Project Description

Overview

Plastic cling films are widely used in food preservation but pose long-term environmental risks due to poor degradability, while microbial spoilage continues to cause significant food waste. To address these challenges, SMS-Shenzhen is designing a biodegradable and antimicrobial cling film using synthetic biology. We introduced the pls gene from Streptomyces albulus into Bacillus subtilis to enable ε-polylysine (PLL) production. To improve biosynthetic efficiency, we introduced PPK2 to regenerate ATP formation. Additionally, to increase the lysine production, we are replacing the native thrD genes with more active variants lysC311 to enhance lysine supply. A kill switch is constructed by knocking out the alrA gene, rendering the strain auxotrophic and limiting its survival to controlled conditions. In parallel, we are developing a bilayer film by combining purified PLL with chitosan and sodium alginate-based materials. Our goal is to create a cling film that is both antimicrobial and biodegradable, offering a safer and more sustainable alternative to traditional plastic packaging.

Problem

Cling film, as a material widely used for food storage and packaging, has a developmental history that can be traced back to the mid-20th century. Initially, synthetic polymers such as polyvinyl chloride (PVC) and polyethylene (PE) were introduced to the market due to their excellent barrier and flexibility properties, significantly improving food storage conditions and extending the shelf life of food. With the advancement of technology, the production processes and types of materials for cling film have continued to diversify, leading to its widespread adoption in everyday households and the food industry. At the household level, cling film is an essential tool in the kitchen, used for packaging leftovers, fruits, and vegetables to maintain their freshness and prevent odor transfer. In the food industry, cling film is widely used for the packaging of meats, dairy products, baked goods, and agricultural products, ensuring the quality and safety of products during transportation and sales.

However, despite the extensive application of traditional cling film, it has two prominent issues that pose severe challenges to the environment and food safety.

Firstly, traditional cling film generally lacks antibacterial properties. Although cling film can physically isolate food from the external environment, the growth and reproduction of microorganisms (such as bacteria and fungi) during food storage remain the main cause of food spoilage and deterioration. Inside the packaging, if the food itself is contaminated with microorganisms or if there are microbial residues during the packaging process, these microorganisms will continue to multiply, thereby shortening the shelf life of the food. The proliferation of microorganisms not only shortens the shelf life of food, leading to significant food waste, but also may trigger foodborne diseases, posing a potential threat to consumer health. According to the World Health Organization (WHO), approximately 600 million individuals fall ill and 420,000 die annually due to the consumption of contaminated food ^[1]. These alarming statistics underscore the urgent need for effective antimicrobial cling films.

Secondly, traditional cling film, especially petroleum-based plastic films, has the problem of being difficult to biodegrade. Materials such as polyethylene (PE), polypropylene (PP), and low-density polyethylene (LDPE) have stable chemical structures that require hundreds of years or even longer to degrade in the natural environment ^[6]. This has led to serious plastic pollution problems, causing long-term harm to ecosystems, such as the accumulation of microplastics in marine ecosystems, as well as pollution of soil and water bodies. Since LDPE and similar materials are the main components of household cling film, the extensive use of such cling film will lead to extremely serious environmental consequences. Research indicates that more than 1,500 species in marine and terrestrial environments are known to ingest plastics, leading to serious health problems ^[2][3]. The development of biodegradable alternative materials has become a focus of global scientists and the industry.

In summary, although cling film plays an important role in modern life, its inherent poor antibacterial properties and non-biodegradability pose significant challenges in terms of sustainable development and food safety, urgently requiring innovative solutions.

Fig1. How the environment and human lives are affected by plastic waste polluted in soil

Fig2. How the environment and human lives are affected by plastic waste polluted in water

Goal

Selection and Large-Scale Production of Antimicrobial Materials

We are committed to finding a suitable antimicrobial material that can be effectively integrated into the production process of cling film and has the potential for large-scale production by using synthetic biology, thereby endowing the cling film with significant antimicrobial properties.

Development of Polyethylene Alternatives and film-forming agents

Another key objective of this project is to develop bio-based materials and film-forming agents to replace traditional polyethylene (PE) plastics, making cling film degradable. This will help address the issue of traditional plastics being difficult to decompose in the environment and causing long-term pollution due to their accumulation.

Material Blending and Production Validation

Ultimately, the project aims to effectively blend the selected antimicrobial materials with degradable alternatives and film formers, and put them into actual production to manufacture a truly viable and high-performance new type of cling film. This stage will involve optimizing material formulations, developing process flows, and testing product performance to ensure that the produced cling film maintains food freshness while having good antimicrobial activity and complete biodegradability, meeting market demands and environmental sustainability requirements.

Fig3. Our goals

Our solution

Finding the suitable antimicrobial material

Principle and antimicrobial mechanism:

Based on these objectives, we systematically screened various potential materials, ultimately focusing our research on ε-poly-L-lysine (ε-PLL). ε-PLL is a water-soluble homopolymer produced by microorganisms like Streptomyces albulus through fermentation. With a molecular weight ranging from 3.2 to 4.5 kDa, ε-PLL is highly water-soluble and biocompatible ^[4][5][6]. Given its natural origin and unique structure, ε-PLL is recognized as a safe, non-toxic, edible antimicrobial agent. It is listed by the FAO/WHO as a food preservative and is approved for food preservation in countries like the United States, Japan, and South Korea ^[10]. Moreover, ε-PLL is biodegradable, breaking down into L-lysine monomers that re-enter the natural cycle, meeting our project's environmental requirements.

The antimicrobial mechanism of ε-PLL is due to its positive charge and ability to disrupt microbial cell membranes ^[5]. ε-PLL molecules, containing multiple free amino groups, are cationic under physiological pH conditions. When they come into contact with negatively charged microbial cell membranes, they adsorb to the membrane surface through electrostatic attraction^[7]. This disrupts the membrane's integrity and stability, increasing permeability and leading to the leakage of essential cellular components (such as ATP, proteins, nucleic acids) and inhibiting intracellular enzyme activity, ultimately causing microbial cell death.

For example, ε-PLL has broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria, including common pathogens like Staphylococcus aureus, Salmonella typhimurium, and Klebsiella pneumoniae. It also shows antifungal activity. This makes ε-PLL highly promising for applications in food packaging, medical devices, wound dressings, and water treatment. Integrating ε-PLL into cling film materials can effectively inhibit microbial growth, extend food storage time, reduce cross-contamination risks, and enhance product safety and hygiene.

Current applications and limitations:

ε-PLL is widely used in the food industry as a natural preservative, for example, to extend the shelf life of bread, noodles, rice, seafood, and meat products. ε-PLL has also shown potential in the biomedical field; for instance, natural silk fiber membranes modified with ε-PLL have been developed for wound dressings, which help address the common issue of bacterial infections during skin wound healing.

Despite the promising prospects of ε-PLL, its application and large-scale production still face challenges:

1. Production Cost and Efficiency: ε-PLL is mainly produced through microbial fermentation. Although co-culture fermentation strategies can increase yield, current production methods still face challenges in achieving a balance between low cost and high production volume ^[7]. The high production cost limits the application of ε-PLL in a broader range of industrial fields.
2. Compatibility and Processability with Other Materials: ε-PLL is a water-soluble polypeptide ^[4][7][10], while traditional food packaging films are mostly hydrophobic polymers. This difference in properties may lead to uneven dispersion of ε-PLL in the polymer matrix, affecting its antimicrobial efficacy and the mechanical properties of the packaging film.
3. Long-term Antimicrobial Effectiveness and Stability: As a bioactive molecule, the long-term stability and antimicrobial effectiveness of ε-PLL in packaging films are susceptible to environmental factors such as humidity, temperature, pH, and ultraviolet light.
4. Impact on Material Mechanical Properties: The addition of ε-PLL may alter the mechanical properties of the packaging film, such as flexibility, tensile strength, and barrier properties. Particularly in biodegradable packaging films, the incorporation of ε-PLL needs to be carefully optimized to avoid negative impacts on the film's mechanical strength, toughness, and selective permeability to gases.
5. Challenges of Environmentally Friendly Materials: Although ε-PLL itself is biodegradable, to achieve the overall biodegradability of the packaging film, it is necessary to find environmentally friendly materials that can replace traditional petroleum-based plastics.

Engineering Bacillus subtilis

Advantages of Bacillus subtilis as a chassis:

Rapid Growth Cycle and High-Efficiency Production:
Bacillus subtilis has an extremely rapid growth rate, which is crucial for enhancing fermentation efficiency and shortening the production cycle. In contrast, Streptomyces, a natural actinomycete that produces ε-PLL, typically has a longer growth cycle and a slower fermentation process, thereby limiting the rapid, large-scale production of ε-PLL ^[8]. The fast-growing Bacillus subtilis can reach high cell density more quickly, thus producing more ε-PLL in the same amount of time.

Strong Extracellular Protein Secretion Capability:
Bacillus subtilis has a naturally robust extracellular protein secretion system, which can efficiently secrete the target product into the culture medium^[9]. This characteristic simplifies the downstream purification process of ε-PLL and reduces production costs. The extracellularly secreted product is easier to separate and purify, avoiding the complex steps of cell lysis and separation of intracellular components.

Tolerance to Extreme Conditions:
Bacillus subtilis can form spores, enabling it to withstand a variety of extreme environmental conditions, such as high temperatures, extreme pH values, desiccation, and radiation^[11]. In contrast, Streptomyces albus has inferior adaptability to extreme environments compared to Bacillus subtilis, which can affect its stable production in complex industrial settings.

Convenience and Safety of Genetic Manipulation:
Bacillus subtilis is a model organism for genetic manipulation, with well-developed genetic tools and techniques that facilitate genetic engineering to optimize the production pathway of ε-PLL. Moreover, many strains of Bacillus subtilis have been recognized as “Generally Recognized As Safe” by the U.S. Food and Drug Administration (FDA), which gives it an advantage in applications in the food and biomedical fields and reduces regulatory barriers.

Fig4. Advantages of bacillus subtilis as a chassis

Genetic engineering strategies for ε-PLL biosynthesis

First Cycle: Construction of the ε-Poly-L-lysine Synthase (PLS) Pathway

The core objective of this phase is to introduce the key gene for ε-PLL biosynthesis—the pls gene—into Bacillus subtilis. We initially sythesized the DNA sequence encoding the pls gene with coden optimazation the natural ε-PLL-producing strain Streptomyces albulus. The optimized pls gene was then cloned into the high-efficiency expression vector pMA5 plasmid to form the recombinant plasmid pMA5-pls. The pMA5 plasmid is an ideal choice for expressing exogenous genes due to its high copy number and stability in B. subtilis. Subsequently, through chemical tranformation, the constructed pMA5-pls plasmid was efficiently transformed into the B. subtilis host strain, thereby giving B. subtilis the ability to synthesize ε-PLL.

Fig5. ε-PLL production in B.subtilis after transformation of pls

Second Cycle: Enhancement of L-Lysine Precursor Supply and ATP Supply Optimization

The synthesis of ε-PLL is an energy-consuming process that requires a large supply of ATP. To further enhance the production efficiency of ε-PLL, we introduced the gene encoding polyphosphate kinase 2 (ppk2) into the pMA5-pls plasmid, forming the recombinant plasmid pMA5-pls-ppk2. The ppk2 gene can enhance the regeneration capacity of ATP within the cell, providing stronger energy support for the synthesis of ε-PLL. Ultimately, the pMA5-pls-ppk2 plasmid was electroporated into B. subtilis.

In addition, in order to relieve the substrate limitation of L-lysine in the biosynthetic pathway of B. subtilis, we knocked out the endogenous thrD gene in B. subtilis and replaced it with the lysC gene from Corynebacterium glutamicum by using CRISPR-Cas9 ^[12]. C. glutamicum is renowned for its efficient production of L-lysine, and the aspartate kinase encoded by its lysC gene can relieve feedback inhibition in the ε-PLL production process, effectively increasing the synthesis of L-lysine and thus providing sufficient substrate for ε-PLL production ^[8].

Fig6. ε-PLL concentration increases after adding ppk2 and genome editing of thrD

Third Cycle: Construction of a Biosafety and Plasmid Stability Assurance Mechanism Based on the lacI/alrA System

To meet the requirements for biosafety and plasmid stability in industrial applications, we constructed a self-control system to ensure that the engineered strains can survive and produce only under specific conditions and create a “suicide” mechanism in unintended environments. To achieve this goal, we checked literature and selected to use a nutrition depletion method to knock out the essential nutrition gene arlA^[13], and introduce a lac operon controlled arlA vector to restore its function ^[14].

We knocked out the alrA gene from the genome of B. subtilis and cloned it into the pHT43 plasmid. The alrA gene plays a key role in the synthesis of peptidoglycan in bacterial cell walls, and its product D-alanine is an essential component for building the tetrapeptide side chain of peptidoglycan.

Simultaneously, we also cloned the lac controlled alrA gene into the pMA5-pls-ppk2 plasmid. The repressor protein encoded by this gene can bind to the promoter and inhibit the expression of downstream genes, with its activity being induced by lactose. In this system, the expression of the alrA gene is placed downstream of the promoter regulated by lacI. This means that only when lactose is added to the culture medium, the inhibitory effect of the LacI repressor is relieved, allowing the expression of the alrA gene and thus enabling the host strain to synthesize D-alanine and grow normally.

Through this suicide switch, the survival of the engineered strains will strictly depend on the presence of IPTG in the culture environment and the stable carriage of the plasmid. Once the strain escapes from the controlled environment or loses the plasmid, the alrA gene will not be expressed, leading to defects in cell wall synthesis and ultimately preventing the strain from surviving. This mechanism not only effectively ensures biosafety, preventing the spread of engineered strains in the natural environment, but also provides an antibiotic-free selection strategy for maintaining plasmid stability in industrial production and avoids the risk of antibiotic contamination.

Fig7. Test result on the ε-PLL yield before and after the cycles are completed

Purification of ε-PLL

Product overview:

After we finish engineering on the bacteria, the next step is to extract the ε-PLL from the bacterial solution in order to utilize it on our cling film product. We are inspired by several studies investigating the purification of the ε-PLL to choose centrifugation to separate the bacteria and the bacteria culture supernatant and use column filtration to extract ε-PLL from the supernatant^{[15] [16][17][18][19]}. The product comprises scafold for support, four filtration columns with different filtration functions, a peristaltic pump for provision of suction force, a collection bottle for product collecting and several tubes to make the filtration a successive process. The four columns are lined up and connected in order to for efficiency and correct step-by-step purification of ε-PLL.

Fig8. ε-PLL Purification hardware

Downstream purification:

ε-PLL is eluted from the supernatant through a step-by-step down stream purification of our hardware product:

1. We add the bacterial culture supernatant to a column packed with Amberlite IRC-50 （a type of weakly acidic cation exchange resin) extraction of ε-PLL from the bacterial culture supernatant. As a polycation, ε-PLL can be adsorbed onto the surface or pores of the Amberlite IRC-50 through electrostatic interactions. Therefore, the ε-PLL in the bacterial culture supernatant can be extracted from solution while the solution flow out of the column under the effect of gravity.
2. After the adsorption is completed, diluteacetic acid is added into the column with Amberlite IRC-50 in order to elute the ε-PLL from the resin. This elution process is achieved mainly through acetic acid's low pH property and the ability to neutralize the functional group of Amberlite IRC-50. As a result, the adsorption effect Amberlite IRC-50 on ε-PLL is undermined and ε-PLL is desorbed from the resin.
3. The eluted solution is pumped into two continuous columns packed with activated carbon for decolorization treatment ^[9]. Activated carbon, with its highly developed pore structure and large specific surface area, can effectively adsorb pigment molecules and other organic impurities in the solution.
4. The solution passes through a column packed with filtration cotton in order to remove the remaining carbon particles left by the active carbon columns from the solution . This is achieved by mechanical filtration characteristic of filtration cotton. The filtration cotton is capable of removing any possible tiny particles in the solution to ensure cleanliness of our ε-PLL solution.

Through the above five steps, we can successfully extract ε-PLL from the bacterial culture supernatant with our hardware device, obtaining the PLL that can be used into out cling film production with only dilution.

Biodegradable Antibacterial Membrane Production

Film material selection and design:

At the initial stage of our project, we extensively reviewed relevant research findings ^[15] and selected three representative film-forming systems for experimental validation: PLA–chitosan–ε-PLL, pullulan–chitosan–ε-PLL, and sodium alginate–chitosan–ε-PLL. Subsequently, we conducted film-forming experiments, antibacterial and mechanical performance tests to evaluate their feasibility in food packaging applications. According to the results, we concluded that sodium alginate–chitosan mambrane was the ideal carrier of ε-PLL.

Bilayer film with ε-PLL incorporation

We adopted a bilayer film preparation strategy, aiming to significantly enhance the tensile strength, mechanical properties, and antimicrobial activity of the food preservation film. By following the membrane production methods from literature and adding ε-PLL, we successfully prepared sodium alginate–chitosan bilayer membranes. At first, their integrated performance was relatively limited. To optimize the final performance, we tried to adjust various variables, such as the concentration of ε-PLL, the drying time and the drying temperature. To enhance the cross-linking performance and stability of the bilayer film and further improve its physicochemical properties, we introduced a 3% calcium chloride solution sprayed between two layers, which enhances the electrostatic interaction between molecules. Eventually we comfirmed the ultimate production process as well as amounts of materials and successfully produced idealized membranes after repetitive experiments and tests.

Results of Antibacterial tests and tensile-strength tests

We then conducted a series of food preservation tests and tensile-strength tests to evaluate the performance of our membrane. Our membrane showed a significant decrease of bacteria compared to common commercial cling membrane.

Fig9. The antibacteria and anti-fungi effect of our ε-PLL membrane vs commercial cling membrane

Fig10. The tensile-strength test of our membrane vs commercial membrane

Our membranes exhibited reliable film-forming capacity, sufficient mechanical strength, and measurable antibacterial effects, meeting the requirements for food packaging applications. In addition, this approach employs biodegradable materials and thereby is eco-friendly.

Bilayer film with ε-PLL incorporation

After successfully addressed the problems, we reach a consensus that our project may hold significant commercial potential, with a substantial global market for cling film and a growing demand for sustainable and antimicrobial solutions. Our biodegradable antimicrobial food wrap is poised to capture a significant share of this market, appealing to both environmentally conscious consumers and businesses seeking to enhance their sustainability profiles. Therefore, we begin to research market and stakeholder’s engagement.

Market Research and Stakeholder Engagement:

To ensure the market success of our product, we conducted extensive market research and stakeholder engagement activities. We distributed electronic questionnaires to individuals across various age groups to gather insights into their perceptions of food safety, environmental concerns, and the potential adoption of our novel food wrap. The feedback received was overwhelmingly positive, indicating a strong demand for a product that addresses both food preservation and environmental sustainability.

In addition to the questionnaires, we conducted interviews with experts in the field of food microbiology, transportation managers, fruit farmers, and other key stakeholders. These interviews provided valuable insights into the practical needs and preferences of potential users, helping us refine our product design and identify key market segments.

Based on the insights gathered, we developed a comprehensive business plan that outlines our strategy for commercializing the product. This plan includes detailed market analysis, product positioning, and a roadmap for scaling up production and distribution. We are confident that our product will not only meet the needs of consumers but also drive significant market adoption through its unique value proposition.

Fig11. Interview with SUTPC Canteen

Real-World Impact and Community Engagement:

Through this project, we have gained a deep appreciation for the real-world applications of iGEM and the potential to positively impact daily life. Our efforts extend beyond the laboratory, as we strive to raise awareness about the importance of food safety and environmental sustainability. To achieve this, we have engaged with the community through various outreach activities, including presentations to middle and high school students, promotional events, and the establishment of an iGEM club at Shenzhen Middle School. These initiatives aim to inspire the next generation of scientists and promote a broader understanding of synthetic biology and its applications.

Fig12. Education at Pingshan Experimental School

Conclusion

Our IGEM project represents a significant step forward in addressing the pressing issues of food safety and environmental sustainability. By developing a biodegradable antimicrobial food wrap, we have created a product that not only solves real-world problems but also holds substantial commercial potential. Through extensive market research, stakeholder engagement, and community outreach, we are well-positioned to bring this innovative solution to market and drive meaningful change. We are excited about the future of our project and its potential to make a lasting impact on both the environment and public health.

References