Taking OmpA as an example, the forward and reverse primers Ompa-F and Ompa-R could bind each other, and the OmpA sequence with a length of 63bp was obtained by PCR, and the Plug-3 '(20bp) and Plug-5' (20bp) were introduced at both ends.

OmpA-F：CGCGCGTGCGTGGCGGTGATGAAAAAGACCGCAATTGCCATTGCAGTTGCACTGGCAGG
OmpA-R：CGCTGATACGGATTGCTTGCCTGTGCAACGGTTGCAAAACCTGCCAGTGCAACTGCAAT

1.4 Conclusion:

(1) Among the four signal peptides, OmpA and PelB were more effective than the other two, so we decided to use OmpA signal peptide as the signal peptide for subsequent turboPETase secretion.

(2) Although the homologous recombination method is a common method in laboratories, a standardized homology arm design was used in construction process, which simplified the homology arm design to a certain extent. A standardized homology-arm design is particularly well suited to large numbers of repeated attempts at a replaceable element (e.g., large-scale exploration of different signal peptides or large-scale validation of mutation-inducing genetic material).

### Period 3: Construction of the MHETase secretion plasmid

1.1 Objective: To construct a plasmid to realize the co-secretion function of turboPETase and MHETase, and to use pET28a plasmid as PV backbone.

1.2.1 Practice: The normal linked OmpA-turboPETase and PelB-turboPETase secretion plasmids were used as the new plasmid backbone, and the designed primers were used to linearly PCR the plasmid. The designed primers were used to introduce a homology arm with the PV backbone at the 3 'end of the synthetic sequence MHETase, and a homology arm with the 3' end of the synthetic sequence Amcut-RBS or PelB-RBS at the 5 'end of the synthetic sequence MHETase. Design primers were used to introduce the homology arm of the PV skeleton at the 5 'end of the Amcut-RBS or PelB-RBS element.

1.2.2 Conclusion: During the construction and sequencing, a large number of single colony sequencing results showed that the construction failed and the MHETase did not connect the plasmid. We suspected that the new sequence was not introduced on the PV backbone during the first primer design, which led to the linear plasmid re-connecting itself to restore the loop.

![Expert Interview](https://static.igem.wiki/teams/5233/engineering/dshc-4.png)

Fig. 4. Sequencing of the transformed plasmid

1.3.1 Second practice: We tried to redesign the primers to introduce heterologous sequences on the PV backbone to prevent its own ligation, designed the homology arm at the PV backbone (10bp), and the 3 'end of MHETase (10bp) or the 5' end of Signal Peptide-RBS (10bp), and once again constructed the plasmid by homologous recombination.

![Expert Interview](https://static.igem.wiki/teams/5233/engineering/dshc-5.png)

Fig. 5. Sequencing of the transformed plasmid (new primers)

1.3.2 Conclusion: Sequencing still showed that MHETase was not connected, and even PETase shed the PV skeleton. We speculated that an RBS sequence was added to the 5'end of MHETase, which was the same as the 5' end RBS sequence of turboPETase on the original turboPetase-PV skeleton. Therefore, the homologous fragments within each fragment were duplicated, resulting in high failure rate at the homologous recombination stage. It is also possible that a new fragment is inserted on the basis of the original plasmid, and at this time, it is impossible to determine whether the new recombinant fragment forms a circular plasmid, so the antibiotic selection is used to exclude the failed recombination results, resulting in a high sequencing failure rate.

1.3.3 Further optimization or adjustment: RBS with different sequences can be assigned to the front end of the two working enzymes, or different restriction sites can be added to each RBS to destroy the homology between the two RBS sequences. Or we can use enzyme digestion and enzyme-linked method, insertinf the SignalPeptide2-MHETase fragment into the SignalPeptide1-PETase-PV. However, due to the limitation of time and effort, this kind of optimization has not been implemented.

1.4 Iteration: Instead of constructing new plasmid, we try to combine two plasmid into our bacterial. We constructed a pET21a plasmid backbone (resistance AmpR) with SignalPeptide2-MHETase, and co-double-transformed into *E. coli* BL21 strain with the original turboPETase secretion plasmid (resistance KanR). And we maintain two plasmids functioning through two different antibiotic pressure.

1.4.1 Practice: Through the design of PCR primers on the backbone, we introduced the homology arm (20bp) of the 3 'end sequence of PV backbone and the homology arm (20bp) of the 5' end of MHETase to the 5 'end of the Enhancer-SignalPeptide element. The 5 '-end sequence (20bp) of the PV backbone was introduced into the 3' -end primer of MHETase as a homology arm, and the target circular plasmid was formed by homologous recombination ligation of the above elements.

![Expert Interview](https://static.igem.wiki/teams/5233/engineering/dshc-6.png)

Fig. 6. pET21a - PT7 - Enhancer- SP -MHETase

## Engineering on *Caenorhabditis elegans (C. elegans)*

### Introduction:

We hope to construct a kind of engineered *C. elegans* which could express TPA transport system on its intestinal cell membrane so that it could enrich TPA after ingesting engineered *E. coli*, achieving the effect of TPA's secondary enrichment.

### Period 1: Construction of TPA transporter plasmid

1. We designed a plasmid that expresses the TPA transport system within the intestinal tract of C.elegans. The basic components(TpiA and TpiB and TphC) are the same as the design of *E. coli* .However, since the expression system is quite different between C.elegans and , the signal peptides are also different. Here we use gbb-1 signal peptide to express TpiA, glr-2 signal peptide to express TpiB and dsl-1 signal peptide to express TphC, the standard of signal peptide selection is based on the number of transmembrane times of the protein structure. Besides, in order to avoid the misfolding of the functional protein, SL2 linker is used between different functional protein sequences. The detailed design of our plasmid is shown as follow.

2. Utilizing homologous recombination techniques, we engineered homologous arms on the primers and employed the highly efficient C116 homologous recombination enzyme to simultaneously recombine four fragments.

3. The vector was linearized, and PCR was performed to obtain the insertion fragments with homologous arms. After gel verification, we conducted gel recovery, followed by homologous recombination and transformation of competent DH5α cells. The resulting colonies were plated, picked, and cultured before plasmid extraction. PCR validation confirmed the presence of the desired bands, which were subsequently sent for sequencing.

![Expert Interview](https://static.igem.wiki/teams/5233/design/celegans-design.png)

Fig. 7. Design for TPA transporter in *C. elegans*

### Period 2: Improvement of TPA transporter plasmid

Upon comparing the company's sequencing results, we discovered a deletion in the tpiA gene. Upon investigation, we found that the linker sequence we used for ligation was SL2, which was employed twice. This resulted in the TPIA sequence being excluded from integration into the plasmid due to homologous recombination occurring between the two SL2 sequences. The issue was resolved by relocating the TPIA gene to the front of tpiB for homologous recombination.

Ultimately, we successfully obtained a complete plasmid shown in figure 8, containing the transport system.

![Expert Interview](https://static.igem.wiki/teams/5233/engineering/c-elegans-plasmid-new.jpg)

Fig. 8. The plasmid we designed for the transportation of TPA. vha-6 is an intestine-specific promoter. TpiA, TpiB are transporter proteins that will be localized on the membrane of the intestine cell by using gbb-1 and glr-2 signal peptides, respectively. Tphc is the TPA binding protein that will be secreted outside the intestine cell by using signal peptide dsl-1. mCherry is the reporter gene used for the detection of expression.

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

# Results

What we have achieved

➤

* [Introduction](#introduction)
* [Plasmid construction](#part1)
* [Degradation ability of PETase in gradient temperature and pH](#part2)
* [Validation of Signal Peptide Function](#part3)
* [Sequence comparison of TTT proteins from E. coli and Comamonas sp. E6](#part4)
* [Validation of Biosensor and Transporter plasmid](#part5)
* [Validation of Signal Peptide-MHETase Function](#part6)
* [Nano PET and TPA toxicity detection on nematode growth medium (NGM)](#part7)
* [Nano PET and TPA toxicity detection on liquid](#part8)
* [Result and discussion for fluorescence detection](#part9)
* [Synthetic biology modification of C. elegans](#part10)

## Results of synthetic biology experiment on ***Escherichia coli (E. coli)***

### Introduction：

We aim to construct an bioremediation system that utilizes **E. coli** to secrete PETase and MHETase
for microplastic PET degradation and to perform primary enrichment of microplastic degradation product TPA,
followed by secondary enrichment by *Caenorhabditis elegans (C. elegans)*, while relying on *C.
elegans* to evaluate the physiological toxicity of each component of the degradation process. For this
purpose, we designed five plasmids, pSec-PETase, pSec-MHETase, pTransporter, and pBiosensor, which were
transformed into **E. coli**, and pTransporter-elegans, which was transformed into *C.
elegans*.

We transformed these plasmids into **E. coli** and *C. elegans* for expression of our target
proteins. Then we will verify that our proteins are properly expressed and perform their specific functions
through a series of experiments.

### Part 1：Plasmid construction

The plasmid containing the target gene was synthesized by company, and primers with homology arms were designed
to amplify the target gene from the corresponding plasmid by PCR. Next, we combined the parts and linear plasmid
vectors through homologous recombination to form a circular plasmid, and then the constructed plasmid was
transformed into *E. coli* Trans-T1 strain. The transformed bacterial were selected by antibiotic and we
designed sequencing primers for sequencing the plasmids to confirm that we obtained expected plasmids with
correct sequences, and the successfully constructed plasmids will be used for subsequent characterization
experiments.

![Expert Interview](https://static.igem.wiki/teams/5233/results/rehc-1.png)

#### 1.1 Construction of pSec-PETase and pSec-MHETase

We constructed plasmids that can secrete PETase and MHETase into the extracellular space of **E.
coli** and verified that the plasmids were successfully constructed and transformed .
pSec-PETase contains TurboPETase, signaling peptides (OmpA, PleB, MalE, LamB) that allow TurboPETase to be
secreted extracellularly, and an enhancer that enhances the effect of the signaling peptides. pSec-MHETase
contains MHETase and signaling peptides that allow MHETase to be secreted extracellularly (PelB, Amcut).

![figure 1.1 ](https://static.igem.wiki/teams/5233/result-synthetic/result-fig1-1.png)

Figure 1.1 a. the agarose gel electrophoresis of signal peptide(SP) fragments M-DL500 marker;
1-LamB; 2-PelB; 3-MalE; 4-OmpA
b. the agarose gel electrophoresis of vector and TurboPETase fragments M-DL5000 marker; 1,2-pET-28a vector;
3,4-TurboPETase

Analysis:The PCR amplification bands of LamB (approximately 110bp), PelB (approximately 100bp),
MalE (approximately 110bp), OmpA (approximately 100bp), pET-28a vector (approximately 5000bp), and TurboPETase
(approximately 750bp) align with the theoretical lengths estimated based on the designed primer positions,
demonstrating that these target genes have been successfully obtained.

![figure 1.2](https://static.igem.wiki/teams/5233/result-synthetic/result-fig1-2.png)

Figure 1.2 a. the agarose gel electrophoresis of signal peptide(SP) and MHETase fragments M-DL500
marker; 1-MHETase; 2-Amcut; 3-PelB b. the agarose gel electrophoresis of vector fragments 1-pET-21a vector;
M-DL5000 marker

Analysis:The PCR amplification bands of MHETase (approximately 500bp), Amcut (approximately
150bp), PelB (approximately 100bp), pET-21a vector (approximately 5000bp) align with the theoretical lengths
estimated based on the designed primer positions, demonstrating that these target genes have been successfully
obtained.

![figure1-3](https://static.igem.wiki/teams/5233/results/result-fig1-3.png)

Figure 1.3 Plates of the different transformed pSec-PETase *E. coli* and pSec-MHETase *E. coli*
a.pSec-PETase with MalE b.pSec-PETase with OmpA c.pSec-MHETase with Amcut d.pSec-PETase with PelB
e.pSec-PETase with LamB f.pSec-MHETase with PelB

Analysis: From the result of plate coating of LB solid medium with antibiotic Amp/Kan, the medium
successfully grew dispersed single colonies, which indicated that the bacterium was successfully screened and
transformed.

![figure1-4](https://static.igem.wiki/teams/5233/result-synthetic/result-fig1-4.png)

Figure 1.4. Sequencing figure of one of the pSec-PETase(The others are also constructed
successfully) a. part of chromatogram b. Elements horizontal sequencing maps comparison for pSec-PETase

Analysis:The sequencing result can show that the target element and the plasmid vector were
correctly connected; there were no heterogeneous peaks in the sequencing result (sequencing sequences were more
accurate in the range of 200-800bp), and the sequence of the actual plasmid is identical with the designed
plasmid.

#### 1.2 Construction of pTransporter coli and pBiosensor

We constructed two plasmids pTransporter and pBiosensor, then we transformed them into one *E. coli* BL21 strain to
apply the detection function. pTransporter is responsible for transporting TPA into intercellular space, and pBiosensor
detects the TPA, indicating the TPA concerntration in the environment by fluorescing.
pTransporter contains TpiA and TpiB, which encode two TPA transporter proteins, as well as TphC, which is a
binding protein that facilitates the transmembrane transport of TPA. pBiosensor contains PtphC, an inducible
promoter that responds to TPA. TphR can encode a regulatory protein of the PtphC promoter, and eGFP for indirect
characterization of the intracellular concentration of TPA.

![figure1-5](https://static.igem.wiki/teams/5233/result-synthetic/result-fig1-5.png)

Figure 1.5 a. the agarose gel electrophoresis of biosensor elements fragments M-DL500 marker;
1-TphR; 2-PtphR; 3-eGFP; 4-terminator

 b. the agarose gel electrophoresis of transporter elements fragments M-DL2000 marker; 1-TpiA; 2-TpiB;
3-TphC

 c.the agarose gel electrophoresis of pET-21a/28a vector fragments M- DL5000 marker; 1-pET21a vector;
2,3-pET28a vector

Analysis: The PCR amplification bands of TphR (approximately 500bp), PtphR (approximately 100bp),
eGFP (approximately 500bp), terminator (approximately 100bp), TpiA (approximately 2000bp), TpiB (approximately
500bp), TphC (approximately 750bp), pET21a vector (approximately 5000bp), pET28a vector (approximately 5000bp)
align with the theoretical lengths estimated based on the designed primer positions, demonstrating that these
target genes have been successfully obtained. However, the plasmid vector pET21a/28a are amplificated by PCR.
The bands at the length we designed were bright and clear, so we think the fragments there is our target gene elements.
PtphC and Terminator fragments both have two bright lines, but at the designed
position it's bright and clear, so we think the fragments here is our target elements.

![figure1-6](https://static.igem.wiki/teams/5233/results/result-fig1-6.png)

Figure 1.6 Plates of the different transformed pBiosensor(c) and pTransporter *E. coli*(b), and dual
plasmid transformed strains of pBiosensor and pTransporter(a)

Analysis: From the result of plate coating of LB solid medium with antibiotic Amp/Kan, the medium
successfully grew dispersed single colonies, which indicated that the bacterium was successfully screened and
transformed.

![figure1-7](https://static.igem.wiki/teams/5233/results/result-fig1-7.png)

Figure 1.7 Elements horizontal sequencing maps comparison for pBiosensor and pTransporter. a.
pBiosensor comparison b. pTransporter comparison

Analysis: The sequencing result show the target element and the plasmid vector of pTransporter
were correctly connected. The sequence of the actual plasmid is identical with the designed plasmid.
But for the pBiosensor sequencing, it failed or appeared bad signal for first several times. We discussed
the reason for the failure sequencing and reached the reason that copy numbers of plasmid vector pET28a are
low for detection. And it might also undermine the biosensor function. So we later redesigned a pBiosensor
using pUC57 plasmid vector.

### Part 2: Degradation ability of PETase in gradient temperature and pH

In the treatment of domestic wastewater, we consider that we may have to deal with different physical and
chemical environments of the treatment object, such as changes in pH in different water samples and seasonally
influenced changes in ambient temperature, leading to changes in treatment efficiency. Therefore, we wished to
explore the optimal pH and optimal temperature of our working enzyme, TurboPETase (hereafter all PETases are
referred to as TurboPETase unless otherwise specified), and to quantify the changes in degradation capacity
beyond the optimal pH and temperature.

We transformed the pBAD-PETase-6His plasmid into **E. coli** BL21 strain, for expressing the working
enzyme by large-scale fermentation. We use high pressure homogenizer to break bacteria as unpurified PETase
samples. And next we purified and concentrated to obtain PETase purified samples.

#### 2.1 Protein purification and SDS-PAGE gel verification:

![Expert Interview](https://static.igem.wiki/teams/5233/results/f2-1.png)

Figure2.1 15% SDS-PAGE gels stained with Coomassie brilliant blue

PETase（#1-#4）refers to each 4mL of PETase samples per tube connected in chronological order during
protein purification by affinity chromatography. Wash Sample is a solution of miscellaneous proteins that
are eluted.

Analysis: #1- 4 PETase showed obvious bands near 25kDa, which is consistent with the theoretical value of
PETase, but the width of the bands indicates that the protein concentration is not high, i.e., E. coli expresses
PETase less efficiently; and the number of stray bands is less indicating that the protein is more pure. While
the eluate showed obvious band missing near 25kDa, which indicated that PETase was purified and the affinity
chromatography method was effective.

#### 2.2 Degradation ability of gradient pH enzymatic samples

(1) The color reaction was performed using p-nitrophenol as a substrate

![Expert Interview](https://static.igem.wiki/teams/5233/results/f2-2-1.png)

Figure2.2.1 Absorbance of PETase at 410nm after purification at different pH

Analysis: The optimum pH of PETase as a lipase is about 5, but the activity of PETase as a lipase is not
necessarily equal to its ability to degrade plastics, and many experiments have shown that this method as an
indicator of the activity of PETase has a large error and the results are not accurate, so we chose to use the
method of high-performance liquid chromatography (HPLC) with Bis (2-hydroxyethyl terephthalate) (BHET) as the
substrate for the subsequent experiments.

(2) Detection of BHET degradation ability in gradient pH
BHET substrate solution of 0.4 g/10 mL was configured, and 0.9 mL of BHET substrate solution plus 0.1 mL of
unpurified/purified enzyme reaction system was configured, and the reaction was carried out at 65°C for 60 min
with a gradient pH setting. After the reaction was completed, the reaction was terminated by the addition of 1
mL of methanol solution, and then the reaction was allowed to stand at 4°C for one night, and the supernatant
was centrifuged for high-performance liquid chromatography (HPLC) to analyze the difference in peak areas of
BHET characteristic peaks.

![Expert Interview](https://static.igem.wiki/teams/5233/results/f2-2-2.png)

Figure2.2.2 Percentage of degradation capacity of unpurified PETase at different pH

![Expert Interview](https://static.igem.wiki/teams/5233/results/f2-2-3.png)

Figure2.2.3 Percentage of degradation capacity of purified PETase at different pH

Analysis: The optimum pH for BHET degradation by PETase was measured twice using unpurified and purified enzyme samples separately.
In the test with unpurified samples, the enzyme exhibited the highest degradation activity at pH 8 but showed no activity at pH 10.
In the test with purified samples, the enzyme displayed optimal degradation activity across the pH range of 8 to 10, with the highest activity observed at pH 10.
Comparing the two results, we assumed that the purified sample results were more accurate due to fewer interfering factors. We hypothesized that an alkaline protease in the unpurified sample might have damaged the PETase. Upon reviewing the original data, it was observed that the lack of activity at pH 10 in the unpurified sample test was caused by high levels of natural degradation in the control group. In contrast, the control group in the purified sample test was consistent with previous results, except for the enzymatic reaction data. This suggests that experimental error occurred in the unpurified sample control group.

Conclusion: We considered the purified sample test results as more reliable and used them in the final analysis. And we apply pH=8 as our experiment condition because the good degradation and more neutral pH.

#### 2.3 For gradient temperature enzyme sample degradation BHET ability detection

Configure 0.9 mL of BHET substrate solution and 0.1 mL of purified enzyme reaction system, and react for 30 min
at a gradient temperature setting of pH=8. At the end of the reaction, terminate the reaction by adding 1 mL of
methanol solution, let it stand overnight at 4℃, and then centrifuge the supernatant for HPLC liquid phase
analysis.

##### 2.3.1 Construct BHET and TPA standard curves in HPLC:

In the experiments to analyze the effect of gradient pH on enzyme degradation ability, we only considered the
difference in the peak area of the substrate BHET to characterize the enzyme degradation ability, but this led
to a potentially incomplete analysis and excessive bias. Therefore, we constructed standard curves for BHET and
TPA to characterize the enzyme degradation capacity by the difference in the consumption of substrate BHET and
the production of product TPA.

#### Gradient concentrations of BHET and TPA standards were configured, and HPLC liquid phase analysis was performed to obtain standard curves of concentrations and characteristic peak areas.

![Expert Interview](https://static.igem.wiki/teams/5233/results/f2-3-1-1.png)
![Expert Interview](https://static.igem.wiki/teams/5233/results/f2-3-1.png)

Figure2.3.1 Standard curves for BHET and TPA under HPLC analysis A,B. TPA and BHET standard
chromatogram peak C,D. BHET and TPA standard curve

##### 2.3.2 Protein concentration determination of two batches of purified PETase samples

Because we purified two batches of purified enzyme samples, considering the difference in enzyme amount between
the two samples led to the property test affected by this factor, we need to carry out the purified enzyme
protein concentration measurement, and can therefore calibrate the enzyme activity.

A gradient concentration PBS protein standard was configured, two batches of purified protein samples were
sampled, and the absorbance of A595 was measured using an enzyme marker. Sample protein concentrations were
calculated from the standard curve using the Calculation Tool.

Standard fit curve: polynomial function fit curve y = 0.264x4 - 0.9697x3 + 0.9756x2 + 0.3684x + 0.5246 R1² =
0.9998; linear fit curve y = 0.5717x + 0.5407 R2² = 0.9799; since R1 > R2, a polynomial function fit was chosen
to calculate the first batch enzyme protein concentration : 0.379 mg/ml and the second batch of enzyme protein
concentration: 0.282 mg/ml.

##### 2.3.3 HPLC analysis of degradation capacity by temperature gradient

The concentration of each component was analyzed by standard curves to calculate the consumption of BHET and
the production of TPA and MHET at different temperatures.

![Expert Interview](https://static.igem.wiki/teams/5233/results/f2-3-2.png)
![Expert Interview](https://static.igem.wiki/teams/5233/results/f2-3-2-1.png)

Figure2.3.2 Degradation ability of PETase in temperature gradient A. Chromatogram comparation of
standard sample and reaction sample B.BHET consumption and TPA production (concentration difference) C. MHET
production (peak area difference)

Analysis: The amount of BHET degradation (concentration of the blank group - concentration of the experimental
group) increased and then decreased with increasing temperature, but considering that the amount of naturally
degraded BHET increased with increasing temperature in the blank group, which in turn affected the analysis of
the enzyme degradation part. To further optimization of the experiment, we should shorten the reaction time to
avoid the substrate being completely consumed; The result showed the tendency that, the amount of TPA net
production(experiment-blank group) increases with the increase of temperture, which can be a stronger indication
of the increased substrate generation capacity in this gradient temperature probe; the intermediate product MHET
was not calibrated with a standard curve, and the difference in
its peak area (concentration of experimental group - concentration of blank group) decreased with increasing
temperature by a relatively small amount, therefore, we suspect that most of the substrate BHET is degraded only
to the intermediate product MHET, rather than to TPA. Next we could work on the optimization of the degradation
efficiency of MHET, which can further increase the working enzyme's degradation ability for PET.

### Part 3: Validation of Signal Peptide Function

#### 3.1 Reaction enzyme sample preparation:

The Enhancer-SP-PETase plasmid ( SP: OmpA, PelB, LamB, MalE ) was constructed and transferred into BL21
strain, while the original pBAD-PETase-6His was transferred as a blank strain. The working enzyme was induced to
be expressed and secreted.

The bacterial fluid was centrifuged and the supernatant was concentrated and fixed to 4 mL to be prepared as
supernatant enzyme sample. 4 mL of pH=8 Tris-HCl Buffer was added to the precipitate and sonicated to break the
bacteria, and prepared as a sample of the intracellular enzyme.

#### 3.2 Reaction system configuration：

The reaction system was configured with 0.8 mL of BHET substrate solution + 0.1 mL of PH=8 Tris-HCl Buffer +
0.1 mL of reaction enzyme samples, and the reaction was carried out for 30 min at a gradient temperature setting
of pH=8. At the end of the reaction, the reaction was terminated by the addition of 1 mL of methanol solution,
and then the reaction was allowed to stand at 4°C overnight, and the supernatant was centrifuged and analyzed by
UPLC (Ultra- Performance Liquid Chromatography (UPLC).

#### 3.3 UPLC Standard Curve re-builted

The analytical speed and resolution of the UPLC instrument, as well as the sensitivity, were superior to that
of HPLC, and we attempted to use UPLC for the next analytical investigations. However, because of the change of
the liquid phase instrument and the column, we need to reconstruct the standard curve of the reaction
components.

![Expert Interview](https://static.igem.wiki/teams/5233/results/f3-1.png)
![Expert Interview](https://static.igem.wiki/teams/5233/results/f3-1-1.png)

Figure 3.1 Standard curves for BHET and TPA under UPLC analysis A,B. BHET and TPA standard
chromatogram peak C,D. BHET and TPA standard curve

![Expert Interview](https://static.igem.wiki/teams/5233/results/f3-2.png)

Figure 3.2 Analysis signal peptide secretion test reaction system with UPLC A.Chromatogram
comparation of standard sample and reaction sample(No SP, OmpA, PelB, MalE, LamB) B. SPs and Blank group
BHET consumption difference

Analysis: As the chromatograms of samples shown above, the peak of TPA and MHET is not effectively separated
in our UPLC method. And the fusion of chromatogram peak might lead to the deviation of integral calculation.
That's why we compare the consumption of BHET between SP groups for the evaluation of SP-enzyme function.

Conclude:
 Based on the data analysis, the best signaling peptide for TurboPETase is OmpA, while MalE
signaling peptide seems to inactivate it completely, we speculate that there may be a shifting mutation in the
plasmid containing MalE signaling peptide, or MalE signaling peptide itself leads to the inactivation of PETase.
Surprisingly, the three others signaling peptide acted more strongly than expected, and the PETase in the
extracellular fluid degraded LamB and OmpA much more than the intracellular one, which was not consistent with
our assumption that the activity of intracellular enzyme was much larger than that of extracellular enzyme. This
is not consistent with our assumption that the activity of intracellular enzymes is much greater than that of
extracellular enzymes. This may be due to several reasons: 1 the expression-secretion balance of intracellular
enzyme levels under prolonged induction, the accumulation of extracellular enzymes due to secretion, 2 the
reduction of enzyme activity by intracellular signal peptide sequences, and 3 the damage to enzymes during the
process of ultrasonication. However, in view of the fact that the stronger the total enzyme activity of the
extracellular fluid and the weaker the total enzyme activity of the broken fluid in the figure can be used as a
supporting evidence for secretion, we believe that the strength of the signal peptide is objectively reasonable,
and that a strong secretion ability of the signal peptide is more conducive to secretion applications in real
environments.

### Part 4: Sequence comparison of TTT proteins from *E. coli* and Comamonas sp. E6

We also found three proteins belonging to the tripartite tricarboxylate transporter (TTT) family in *E.
coli*. We compared the three proteins of *E. coli* with those of Comamonas sp. E6 to confirm whether the TTT
proteins in *E. coli* are similar to TpiA, TpiB, and TphC in structure and function, which will guide us to
partially modify transmembrane proteins for heterologous expression in the following experiments.

We used BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to perform the sequence comparison of the proteins, and
the results were as follows:

![table1](https://static.igem.wiki/teams/5233/results/result-table1.png)

Table 1. Table of protein sequences sources and percentage of identity

![figure4-1](https://static.igem.wiki/teams/5233/results/result-fig4-1.png)

Figure 4.1 Dotplot figure between proteins a.TctA of Escherichia coli vs. TctA of Comamonas sp. E6
b. TctC of Escherichia coli vs. TctC of Comamonas sp. E6

Analysis：From the results of blast, for the TTT of *E. coli* and Comamonas sp. E6, the
similarity of proteins from TctA family or TctC family was high, but there is no significant identity of
proteins from TctB family.This similarity in some degree provides a partial basis for the possibility of
heterologous expression of membrane proteins in Escherichia coli, and the low similarity of TctB provides
support for further optimization of heterologous expression of membrane proteins in the future.

### Part 5: Validation of Biosensor and Transporter plasmid

In environmental wastewater treatment, there are difficulties in the application of LC (Liquid Chromatography,
including HPLC and UPLC), which is currently the most commonly used means of detecting PET degradation, for
large-scale continuous monitoring. We designed and constructed a transporter plasmid that can transport the PET
degradation product TPA, and a detection plasmid with the TPA-specific promoter Ptpa. Transforming them into the
BL21 strain to verify the ability of the transporter as well as the ability to detect the fluorescence of TPA
expression in the samples.

Strains transformed with pBiosensor, pTransporter, and both plasmids were cultured for fluorescence detection at
12h, 18h, and 24h under TPA induction.

![figure5-1](https://static.igem.wiki/teams/5233/results/f4-1.png)

Figure 5.1 Fluorescence intensity per unit OD600 value for 12h,18h,24h

![Expert Interview](https://static.igem.wiki/teams/5233/results/f4-2.png)

Figure 5.2 Quantification graph of validation biosensor and transporter. n=3. Error bars represent
SD of mean. ns indicates no significant difference. \*p<0.05, \*\*p<0.01, \*\*\*p<0.001 (one-way ANOVA
test)

Analysis：Fluorescence intensity per unit OD600 value increases from 12h to 18h, but it decrease
from 18h-24h, we guess it’s because the nutrition of the LB medium went out, and the bacterial would consume the
heterologous protein eGFP in starvation, which leads the decreasing. And in the Figure 5.1, the
Biosensor+Transporter+ inducer TPA is slightly higher than the two others group. It might meets our first
expectation—*E. coli* BL21 strain don’t have the orginal ability to transport the TPA, and specific inducible
promoters PtphC reacts to the TPA transported inside bacterial. But the ANOVA analysis in Figure 5.2 tells us
the difference among the groups at 12h, 24h is not significant enough to support our expectation.

Conclude：
In fact, we repeated this experiment twice. In the first experiment, there were also some
differences among the three groups, but because there might be some problems in the operation during our first
experiment, which led to large differences among the data within the groups and the data were not credible, so
we repeated the fluorescence detection experiment once.

In the data of the second experiment, although the data still show the trends that there exists the difference
between three groups. However, due to the non-significant difference between the experimental groups and the
fact that there is still a certain error between the parallel groups (there is a non-normal data). So, we still
lack strong evidence of the actual effect of our components (or the effect of our components still needs to be
optimized).

When analyzing the reasons for the unsuccessful characterization experiments of pBiosensor and pTransporter, we
considered that Transporter is a heterologous Transporter element, so we predicted the protein structure of the
membrane transporter protein elements in Transporter, and two of the elements are heterologous multiple
transmembrane proteins (the number of transmembrane transitions for tpiA is 11, tpiB is 5), we think that the
multiple transmembrane crossing is likely to lead to unsuccessful construction or difficult to function properly
in our **E. coli** engineering bacteria. Due to the tight experimental time, we continued the
subsequent experiments after the initial characterization of pBiosensor and pTransporter. Afterwards, we will
continue to explore the function of this transmembrane protein and enhance its transporter ability to achieve
and optimize the enrichment of TPA in our engineered bacteria **E. coli**.

### Part 6: Validation of Signal Peptide-MHETase Function

In order to optimize the MHET accumulation problem analyzed in Part2 section, we tried to construct
pET28a-SP-PETase-SP-MHETase (SP: PelB, Amcut) plasmid. However, this dual secretion plasmid was unsuccessful in
designing primers for homologous recombination construction twice. Considering the time constraints for
enzyme-linked design, we decided to construct a single secretion plasmid using a new plasmid backbone with
different resistance from the pSec-PETase (pET28a, Kan resistance) plasmid: pSec-MHETase (pET21a, Amp
resistance). Subsequently, a double plasmid double secretion BL21 strain can be constructed by double resistance
selection.

Transform the newly constructed single-secretory plasmid pSec-MHETase, induce expression and secretion of the
working enzyme, centrifugation of the bacterial fluid to take the supernatant for concentration and volume to
4mL, to the precipitate add 4mL pH=8 Tris-HCl Buffer and ultrasonic broken bacteria, prepared for the treatment
of MHETase enzyme samples.

A complete blank reaction system was configured with 0.8mL of BHET substrate solution + 0.1mL of purified PETase
sample + 0.1mL of treated MHETase enzyme/0.1mL of pH=8 Tris-HCl Buffer sample and no PETase or MHETase reaction
enzyme samples, and the reaction was carried out for 60min at pH=8 and 65°C. The reaction was completed by
adding 1mL of methanol to the precipitate and sonicated to break the bacteria. At the end of the reaction, 1 mL
of methanol solution was added to terminate the reaction, which was allowed to stand overnight at 4°C, and the
supernatant was centrifuged for HPLC liquid phase analysis.

![Expert Interview](https://static.igem.wiki/teams/5233/results/f5-1.png)

Figure 6.1 HPLC Analysis of the MHETase function A. Chromatogram comparation of standard sample
and experiment group (No MHETase, PelB-MHETase Supernatant, Amcut-MHETase Supernatant) B. MHETase Experiment
and PETase blank group (BHET consumption, MHET and TPA production difference)

In this experiment, there is no different between PETase blank group and MHETase experiment group, at first we
thought that it might be the function of MHETase has some problems. But later we realized that our reaction
condition is 65℃ (The best working temperature for modified TurboPETase), and it’s might be fatal to MHETase. We
made a really big mistake here. But the freezing time is approaching, we could only correct this reaction
experiment for MHETase in the future…….

## Result of *Caenorhabditis elegans* (*C. elegans*) part

### Pure nano PET and TPA toxicity detection on nematode growth medium (NGM)

Compared with control group, exposure to 50, 100, 200, 400, 600, 800, 1000 μg/mL of polyethylene terephthalate
(PET), or 125, 250, 500, 750, 1000 μg/mL terephthalic acid (TPA) on NGM show no adverse effects on
*Caenorhabditis elegans* (*C. elegans*) intestinal ROS production, indicating that pure nano-size PET
(Figure 1) and pure TPA (Figure 2) of these concentrations has almost no toxicity to *C. elegans* on NGM.

![Expert Interview](https://static.igem.wiki/teams/5233/results/pet-s.png)

Fig. 1. Effect of pure PET on oxidative stress in P0 generation of *C. elegans* on
NGM.
(A) Microscopy images of *C. elegans* fed on **E. coli** OP50 with different
concentrations of pure PET under Differential Interference Contrast (DIC) and fluorescence excitation with
light of wavelength of 488nm.
(B) Quantification graph of intestinal fluorescence intensity of *C. elegans*. Scale bars, 200µm. n=21.
Error bars represent SD of mean. ns indicates no significant difference. \*\*p<0.01 (one-way ANOVA test).

![Expert Interview](https://static.igem.wiki/teams/5233/results/tpa-s.png)

Fig. 2. Effect of pure TPA on oxidative stress in P0 generation of *C. elegans* on
NGM.
(A) Microscopy images of *C. elegans* fed on **E. coli** OP50 with different
concentrations of pure TPA under Differential Interference Contrast (DIC) and fluorescence excitation with
light of wavelength of 488nm.
(B) Quantification graph of intestinal fluorescence intensity of *C. elegans*. Scale bars, 200µm. n=21.
Error bars represent SD of mean. ns indicates no significant difference. \*\*p<0.01, \*\*\*\*p<0.0001
(one-way ANOVA test).

### Pure nano PET and TPA toxicity detection in liquid culture system

Compared with control group, exposure to 125, 250, 500, 750, 1000 μg/mL pure PET and 125, 250, 500, 750,
1000μg/mL TPA in liquid culture system show no adverse effects on *C. elegans* intestinal ROS production,
indicating that pure nano-size PET (Figure 3) and TPA (Figure 4) of these concentrations has almost no toxicity
to *C. elegans* in liquid culture system.

The survival of *C. elegans* was examined under stereomicroscope before sampling and all *C. elegans*
we observe survived.

![Expert Interview](https://static.igem.wiki/teams/5233/results/pet-l.png)

Fig. 3. Effect of pure PET on oxidative stress in P0 generation of *C. elegans* in liquid
culture system.
(A) Microscopy images of *C. elegans* fed on **E. coli** OP50 with
different concentrations of pure PET under Differential Interference Contrast (DIC) and fluorescence
excitation with light of wavelength of 488nm.
(B) Quantification graph of intestinal fluorescence intensity of *C. elegans*. Scale bars, 200µm. n=21.
Error bars represent SD of mean. ns indicates no significant difference. (one-way ANOVA test).

![Expert Interview](https://static.igem.wiki/teams/5233/results/tpa-l.png)

Fig. 4. Effect of pure TPA on oxidative stress in P0 generation of *C. elegans* in liquid culture
system.
 (A) Microscopy images of *C. elegans* fed on **E. coli** OP50 with different
concentrations of pure TPA under Differential Interference Contrast (DIC) and fluorescence excitation with
light of wavelength of 488nm.
(B) Quantification graph of intestinal fluorescence intensity of *C. elegans*. Scale bars, 200µm. n=21.
Error bars represent SD of mean. ns indicates no significant difference. (one-way ANOVA test)

#### Conclusion:

**Based on our result, both pure nano-size PET and TPA are not toxic to *C. elegans*, which indicate that
it can function normally in our liquid co-culture system.**

### Result and discussion for fluorescence detection

We examined the intestinal fluorescence intensity of Caenorhabditis elegans (*C. elegans*) at different
stages. For *C. elegans* N2 worms at different stages (L4 Larva/day 1 adult) fed with different strains of
Escherichia coli (BL21/OP50) expressing the fluorescent protein tdTomato, we took 21 worms from each group,
capturing fluorescence with MetaMorph (Molecular Devices Inc.) software on an IX73 invert microscope (Olympus),
and analyzing fluorescence intensity with ImageJ software.

Then, we performed data analysis. For *C. elegans* of both L4 stage and day 1 stage, the data are not
normally distributed, so we performed nonparametric test - Mann Whitney test, to detect whether there is
significant difference of *C. elegans* intestinal fluorescence between different **E. coli**
feeding strains.

The results are shown in Figure 5 and Figure 6 for L4 stage and day 1 adult stage, respectively, with
representative images on the left and quantification graph on the right. N2 worms fed on normal OP50 without
tdTomato expressing was set as negative control, showing no visible fluorescence as expected. For worms fed on
**E. coli** expressing tdTomato both at the L4 (Figure 5) and day 1 adult stage (Figure 6), the BL21
group shows stronger fluorescence compared with OP50 group, suggesting that **E. coli** BL21 strain
may be more effectively than OP50 strain colonized or retained within intestine of *C. elegans*.

![Expert Interview](https://static.igem.wiki/teams/5233/results/l4-newwwww.png)

Fig. 5. Intestinal fluorescence intensity in F3 generation L4 stage larva *C. elegans* on NGM.
(A) Microscopy images of *C. elegans* fed on non-fluorescent ***E. coli** OP50*
(negative control), ***E. coli** OP50* tdTomato and ***E. coli** BL21*
tdTomato under Differential Interference Contrast (DIC) and fluorescence excitation with light of wavelength
of 555nm.
(B) Quantification graph of intestinal fluorescence intensity of *C. elegans*. Scale bars, 200µm.
n=~21. Error bars represent SD of mean. \*\*\*p<0.001 (Mann Whitney test).

![Expert Interview](https://static.igem.wiki/teams/5233/results/day-1.png)

Fig. 6. Intestinal fluorescence intensity in F3 generation Day 1 stage adult *C. elegans* on NGM.
(A) Microscopy images of *C. elegans* fed on non-fluorescent ***E. coli** OP50*
(negative control), ***E. coli** OP50* tdTomato and ***E. coli** BL21*
tdTomato under Differential Interference Contrast (DIC) and fluorescence excitation with light of wavelength
of 555nm.
(B) Quantification graph of intestinal fluorescence intensity of *C. elegans*. Scale bars, 200µm. n=21.
Error bars represent SD of mean. \*p<0.05 (Mann Whitney test).

These results above suggest that **E. coli** BL21 strain may be more effectively colonized or
retained within intestine of *C. elegans* than OP50 strain.

### *C. elegans* intestine lysis spreading assay

After detecting the fluorescence intensity inside the intestine of *C. elegans*, we wonder about the
feasibility of engineered **E. coli** to colonize in the worm intestine. To address this issue, we
performed the spread plate assay to evaluate the number of living bacteria in the worm intestine.

Since the plasmids carrying the tdTomato gene also carry the ampicillin resistance gene, by adding the
antibiotic ampicillin to the solid LB medium, we can avoid the contamination of undesired microbe from the
medium. Through a series of gradient dilutions, we obtained the following results shown in Table 1 and Figure
7.

Table 1 Intestine lysis spreading assay result

![Expert Interview](https://static.igem.wiki/teams/5233/results/table1.png)

![Expert Interview](https://static.igem.wiki/teams/5233/results/.png)

Fig. 7. Result of colony growth of **E. coli** on solid LB medium. 0 represents no dilution, 2
represents 102 dilution and 4 represents 104 dilution.

Then we analyzed the data. Colony-forming unit (CFU) per 10 worms were normalized by subtracting the number of
colonies observed in plates from the wash control.

For the OP50 group, we chose the number of colonies in group 0 as the reference for calculating CFU. For the
BL21 group, we chose the number of colonies in group 4 as the reference for calculating CFU.

N (OP50) = N (group 0) \*n (dilution ratio) -N (group supernatant) = 208\*1-7 = 201 CFU/10 worms
N (BL21) = N (group 4) \*n (dilution ratio) -N (group supernatant) = 115\*104-0 = 1.15\*106
CFU/10 worms

The results showed that the number of living bacteria inside the intestine of *C. elegans* in the BL21
group was much higher than that in the OP50 group, consistent with the previous fluorescence results.

#### Conclusion:

**Based on the results of *C. elegans* fluorescence intensity detection and intestinal lysis spreading
assay, we have reached the conclusion: The intake of **E. coli** BL21 strain by *C. elegans* is
much higher than that of OP50 strain. As an uncommon food for *C. elegans*, **E. coli** BL21
strain is able to colonize in the intestinal tract of *C. elegans* and the ability is much better than
that of OP50 strain. This proves that our modified BL21 strain with the TPA transportation ability is suitable
to colonize in the *C. elegans* intestine for further accumulation.**

### Synthetic biology modification of *C. elegans*

We have designed a plasmid that enables the expression of the TPA transport system specifically within the
intestinal tract of *C. elegans*. The detailed design of this plasmid is presented below.

![Expert Interview](https://static.igem.wiki/teams/5233/engineering/c-elegans-plasmid-new.jpg)

Fig. 8. The plasmid we designed for the transportation of TPA. vha-6 is an intestine-specific promoter.
TpiA, TpiB are transporter proteins that will be localized on the membrane of the intestine cell by using
gbb-1 and glr-2 signal peptides, respectively. Tphc is the TPA binding protein that will be secreted outside
the intestine cell by using signal peptide dsl-1. mCherry is the reporter gene used for the detection of
expression.

Using homologous recombination, we designed homology arms on the primers and simultaneously recombined four
fragments using the efficient C116 homologous recombinase.

The vector was linearized and the insertion fragments with homology arms were obtained by PCR amplification. Gel
validation was followed by gel extraction, homologous recombination and transformation of competent DH5α cells.
The resulting colonies were plated, picked, and cultured before plasmid extraction. PCR validation (Figure 9)
confirmed the presence of the desired band, which was subsequently confirmed by sequencing.

![Expert Interview](https://static.igem.wiki/teams/5233/results/2024-10-01-20-43-20.png)

Fig. 9. PCR validation results of different fragments. The agarose gel showed PCR products for the
amplification of different fragments. Lanes are organized from left to right as follows: TphC, sl2, TpiA,
sl2, TphC, sl2, TpiA, sl2. The molecular weight marker is at the middle, with bands indicating approximate
sizes at 1000 bp, 1500 bp, and 2000 bp.

### Outlook

Although we have successfully constructed a new plasmid used for the enrichment of TPA inside the intestinal
tract of *C. elegans*, we have not yet validated the function of it in vivo. Our future work is to
microinject and integrate our constructed plasmid into *C. elegans* to study the actual effect of this
plasmid on TPA enrichment.