iGEM 2023 — Team xhd-wuhan-pro-china

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CONTENTS

[week1：learn basic experimental operation.](#a1)
[week2 Overexpression and Activity Analysis of PETase in Escherichia coli Rosetta](#a2)
[week3 Enhancing the Efficiency of Whole-Cell Catalysts Using Surface Display Technology](#a3)
[week4 Identification of Effective Components of Whole-Cell Biocatalysts](#a4)
[week5 Surface Display of Cellulose Binding Domain (CBD) on E. coli for Enhanced Adhesion to Cellulose Membrane](#a5)
[week6 Verification of experiments and processing of data](#a6)
[Photos:](#a7)

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week1：learn basic experimental operation.

1. Extract plasmid: extract 1 tube of pET23b and 1 tube of pSB1A3-insert according to the instructions of plasmid extraction kit.

(1) column balance: put the adsorption column CP3 into the collection tube, add 500 μ l equilibrium liquid BL to the adsorption column CP3, centrifuge 12000rpm for 1 minute, pour out the waste liquid and set aside.

(2) add 2mL bacteria to the 2mL centrifuge tube and centrifuge 12000rpm for 1 minute. The bacteria are deposited at the bottom of the tube, then pour out the liquid and use the liquid transfer gun to absorb the residual liquid.

(3) add 250 μ l of solution P1 and blow with a liquid transfer gun to mix well.

(4) add 250 μ l solution P2, turn it up and down gently for 8 times, and the liquid becomes clear and sticky.

(5) add 350 μ l solution P3 and turn it up and down gently for 8 times at once to produce white flocculent precipitation. centrifuge with 12000rpm for 10 minutes.

(6) transfer the supernatant (about 800 μ L) to the adsorption column CP3 in step (1), centrifuge with 12000rpm for 1 minute, pour out the waste liquid from the collection tube, and put the adsorption column CP3 back into the collection tube.

(7) add 600 μ L rinse solution PW,12000rpm to the adsorption column CP3 and centrifuge for 1 minute, then pour out the waste liquid.

(8) repeat steps (7).

(9) put the adsorption column CP3 into the collection tube and centrifuge for 2 minutes to remove the residual liquid from the adsorption column.

(10) uncover the adsorption column CP3 for 10 minutes and thoroughly dry the residual liquid in the adsorption column.

(11) put the adsorption column CP3 into a clean 1.5mL centrifuge tube, drop 80 μ L elution buffer EB in the middle, and centrifuge at room temperature for 2min and 12000rpm for 2 minutes.

2. PCRreaction

(1) using high fidelity enzyme Pfu DNA Polymerase to amplify the target gene from pSB1A3-insert.

Take out the 200 μ l PCR tube and add the following solution:

![](https://static.igem.wiki/teams/4995/wiki/note/img1.png)

Centrifuge the liquid to the bottom of the pipe with a small centrifuge.

(2) PCR amplification program.

94 ℃, 3 min.

The following three steps are followed by 30 cycles.

94 ℃ for 30s.

55 ℃ for 30s.

72 ℃ 45s.

72 ℃ 10 min.

The plasmids, primers and products required for PCR are shown in the following table:

![](https://static.igem.wiki/teams/4995/wiki/note/img2.png)

3. Preparation of 50 mL 1.2% agarose gel.

(1) dissolve 0.6 g agarose (Agarose) in 50 mL 1 X TAE buffer, use microwave oven to boil for 1-2 minutes, then boil twice for 10 seconds, cool down (not hot hands as the standard).

(2) prepare the dispensing board when cooling, and insert the 25 μ L comb into the 50 mL dispensing device.

(3) add 5 μ L 10 mg/mL EB solution, shake evenly, pour into the dispensing plate and wait for about 30 min to solidify.

4. Agarose gel electrophoresis.

(1) the above PCR products were added to the agarose gel and 5 μ L DL 5000 DNA marker was added for electrophoresis (150V DL 15 min).

(2) after the end of electrophoresis, the gel was observed by gel imaging system, and the DNA gel was cut and placed in the 1.5mL centrifuge tube.

5. Recovery of PCR products from glue.

The gel of DNA gel of PCR product was recovered according to the instructions of gel recovery kit (1 h), and the concentration of DNA was determined by Nanodrop.

(1) turn on the water bath or metal bath and adjust the temperature to 55 ℃.

(2) add 500 μ L solution to the glue block and bathe at PN,55 ℃ for 10-15 minutes, during which the centrifuge tube should be turned up and down until the glue block is completely dissolved.

(3) column balance: put the adsorption column CA2 into the collection tube, add 500 μ l equilibrium liquid BL to the adsorption column CA2, centrifuge 12000rpm for 1 minute, pour out the waste liquid and set aside.

(4) transfer the supernatant (about 800 μ L) to the adsorption column CA2 in step (1), centrifuge with 12000rpm for 1 minute, pour out the waste liquid from the collection tube, and put the adsorption column CA2 back into the collection tube.

(5) add 600 μ L rinse solution PW,12000rpm to the adsorption column CP3 and centrifuge for 1 minute, then pour out the waste liquid.

(6) repeat steps (5).

(7) put the adsorption column CA2 into the collection tube and centrifuge for 2 minutes to remove the residual liquid from the adsorption column.

(8) uncover the adsorption column CA2 for 10 minutes and thoroughly dry the residual liquid in the adsorption column.

(9) put the adsorption column CA2 into a clean 1.5mL centrifuge tube, drop 80 μ L elution buffer EB in the middle, and centrifuge at room temperature for 2min and 12000rpm for 2 minutes.

(10) the recovered products should be stored at-20 ℃.

6. Double enzyme digestion:

The extracted plasmid pET23b and the amplified target DNA fragment were digested by EcoRI and XhoI.

Take out the 200 μ l PCR tube and add the following solution:

![](https://static.igem.wiki/teams/4995/wiki/note/img3.png)

Enzyme digestion at 37 ℃ for 1 hour

7. Connection reaction.

Take out the 200 μ l PCR tube and add the following solution connection system as follows:

![](https://static.igem.wiki/teams/4995/wiki/note/img4.png)

Incubate the connection overnight with 16 °C to get pET23b-insert.

8. Conversion and connection products.

(1) turn on the water bath and adjust the temperature to 42 ℃.

(2) add 10 μ L ligated products to 50 μ L competent cells on ice, mix lightly and 20min in ice bath.

(3) Water bath at 42 ℃ for 90s, ice bath 2min, add 600 μ LLB liquid medium to the super clean table, culture 40min for resuscitation (37 ℃, 150rpm) [pay attention to aseptic operation].

(4) centrifuge with 8000rpm for 1 minute.

(5) in the super purification platform, the 500 μ L supernatant is sucked away with the gun head, and the remaining 100 μ L liquid is left, and the deposited bacteria are blown with the gun head to re-suspend it.

(6) add all the remaining liquid to the Amp antibiotic plate and spread the bacterial liquid evenly with an aseptic coating stick.

(7) the inverted plate was cultured overnight in the incubator at 37 ℃.

9. Culture medium and sterilization:

(1) 100mL LB liquid medium.

Tryptone peptone: 1g.

Yeast extract yeast powder: 0.5g.

NaCl:1g.

(2) 100 mL LB solid medium: (add 1.5g additional Agar).

(3) autoclave: 121C, 20min (the whole process is about 1.5h).

(4) inverted plate: pour 20mL around each petri dish (covered with a bottom), open the cover and blow 15min, and then use the sealing plate to seal the plate.

10. PCR verification:

A colony was selected as a template and Taq enzyme was used to PCR the colony. The primer was TF,TR, and the reaction system and procedure were carried out according to the instructions.

(1) using high fidelity enzyme Pfu DNA Polymerase to amplify the target gene from pSB1A3-insert.

![](https://static.igem.wiki/teams/4995/wiki/note/img5.png)

(2) PCR amplification program.

94 ℃, 3 min.

The following three steps are followed by 30 cycles.

94 ℃ for 30s.

55 ℃ for 30s.

72 ℃ 45s.

72 ℃ 10 min

11. Shaking bacteria.

(1) add 5mLLB liquid medium (Amp+) to the shaker tube, pick up the recombinant colony with a small gun head, and shoot the gun head directly into the shake tube.

(2) add 5mLLB liquid medium (no resistance) to the shake bacteria tube, and put the E.coliRosetta strain 20 μ l into the shake bacteria tube.

12. Fluorescence microscope verification experiment:

(1) sample preparation: take appropriate amount (about 30 μ l) of Rosetta-pET23b- gene bacteria and wild type Rosetta onto the slide and cover it with the cover slide.

(2) turn on the power control box switch of the high pressure mercury lamp.

(3) insert the light shield to interrupt the light path.

(4) preheat 5-10min.

(5) put the slide containing the sample on the loading platform.

(6) choose the objective lens (in the order of low power first, then high power).

(7) rotate the turntable of the spectroscope assembly and select the spectroscope assembly that observes the mRFP (red fluorescence).

(8) adjust the focal length by coarse and fine spirals.

(9) turn on the computer connected to the microscope and click on the digital imaging system software to collect digital images.

13. The expression of fluorescent protein was detected by enzyme labeling instrument: 100 μ L recombinant bacterial liquid and E. coli Rosetta were added to 96-well plate and repeated for 4-6 times. The detection conditions of enzyme labeling instrument (mRFP excitation λ: 584nm/10 nm, emission λ: 607max 10 nm) were set and read. Use Excel to record data.

preparation before the experiment.

Synthesis of INP-PEAase sequence and INP-CBD sequence in Azenta Company.

Consult the map of pET23b vector and design the corresponding primers according to the target gene and vector information.

Configuration of LB liquid medium.

Configuration of PBS，1mM pNPB

Purchase and preparation of experimental consumables and experimental reagents

week2 Overexpression and Activity Analysis of PETase in Escherichia coli Rosetta

To study the expression and function of PETase in Escherichia coli Rosetta, we synthesized the PETase gene (Azenta life science, USA) and cloned it into the pET23b vector, allowing the target protein to be continuously expressed in E. coli Rosetta without the need for IPTG induction. The recombinant vector was transformed into E. coli Rosetta cells, and positive clones were selected on LB agar plates containing ampicillin. Positive clones were verified by DNA sequencing (Generalbiol, China). Positive clones were cultured overnight in LB medium, and cell pellets were collected by centrifugation and resuspended in Tris-HCl (pH 7.4). Cells were then lysed by sonication (150 W, sonication for 1s, interval 3s, for a total of 20 minutes) to obtain cell lysate. The total protein concentration in the lysate was measured using a Bradford reagent kit (Beyotime, China). Enzyme activity was measured using para-nitrophenyl butyrate (pNPB) as a substrate. A 100 mM pNPB stock solution (Merck, Germany) was prepared in acetonitrile. PETase activity was tested in a 100 μL reaction system: 1 mM pNPB, 50 mM Tris-HCl buffer (pH 7.4), 20 μL crude enzyme solution. The reaction was carried out at 30°C for 30 min. The release of para-nitrophenol ester from PETase cleavage of pNPB was measured at 405nm using a microplate reader. The content of para-nitrophenol ester was calculated based on a standard curve. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of para-nitrophenol ester per minute. The specific activity of PETase enzyme is defined as U/mg. All experiments were performed in triplicate, and data are presented as mean values with SD.

![](https://static.igem.wiki/teams/4995/wiki/note/img6.png)
![](https://static.igem.wiki/teams/4995/wiki/note/img7.png)
![](https://static.igem.wiki/teams/4995/wiki/note/img8.png)

week3 Enhancing the Efficiency of Whole-Cell Catalysts Using Surface Display Technology

To improve the efficiency of engineered E. coli as whole-cell biocatalysts for PET degradation, the ice nucleation protein encoding gene INP was fused upstream of the PETase gene. The recombinant plasmid was transformed into E. coli Rosetta. Positive clones were cultured overnight in LB medium (37°C, 180 rpm), and 1 mL of bacterial culture was taken, adjusted to OD600=1, and centrifuged (10,000 rpm, 1 min) to collect cell pellets, which were resuspended in 1 mL PBS (pH 7.0). In 1 mL of bacterial suspension, 1 mM pNPB was added, and the reaction was incubated at 30°C for 30 min. The supernatant was collected by centrifugation, and the absorbance at 405nm was measured using a microplate reader. A standard curve was prepared using 0-1 mM para-nitrophenol ester dissolved in PBS. All experiments were performed in triplicate, and data are presented as mean values with SD.

![](https://static.igem.wiki/teams/4995/wiki/note/img9.png)

week4 Identification of Effective Components of Whole-Cell Biocatalysts

E. coli Rosetta/pET23a-INP-PETase was cultured overnight in LB medium (37°C, 180 rpm), and 1 mL of bacterial culture was taken, adjusted to OD600=1. The culture medium was collected by centrifugation (10,000 x g, 1 min). The cell supernatant was transferred to a new centrifuge tube as an extracellular component sample. The cell pellet was washed with PBS to remove residual medium and lysed by sonication. The lysed cell suspension was transferred to a new centrifuge tube. Intracellular components and cell membranes were separated by centrifugation (e.g., 10,000 x g, 10 min). The supernatant was transferred to a new centrifuge tube as an intracellular component sample. The cell pellet was retained as a cell membrane sample. The cell pellet was resuspended in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100. The suspension was gently shaken or vortexed to dissolve the cell membrane proteins. Insoluble material was removed by centrifugation at 14,000 x g for 30 min. The supernatant was transferred to a new centrifuge tube as a cell membrane protein sample. The concentrations of extracellular, intracellular, and cell membrane proteins were measured using a Bradford reagent kit. Enzyme activity was measured using para-nitrophenyl butyrate (pNPB) as a substrate.

![](https://static.igem.wiki/teams/4995/wiki/note/img10.png)

week5 Surface Display of Cellulose Binding Domain (CBD) on E. coli for Enhanced Adhesion to Cellulose Membrane

In order to enable E. coli to adhere to the cellulose membrane, the INP-CBD sequence was synthesized and cloned into the pET23b vector. The recombinant plasmid pET23b/INP-CBD was then transformed into E. coli Rosetta. Subsequently, the adhesion effect of the recombinant strain to bacterial cellulose was tested. The purchased sterile bacterial cellulose membrane (BC membrane) was cut into 5 cm x 5 cm pieces. The recombinant E. coli was cultured overnight in 5 mL LB medium containing 100 μg/mL ampicillin. The following day, bacterial pellets were collected by centrifugation, washed with PBS (pH 7.4), and the recombinant E. coli was resuspended to adjust the bacterial OD600 = 0.5. The recombinant E. coli suspension was then incubated with the BC membrane for 1 hour. Afterwards, the membrane was gently rinsed with 10 mL PBS to remove non-specifically adhered cells, and the rinse solution was collected. The rinsing solution was subsequently diluted in a gradient and spread onto LB AGAR plates containing ampicillin.. For comparison, wild-type E. coli was processed in the same way. After 24 hours of incubation, we calculated the CFU/mL for each sample. By comparing the non-specific adhesion of the INP-CBD fusion protein E. coli with that of the wild type on the bacterial cellulose membrane, we evaluated the adhesive effect of INP-CBD. Finally, the results were statistically analyzed using the student t-test.

![](https://static.igem.wiki/teams/4995/wiki/note/img11.png)

week6 Verification of experiments and processing of data

Statistical Analysis

All experiments were repeated three times and the results are presented as means ± SD. The study data were statistically analyzed using Student's t-test (two-tailed, unpaired). Significance was denoted as \*P < 0.05, \*\*P < 0.01 and \*\*\*P < 0.001.

Photos:

![](https://static.igem.wiki/teams/4995/wiki/note/img12.jpeg)
![](https://static.igem.wiki/teams/4995/wiki/note/img13.jpg)

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CONTENTS

[Overview](#a1)
[Cycle 1: The first-generation microplastic processor](#a2)
[Design](#b1)
[Build](#b2)
[Test](#b3)
[Learn](#b4)
[Cycle 2： The sencond-generation microplastic processor](#a3)
[Design](#b5)
[Build](#b6)
[Test](#b7)
[Learn](#b8)
[Cycle 3：The third-generation microplastic processor](#a4)
[Design](#b9)
[Build](#b10)
[Test](#b11)
[Learn](#b12)
[Conclusion](#a5)
[References](#a6)

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Overview

In recent years, the pollution caused by microplastics in the ocean has garnered global attention. Especially in coastal areas, where human activity is frequent, the accumulation of microplastics has severely impacted the health and stability of marine ecosystems. Among the various types of plastics, PET (polyethylene terephthalate) is one of the most common, widely used in everyday items such as beverage bottles and food containers. However, the degradation and recycling of PET have always posed significant challenges, as traditional physical and chemical methods have proven ineffective in addressing this issue.

Fortunately, scientists have discovered a potential solution from nature. Researchers in Japan found a bacterium (Ideonella sakaiensis) capable of degrading PET and isolated an enzyme from it called PETase. This enzyme has the ability to break down hcPET (high crystallinity PET), effectively dismantling its long-chain molecules, enabling degradation.

To apply this discovery in practice and further enhance the activity of PETase, we employed a surface display technique to present PETase on the surface of E. coli bacteria. This approach significantly boosted the activity of the whole-cell catalyst, making it more suitable for real-world application. Importantly, to anchor the engineered bacterial strains in open waters and to address potential bio-safety concerns, we adopted the Part (http://parts.igem.org/Part:BBa\_K4380000 ) from the 2022 Vilnius-Lithuania. Using cellulose-binding domains, we fixed the engineered strains onto bacterial cellulose surfaces. Through this method, we successfully developed a hardware system capable of effectively treating PET in open water environments.

The promotion and application of this technology undeniably offer a powerful tool in addressing the issue of marine microplastic pollution. We hope that with continuous research and innovation, we can make even more significant contributions to the protection of marine environments.

Cycle 1: The first-generation microplastic processor

Design

![](https://static.igem.wiki/teams/4995/wiki/engi/img1.png)

Figure 1 Design of gene circuit of PETase overexpression system.

In the first-generation microplastic processor, we conducted an extensive review of relevant literature and designed the genetic circuitry depicted above. This circuitry comprises elements such as the T7 promoter, ribosome binding site (RBS), PETase gene, and terminator. In this configuration, engineered bacteria are capable of continuously expressing PETase, which subsequently enables the degradation of microplastics present in the environment. This approach aligns with the overarching goals of environmental protection and sustainable development.

Build

To study the expression and function of PETase in Escherichia coli Rosetta, we synthesized the PETase gene (Azenta life science, USA) and cloned it into the pET23b vector, allowing the target protein to be continuously expressed in E. coli Rosetta without the need for IPTG induction. The recombinant vector was transformed into E. coli Rosetta cells, and positive clones were selected on LB agar plates containing ampicillin. Positive clones were verified by DNA sequencing (Generalbiol, China). Positive clones were cultured overnight in LB medium, and cell pellets were collected by centrifugation and resuspended in Tris-HCl (pH 7.4). Cells were then lysed by sonication (150 W, sonication for 1s, interval 3s, for a total of 20 minutes) to obtain cell lysate. The total protein concentration in the lysate was measured using a Bradford reagent kit (Beyotime, China).

Test

Enzyme activity was measured using para-nitrophenyl butyrate (pNPB) as a substrate. A 100 mM pNPB stock solution (Merck, Germany) was prepared in acetonitrile. PETase activity was tested in a 100 μL reaction system: 1 mM pNPB, 50 mM Tris-HCl buffer (pH 7.4), 20 μL crude enzyme solution. The reaction was carried out at 30°C for 30 min. The release of para-nitrophenol ester from PETase cleavage of pNPB was measured at 405 nm using a microplate reader. The content of pNPB was calculated based on a standard curve. One enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of para-nitrophenol ester per minute. The specific activity of PETase enzyme is defined as U/mg.

![](https://static.igem.wiki/teams/4995/wiki/engi/img2.png)

Figure 2 Activity test of PETase. A: PETase enzyme kinetics curve; B: Effect of temperature on PETase activity. C：Effect of pH on the activity of PETase.

Learn

By varying the substrate pNPB concentration, we observed an increase in PETase activity. However, at a substrate concentration of 1000 μM, the enzyme activity growth rate plateaued, suggesting that the engineered bacterial strain successfully expressed PETase. Further analysis of the enzyme kinetics curve using Graphpad Prism 8.0 revealed a maximum reaction rate (Vmax) of 0.92 U/min and a Km value of 329.5 μM, calculated using the Michaelis-Menten equation (Figure 2A). Additionally, we explored the impact of temperature and pH on the microplastic processor's performance. The results indicated that the processor effectively operated within a temperature range of 25°C to 45°C, with optimal efficiency observed at 40°C. For pH, the processor functioned well within the pH range of 7.4 to 9.8, with peak efficiency at pH 9.2. This finding suggests that the microplastic processor can operate effectively in slightly alkaline environments, making it suitable for deployment in seawater environments without the need for additional alkaline conditions (see Figure 2B and 2C).

However, in our first-generation microplastic processor, we faced a cumbersome challenge: the need to disrupt bacteria to release PETase enzyme. This process was overly complex and inefficient. To enable the direct application of the microplastic processor in Escherichia coli, we conducted an extensive literature review. Ultimately, we decided to employ surface display technology to present the PETase enzyme on the surface of Escherichia coli. This approach allows engineered Escherichia coli to efficiently and conveniently access PETase.

Cycle 2： The sencond-generation microplastic processor

Design

Cell surface display technology, also known as microbial surface display technology, is a biotechnological approach that enables the expression and localization of target proteins on the surface of microbial cells. This technology allows researchers to manipulate and alter proteins directly on the surface of living cells, making them more amenable to studies of biochemical and biophysical properties, enzyme catalysis, and cellular engineering. At the core of this technique is the utilization of naturally occurring signal peptides or carrier proteins capable of directing proteins to the cell surface. Typically, these proteins transport the target protein to the cell surface through specific pathways. In the field of genetic engineering, we can link the gene sequence of the target protein with the gene sequence of these signal peptides or carrier proteins through genetic engineering methods, creating fusion genes. These fusion genes are then introduced into microbes, allowing for the expression and localization of the fusion protein on the cell surface. Ice Nucleation Protein (INP) is a commonly used protein carrier in cell surface display technology, and it can display the target protein in the periplasmic space of bacteria.

![](https://static.igem.wiki/teams/4995/wiki/engi/img3.png)

Figure 3 Design of gene circuit of INP-PETase expression system.

In the design of the second-generation microplastic processor, we constructed the following gene circuitry. This circuitry comprises the T7 promoter, ribosome binding site (RBS), INP-PETase gene, and terminator. In this design paradigm, we successfully achieved continuous expression of INP-PETase by the engineered bacteria. INP is capable of anchoring itself to the cell membrane's surface, thereby presenting PETase outside the cell, enabling it to efficiently degrade microplastics present in the environment.

Build

The ice nucleation protein encoding gene INP was fused upstream of the PETase gene. The recombinant plasmid based on PET23b was transformed into E. coli Rosetta. Positive clones were selected in LB medium contaning 100 μg/mL ampicillin antibiotic under controlled conditions (37°C, 180 rpm).

Test

To explore the catalytic activity of whole cell catalysts. The bacterial culture of engineered E. coli and control group (wild type E. coli) were harvested, adjusted to an OD600 of 1, and subjected to centrifugation (10,000 rpm, 1 min) to collect cell pellets. These pellets were then resuspended in 1 mL of PBS (pH 7.0). In a 1 mL bacterial suspension, 1 mM pNPB was added, and the reaction was allowed to incubate at 30°C for 30 minutes. Subsequently, the supernatant was collected by centrifugation, and the absorbance at 405nm was measured using a microplate reader. A standard curve was generated using para-nitrophenol ester concentrations ranging from 0 to 1 mM dissolved in PBS.

![](https://static.igem.wiki/teams/4995/wiki/engi/img4.png)

Figure 4: A: Comparison of whole-cell catalytic activity of wild-type Escherichia coli (WT), PETase overexpressing E. coli and INP-PETase overexpressing E. coli; B: Difference analysis of WT and engineered E. coli that express INP-PETase；C：Agarose gel electrophoresis image of INP-PETase.

![](https://static.igem.wiki/teams/4995/wiki/engi/img5.png)

Figure 5: Exploring the effective components of whole-cell catalyst.

To further investigate the effective components of the whole-cell catalyst, we cultured E. coli Rosetta overexpressing INP-PETase overnight. From this culture, we separated extracellular and intracellular components through centrifugation. The cell pellet underwent a series of processes, including washing, sonication, and additional centrifugation, to segregate intracellular components and cell membranes. We then extracted cell membrane proteins using a Tris-HCl solution with Triton X-100, followed by centrifugation to remove insoluble fractions. The protein concentrations of all three components (extracellular, intracellular, and cell membrane) were determined using a Bradford reagent kit.

Learn

The findings revealed that E. coli/INP-PETase is capable of degrading 1 mM p-nitrophenol butyrate (pNPB), resulting in the production of 0.3 mM p-nitrophenol ester. In contrast, both the wild-type Escherichia coli Rosetta and E. coli/PETase yield negligible amounts of para-nitrophenol esters. These experimental outcomes demonstrate that the catalytic efficiency of PETase can be substantially enhanced through the application of surface display technology (Figure 4). On the othre hand, we found that there was almost no PETase activity outside the cell, but in the cell contents, the activity of PETase was about 0.15U/mg, and the activity of PETase on the cell membrane was the highest, reaching 0.35U/mg. Thus, through the surface display, the main active components of PETase are located in the cell membrane and bacterial contents (Figure 5).

However, biosafety experts have cautioned us that if we intend to release this strain into lakes or oceans, we would face significant biosafety concerns. It is not advisable to introduce a genetically modified organism into an open environment without effective control mechanisms in place. We need to consider strategies for immobilizing the engineered bacterial strain in open water bodies to address these biosafety concerns and ensure environmental stability.

Cycle 3：The third-generation microplastic processor

Design

We found 2022 Vilnius-Lithuania igem team representation of a cellulose binding protein (CBD, http://parts.igem.org/Part:BBa\_K4380000), We decided to use part of it as a binding protein for a microbial fixation technology based on bacterial cellulose (Figure 6). We also received the selfless help of the team at Squirrel-Beijing-I, who donated their bacterial cellulose to help us verify the feasibility of their fixation technology.

![](https://static.igem.wiki/teams/4995/wiki/engi/img6.png)

Figure 6 Protein structure of CBD（This image derived from Part:BBa\_K4380000，pictured by 2022 Vilnius-Lithuania).

![](https://static.igem.wiki/teams/4995/wiki/engi/img7.png)

Figure 7 Design of gene circuit of INP-PETase and INP-CBD co-expression system.

We designed the third generation of microplastic processors. The first module is the anchoring and degradation module, which continuously expresses INP-PETase inside the engineered bacteria（Figure 7）. The second module is the anchoring module, which continuously expresses INP-CBD and can anchor the engineered bacteria on the cellulose membrane to prevent the engineered bacteria from flowing into the water and causing biological pollution (Figure 8).

![](https://static.igem.wiki/teams/4995/wiki/engi/img8.png)

Figure 8 Illustration of hardware for water microplastic treatment.

Build

To enhance the adhesion of Escherichia coli to cellulose membranes, we have devised a plan to introduce INP-CBD onto the foundation of the INP-PETase engineered strain, utilizing a polycistronic approach to achieve this project (Figure 7). Prior to embarking on this endeavor, we first conducted individual tests to assess the functionality of INP-CBD. We initiated the process by synthesizing the INP-CBD gene sequence, followed by cloning it into the pET23b vector. To ensure a robust fusion, we incorporated a proline-based 17 x helixl peptide (AEAAAKEAAAKEAAAKA) as a connector between INP and CBD. Subsequently, the resulting recombinant plasmid was named pET23b/INP-CBD and was introduced into Escherichia coli Rosetta. Positive clones were selected using the selective antibiotic ampicillin at a concentration of 100 μg/ml.

Test

Subsequently, the adhesion effect of the recombinant strain to bacterial cellulose was tested. The sterile bacterial cellulose membrane (BC membrane) was cut into 5 cm x 5 cm pieces. The recombinant E. coli was cultured overnight in 5 mL LB medium containing 100 μg/mL ampicillin. The following day, bacterial pellets were collected by centrifugation, washed with PBS (pH 7.4), and the recombinant E. coli was resuspended to adjust the bacterial OD600 = 0.5. The recombinant E. coli suspension was then incubated with the BC membrane for 1 hour. Afterwards, the membrane was gently rinsed with 10 mL PBS to remove non-specifically adhered cells, and the rinse solution was collected. The rinsing solution was subsequently diluted in a gradient and spread onto LB Agar plates containing ampicillin.. For comparison, wild-type E. coli was processed in the same way. After 24 hours of incubation, we calculated the CFU/mL for each sample. By comparing the non-specific adhesion of the INP-CBD fusion protein E. coli with that of the wild type on the bacterial cellulose membrane, we evaluated the adhesive effect of INP-CBD.

![](https://static.igem.wiki/teams/4995/wiki/engi/img9.png)

Figure 9 A: Explore the adhesion of CBD enhancement to cellulose membrane; B: Difference analysis of experimental results；C：Agarose gel electrophoresis image of INP-linker-CBD.

Learn

After the recombinant Escherichia coli strain was incubated with bacterial cellulose membrane, the CFU value of the washing solution was counted. The wild type strains without INP-CBD fusion had the least adhesion to bacterial cellulose membrane. The average CFU of flushing solution is 1.2 × 10 ^ 6CFU / mL. The average CFU of the rinse solution of recombinant Escherichia coli strain was 7.6 × 10 ^ 5 CFU / mL (Figure 9). T-test was used for comparison. The p values obtained were all less than 0.05, indicating that there was a statistically significant difference in the adhesion ability of the two strains. This significantly enhanced adhesion confirmed the successful display of CBD on the surface of Escherichia coli and its function of binding to cellulose membrane.

Conclusion

We have successfully constructed the PETase overexpression recombinant plasmid in the pET23b vector and produced PETase in E. coli Rosetta. Through enzymatic kinetics studies, we determined that the crude enzyme solution has a maximum catalytic rate of 0.92 U/min, with a Km value of 325.5 μM. To enhance efficiency, we employed INP, a surface display protein, to deliver PETase into the periplasmic space of E. coli. Experimental data conclusively show that this display technique significantly boosts the performance of whole-cell catalysts. Lastly, to evaluate the potential application of the engineered strain in open waters, we tested the adhesion capability of the INP-CBD engineered E. coli to bacterial cellulose.

References

Chen, Zhuozhi, et al. "Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase." Science of The Total Environment 709 (2020): 136138.

Dou, Jian-lin, et al. "Surface display of domain III of Japanese encephalitis virus E protein on Salmonella typhimurium by using an ice nucleation protein." Virologica Sinica 26 (2011): 409-417.

Morag, Ely, et al. "Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum." Applied and environmental microbiology 61.5 (1995): 1980-1986.

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![](https://static.igem.wiki/teams/4995/wiki/cover/experiments.jpg)

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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