iGEM 2023 — Team nis-kazakhstan

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{% block title %}Notebook{% endblock %}
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# Notebook

## Our Notebook

### NIS-Kazakhstan

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Innovative educational tools and outreach activities have the ability to establish a two-way dialogue with new communities by discussing public values and the science behind synthetic biology.{% endblock %}
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# Engineering Success

Analysis

Enzymatic Modification

References

Analysis of IsPETase\_W159H\_S238F with
6xHis-tag gene expression

The gene IsPETase\_W159H\_S238F with 6xHis-tag, which had two mutations and was 3.5-fold more efficient than wild-type PETase, was selected from the listed genes [2]. This gene sequence was located in the vector pSB1C5C (Registry Part ID: Bba\_k2910000).

At the initial stages of work, we needed to develop the plasmid pSB1C5C-IsPETase\_W159H\_S238F-6xHis-tag and the genes of the promoter (BBa\_J23118), strong RBS (BBa\_K2680529) and constitutive terminator (Bba\_J428091) for further work with them. For this purpose, electrocompetent cells of E. coli strain DH5a were used. For analysis, 1 μl of diluted plasmid was taken and mixed  in a pre-chilled electroporation cuvette with 40 μl of cells. Next, the electroporation program was set: voltage 1700 V (field strength 17 kV/cm), resistance 200 Ohm, and capacitance 25 μF. The sample was then pulsed once, the cuvette was quickly removed, and 960 μL of SOC medium (kept at 37°C) was immediately added to resuspend the cells. Transfer the cells to a sterile BD Falcon 14 mL conical bottom polypropylene tube and incubate the tube at 37°C for 1 hour with shaking at 225-250 rpm. Next, 100 μl of the transformation mixture was applied to LB agar plates containing the appropriate antibiotic (Chloramphenicol 25 μg/ml).

The colonies obtained the next day were subcultured into 5 ml of LB medium with antibiotic and incubated overnight at 37°C with shaking at 180 rpm. The next morning, plasmids were isolated using the GeneJET Plasmid Miniprep Kit (Thermo Scientific™, K0503). The concentration of the resulting plasmids and purity were checked on Nanodrope One C. The concentration of plasmids was about 150 ng/μl and the purity was 1.8-1.9 (Ratio 260/280).

The resulting plasmids were treated with BsaI restriction enzymes in the Green buffer. 2000 ng of plasmids and 2 unit each were taken for restriction. enzymes for a total volume of 20 µl. The reaction lasted 1 hour at 37°C followed by inactivation at 65°C for 20 minutes. The processed products were then analyzed on a 1.0% agarose gel. After confirmation of the restriction products, ligation was carried out using T4 DNA ligase (Thermo Scientific™, EL0014) at 22 °C for 1 hour. The ligation products were used for transformation. Ligation products (10 μl) were transformed into strain DH5a by heat shock and plated on selective LB medium with ampicillin (100 μg/ml).

The resulting plasmids were treated with BsaI restriction enzymes in the Green buffer. 2000 ng of plasmids and 2 unit each were taken for restriction. enzymes for a total volume of 20 µl. The reaction lasted 1 hour at 37°C followed by inactivation at 65°C for 20 minutes. The processed products were then analyzed on a 1.0% agarose gel. After confirmation of the restriction products, ligation was carried out using T4 DNA ligase (Thermo Scientific™, EL0014) at 22 °C for 1 hour. The ligation products were used for transformation. Ligation products (10 μl) were transformed into strain DH5a by heat shock and plated on selective LB medium with ampicillin (100 μg/ml).

PCR products were analyzed in a 1.0% agarose gel with the addition of ethidium bromide and visualized under transmitted UV light using a transilluminator. As a result, a DNA fragment of 890 nucleotide pairs in size was amplified, corresponding to the length of the IsPETase\_W159H\_S238F gene with 6xHis-tag (Figure 5).

![HP layout competition1](https://static.igem.wiki/teams/4901/wiki/projects/image-87.png)

Figure 5. Analysis of IsPETase\_W159H\_S238F gene amplification product with 6xHis-tag. M - GeneRuler 1 kb DNA Ladder marker; 1-2 - PCR amplification product.

After confirmation of the correctness of the obtained plasmid, transformation into expression strain TG1 was performed. The obtained colonies were also tested for the presence of the plasmid using specific primers: Direct 5'-AACTTCCCCCCCGTGCC-3' and Reverse 5'-CTCGAGGGAACAGTTCGC-3'. Positive colonies were used for protein induction. Induction conditions: Overnight bacterial culture was crossed into 50 ml at a ratio of 1:50 and incubated at 37°C until OD600 was 0.6. The culture temperature was then reduced to 30°C and 0.5 mM IPTG was added and left overnight at 30°C on a shaker.

The next morning, protein extraction was performed by sonication and the cell extract was analysed by SDS-PAFE electrophoresis using 15% gel. After confirming the presence of protein in the cell extract, induction was carried out in 1L of LB medium. Protein extraction was performed in lysis buffer (50 mM Tris-HCl (pH 9.0), 50 mM NaCl, 20 mM imidazole, 1 mM EDTA, 5 mM β-mercaptoethanol, 1 mM DTT, 2% of Triton X-100, and 5% of glycerol) supplemented with Complete Protease Inhibitor Cocktail (Roche Diagnostics, Switzerland). The resulting supernatant after centrifugation was used for protein purification on an FPLC ÄKTA start chromatography protein purification system using a 1 ml HisTrap column charged with Ni2+. The purified fractions were subjected to gel electrophoresis. The result of gel electrophoresis is shown in Figure 7.

![HP layout competition1](https://static.igem.wiki/teams/4901/wiki/projects/88.png)

Figure 6. SDS-PAGE gel electrophoresis of IsPETase\_W159H\_S238F protein with 6xHis-tag. M, protein marker; 2-14, purified fractions from tubes 1 to 13

From the gel electrophoresis result, we can see that we were able to express and purify IsPETase\_W159H\_S238F with 6xHis-tag (32.4 kDA). This protein can be further analysed for enzymatic activity and further used for plastic recycling.

Enzymatic Modification of PET: Surface Hydrophilization
and Polymer Chain Analysis.

![HP layout competition1](https://static.igem.wiki/teams/4901/wiki/projects/image-89.png)
![HP layout competition1](https://static.igem.wiki/teams/4901/wiki/projects/image-90.png)

Figure 7. Investigating PET hydrolase-mediated hydrolysis for surface hydrophilization and analyzing interactions with polymer chain ends, enhancing our understanding of enzymatic PET modification.

1. Hydrolysis of PET Film to Ends/Loops of Polymer Chains:
Polyethylene terephthalate (PET) is a synthetic polymer used in various applications, including plastic bottles and films. The hydrolysis of PET film involves breaking down the polymer chains through a chemical reaction with water. This process can occur in two steps:

a. Hydrolysis: In this step, water molecules (H2O) interact with the ester linkages in PET. The ester linkages are the bonds that hold the polymer chains together. Hydrolysis breaks these ester bonds, resulting in shorter polymer chains, as well as the release of molecules like ethylene glycol (EG) and terephthalic acid (TPA).

b. Ends/Loops of Polymer Chains: The result of hydrolysis is the creation of polymer chains with open ends and loops. The ends of the chains may have functional groups exposed, making them more reactive for subsequent chemical reactions or recycling processes. These ends and loops can be further processed into monomers or intermediates for PET recycling or used to create new PET-based materials.

2. Surface Hydrophilization: PET is known for its hydrophobic nature, meaning it repels water. Surface hydrophilization is the process of modifying the surface of PET to make it more water-attractive or hydrophilic. This can be achieved in several ways:

a. Chemical Treatment: Chemical treatments involve applying substances or coatings to the PET surface that have hydrophilic properties. For example, plasma treatment or chemical grafting can introduce hydrophilic functional groups to the surface, making it more receptive to water.

b. Physical Methods: Physical methods can also be used to enhance hydrophilicity. These methods include roughening the surface through techniques like sandblasting or creating micro/nanostructures that can trap water molecules.

c. Additives and Coatings: Sometimes, hydrophilic additives or coatings are applied to the PET surface to change its properties. These coatings can include surfactants, coatings with hydrophilic nanoparticles, or other materials that interact favorably with water.

By hydrophilizing the PET surface, it becomes more suitable for applications where contact with water is necessary, such as in medical devices, food packaging, or membranes used for water purification.

![HP layout competition1](https://static.igem.wiki/teams/4901/wiki/projects/image-91.png)

Hydrolysis of p-nitrophenyl butyrate (pNPB)

In the experiment described, the hydrolysis of p-nitrophenyl butyrate (pNPB) was studied using two enzymes: IsPETase and wild-type (WT) enzyme. The reaction conditions included a substrate concentration of 2500 uM (micromolar), a slightly basic pH of 9.0, and a temperature of 30°C.

During the experiment, the enzymes catalyzed the hydrolysis of pNPB, breaking it down into p-nitrophenol and butyric acid. We used this reaction to understand the kinetics and substrate specificity of enzymes. The specific purpose of this experiment is investigating the efficiency of IsPETase compared to the wild-type enzyme, studying the enzyme's activity under specific pH and temperature conditions, and exploring potential applications in biotechnology.

In the experiment comparing PETase wildtype with the variant BBa\_J435500\_W159H\_S238F, the researchers assessed their respective enzymatic activities under specific conditions. The experiment focused on the hydrolysis of p-nitrophenyl butyrate (pNPB) at a concentration of 2500 nanomolar, with the reaction taking place at pH 9.0 and a temperature of 30 degrees Celsius. The results demonstrated a remarkable difference in enzymatic activity between the two variants.

Wildtype PETase: The wildtype PETase exhibited a relatively lower enzymatic activity under the specified conditions. The graph representing the wildtype PETase's activity showed a gradual increase in product formation (p-nitrophenol) over time. However, the rate of reaction was comparatively slower.

BBa\_J435500\_W159H\_S238F: In contrast, the variant BBa\_J435500\_W159H\_S238F displayed significantly enhanced enzymatic activity. The graph depicting its activity revealed a much steeper slope, indicating a rapid conversion of pNPB into p-nitrophenol. The variant exhibited a catalytic efficiency that was seven times higher than that of the wildtype PETase under the same conditions.

This significant improvement in enzymatic activity in the BBa\_J435500\_W159H\_S238F variant can be attributed to the specific mutations (W159H and IsPETase) incorporated into its structure. These mutations likely enhance the substrate-binding affinity and catalytic efficiency of the enzyme, leading to the observed increase in activity. Overall, the results highlight the potential for using BBa\_J435500\_W159H\_S238F variant in applications requiring efficient enzymatic hydrolysis of PET (polyethylene terephthalate) or similar substrates, as it outperforms the wildtype PETase by a substantial margin under the specified experimental conditions.

To evaluate the activity of expressed PETase enzyme in comparison to the original wild type and our BBa\_J435500\_W159H\_S238F PETase variant, we conducted assays using p-nitrophenyl butyrate. This compound mimics the ester bonds found in PET plastic. When PETase enzymes break these ester bonds, they release a signal at 405 nm, so we observed absorbance at 405 nm. Additionally, to compare the activity of wild type, we used the result of wild-type PETase activity from iGEM registry parts (Part: BBa\_K2910000) designed by iGEM 2019 Toronto team. The graph above demonstrates the outcomes of these assays for the mentioned PETase variants.

References

1. Majid H. Al-Jailawi, Rasha S. Ameen and Ali A. Al-Saraf (2015). Polyethylene degradation by Pseudomonas putida S3A. Int. J. Adv. Res. Biol.Sci. 2(1): (2015): 90-97

2. Writtik Maity, Subhasish Maity, Soumen Bera, Amrita Roy (2022). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes. Applied Biochemistry and Biotechnology <https://doi.org/10.1007/s12010-021-03562-4>

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# Parts Library

LB Agar

LB-Ampicillin Agar

SOB Medium

SOC Medium

TE Buffer

50x TAE

SDS-PAGE

Tris-Glycine-SDS

Protein extraction

A (Wash) buffer

B (Elution) buffer

Protocols:

LB Agar (per Liter)

● 10 g of NaCl

● 10 g of tryptone

● 5 g of yeast extract

● 20 g of agar (only for solid media)

● Add deionized H2O to a final volume of 1 liter

● Adjust pH to 7.0 with 5 N NaOH

● Autoclave

LB-Ampicillin Agar (per Liter)

● 1 liter of LB agar, autoclaved

● Cool to 55°C

● Add 10 ml of 10 mg/ml filter-sterilized ampicillin

● Pour into petri dishes (~25 ml/100-mm plate)

SOB Medium (per Liter)

● 20.0 g of tryptone

● 5.0 g of yeast extract

● 0.5 g of NaCl

● Add deionized H2O to a final volume of 1 liter

● Autoclave

● Add 10 ml of filter-sterilized 1 M MgCl2 and 10 ml of filter-sterilized 1 M MgSO4 prior to use

SOC Medium (per 100 ml)

● Note This medium should be prepared immediately before use.

● 2 ml of filter-sterilized 20% (w/v) glucose or 1 ml of filter-sterilized 2 M glucose

● SOB medium (autoclaved) to a final volume of 100 ml

TE Buffer

● 10 mM Tris-HCl (pH 7.5)

● 1 mM EDTA

50 x TAE buffer recipe

● 2 M Tris base

● Cool to 55°C

● 0.05 M of 0.5 M EDTA0

● Add deionized H2O water to a final volume of 1 liter

SDS-PAGE Sample 5X Loading Buffer

● 250 mM Tris·HCl, pH 6.8

● 10% SDS

● 30% (v/v) Glycerol

● 10 mM DTT

● 0.05% (w/v) Bromophenol Blue

● Add deionized H2O water to a final volume of 50 ml

Tris-Glycine-SDS running Buffer X10

● Dissolve 30.0 g of Tris base

● 144.0 g of glycine

● 10.0 g of SDS in 1000 ml of H2O

● The pH of the buffer should be 8.3

● No pH adjustment is required

● Store the running buffer at room temperature

● Dilute to 1X before use.

Protein extraction lysis buffer

● 50 mM Tris·HCl, pH 8.0

● 300 mM NaCl

● 10 mM Imidazole

● 2% Triton X-100

● 5% (v/v) Glycerol

● 5 mM DTT

● 1 mM β-mercaptoethanol

● Add deionized H2O water to a final volume of 100 ml

A (Wash) buffer

● 50 mM Tris·HCl, pH 8.0

● 300 mM NaCl

● 10 mM Imidazole

B (Elution) buffer

● 50 mM Tris·HCl, pH 8.0

● 300 mM NaCl

● 500 mM Imidazole

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# Results

Absract

Contribution

Parts

Enginering Success

Absract

In our ever-evolving world, where plastic pollution poses a formidable challenge, our team stands undeterred, united by a shared thirst for knowledge and a commitment to making a difference. As we participate in the IGEM competition for the second time, our focus remains sharp: addressing the pervasive issue of polyethylene terephthalate (PET) pollution. PET, a widespread polymer foundational to countless consumer products, particularly in packaging, lingers in our environment, refusing to degrade for centuries.

Despite the daunting challenges and our previous bouts with the limitations of knowledge, our passion has served as our North Star, guiding us forward. Our collective determination has brought to our attention the incredible potential of two enzymes: PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalate hydrolase (MHETase). Working synergistically, these enzymes can decompose PET into its basic building blocks, offering a revolutionary solution to the persistent issue of plastic pollution.

Despite the daunting challenges and our previous bouts with the limitations of knowledge, our passion has served as our North Star, guiding us forward. Our collective determination has brought to our attention the incredible potential of two enzymes: PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalate hydrolase (MHETase). Working synergistically, these enzymes can decompose PET into its basic building blocks, offering a revolutionary solution to the persistent issue of plastic pollution.

Despite the daunting challenges and our previous bouts with the limitations of knowledge, our passion has served as our North Star, guiding us forward. Our collective determination has brought to our attention the incredible potential of two enzymes: PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalate hydrolase (MHETase). Working synergistically, these enzymes can decompose PET into its basic building blocks, offering a revolutionary solution to the persistent issue of plastic pollution.

Our journey is centered on enhancing these enzymes' capabilities. By understanding existing mutations and exploring the potential for new ones, we aim to create a biocatalytic process that is both efficient and transformative. We picture a world where waste PET is not just discarded but is seen as a valuable resource, thanks to a process that can convert this waste into indispensable raw materials.

Contribution

In our mission to address PET degradation, we understand the value of building upon established knowledge. Rather than starting afresh, we're diving deep into previously discovered mutations, harnessing their insights to advance our work. That is why our team started from modelling and analysing the efficiency of mutations for degradation of plastic materials. We successfully performed a comparative analysis of various mutations including S238F, R61A, L88F, R280A, I179F, S121E, D280A for PETase enzyme and Phe424D, Phe424H, Phe424V, Phe424E, R411K for MHETase enzyme.

![HP layout competition1](https://static.igem.wiki/teams/4901/wiki/projects/image-77.png)

![HP layout competition1](https://static.igem.wiki/teams/4901/wiki/projects/image-78.png)

Our analysis showcased that, at 37°C with concentrations of 10 μg/mL, the combined action of PETase and MHETase resulted in a 40% reduction of a 100 μm PET film in 24 hours. Crucially, mutations such as S131G, E226T, and F495I impacted degradation negatively, underscoring their pivotal role in enzymatic action. This combined enzymatic approach, notably more effective than PETase alone, hints at the vast potential for efficient PET recycling, emphasising the importance of these specific mutations in the degradation process.

Parts

In biotechnology research and innovation, a key focus should be the potential modifications of the enzymes PETase and MHETase, which play an important role in the degradation of polyethylene terephthalate (PET). To this end, our team has developed a significant number of parts and constructs this year, although not all have been fully validated due to time and resource constraints

Our main goal was to clone the studied genes into appropriate vectors for expression in Pseudomonas putida. There are several plasmids with dual expression systems that can be compatible with Pseudomonas putida for co-expression of multiple genes. So, 2 vectors J435500 and pSEVA-25 were used to clone PETase and MHETase genes. During the research process, we have created and provided many designs and parts that, although not fully tested, may be useful to future researchers. We hope that our work will be a starting point for further research and bring new ideas and approaches to the study of plastic decomposition.

![HP NIS Talks1](https://static.igem.wiki/teams/4901/wiki/projects/image-81.png)

Figure 1: Map J435500 vector with differentially cloned genes.

![HP NIS Talks1](https://static.igem.wiki/teams/4901/wiki/projects/image-82.png)

Figure 2. Map of pSEVA-251 dual expression plasmid with CJ-MHETase and PETase genes.

Engineering Success

We had to overcome significant challenges on the way to beginning our experimental work. In the beginning, we were faced with waiting for permission from the university, which took more than four months. This wait was not an easy ordeal, requiring patience and perseverance, but we moved forward steadily, eager to begin our work. At the same time we had limited resources and were not able to order MHETase enzyme to fully test out our constructs. So, we tried to use everything available to conduct experiments and came up with the idea to build a part out of materials we had in the Distribution Kit 2023.

So, we used BBa\_K2910000 and BBa\_J435500 parts to test out the protein expression and enzymatic activity of IsPETase-W159H-S238F with C-terminal Hexahistidine Tag and created a new part BBa\_K4901036 (http://parts.igem.org/Part:BBa\_K4901036).

Emphasis was placed on studying the activity of the BBa\_J435500\_W159H\_S238F variant compared to the wild-type enzyme. The experiment focused on the hydrolysis of pNPB at a substrate concentration of 2500 µM, at a slightly alkaline pH of 9.0 and a temperature of 30°C. The results of this part of the study showed a significant difference in enzymatic activity between the two variants.

![HP NIS Talks1](https://static.igem.wiki/teams/4901/wiki/projects/image-91.png)

The results demonstrated a remarkable difference in enzymatic activity between the two variants.
Wildtype PETase: The wildtype PETase exhibited a relatively lower enzymatic activity under the specified conditions. The graph representing the wildtype PETase's activity showed a gradual increase in product formation (p-nitrophenol) over time. However, the rate of reaction was comparatively slower.
BBa\_J435500\_W159H\_S238F: In contrast, the variant BBa\_J435500\_W159H\_S238F displayed significantly enhanced enzymatic activity. The graph depicting its activity revealed a much steeper slope, indicating a rapid conversion of pNPB into p-nitrophenol. The variant exhibited a catalytic efficiency that was seven times higher than that of the wildtype PETase under the same conditions.
In our research, we meticulously engineered dual expression plasmids harbouring both PETase and MHETase enzymes. The objective was to optimise PET degradation efficacy, with an eye on scalability for potential implementation in large-scale PET degradation facilities. This endeavour aimed to facilitate enhanced PET recycling by leveraging the synergistic capabilities of these enzymes. Particularly notable was the BBa\_J435500\_W159H\_S238F variant of PETase, which showcased a sevenfold efficiency increase compared to its wildtype counterpart. Despite challenges, we innovated with available resources, setting the stage for scalable PET degradation solutions in industrial settings. Our findings illuminate a promising avenue for sustainable plastic waste management in the future.