In our commitment to upholding the highest safety standards, we approach safety from multiple dimensions throughout our project. From a design perspective, we have thoroughly considered and integrated biosafety measures. With an eye toward future commercialization and industrial application, we have adopted—and recommend to the iGEM community—a novel Pichia pastoris genome integration vector system. This system is designed to automatically excise antibiotic resistance markers upon induction, thereby preventing potential antibiotic misuse during high-copy screening for high-yield strains in industrial settings.

In parallel, we have established stringent laboratory safety protocols to ensure all experiments are conducted in full compliance with specifications. All team members have undergone comprehensive safety training and regularly pass lab safety assessments. In terms of human practices, we adhere to strict ethical procedures to safeguard the privacy and security of every interviewee who provides information support. Furthermore, we emphasize the importance of biosafety in synthetic biology during educational activities, placing safety at the core of all our public engagement initiatives.

Our laboratory is classified as a Level 2 Containment Facility, and primarily handles Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris in our projects.We have comprehensive Personal Protective Equipment (PPE), including disposable gloves, masks, lab coats, goggles, and other gear. By establishing different isolation areas, such as rest areas and experimental zones, we effectively prevent contamination and infection. Additionally, the establishment of electrophoresis rooms also effectively prevents the leakage of gel dyes or direct contact with people.

Our Goal

To achieve the conversion of straw waste into high-value textile fibers, we have developed a comprehensive synthetic biology strategy that simultaneously addresses lignin degradation and fiber performance enhancement. Our technical approach features a dual-system design: First, we have created an enzyme immobilization platform where Pichia pastoris (GS115) expresses three lignin-degrading enzymes (laccase, versatile peroxidase, and lytic polysaccharide monooxygenase), which are subsequently displayed on the Saccharomyces cerevisiae (EBY100) cell surface via the AGA1/AGA2 system. Second, we utilize Escherichia coli (DH5α/BL21) to express high-performance proteins (HBP and EV1) fused with cellulose-binding module 3 (CBM3), which are incorporated during the spinning process to reinforce the straw fibers.

From the initial design phase, biosafety considerations were proactively integrated into our selection of biological components. All microbial chassis (S. cerevisiae EBY100, P. pastoris GS115, and E. coli DH5α/BL21) were specifically chosen for their well-established non-pathogenic nature and safety profiles. Similarly, all enzymatic and structural protein components originate from naturally occurring, biologically safe sources.

While these fundamental biological components demonstrate inherent safety, our forward-looking risk assessment has identified potential challenges in both laboratory operations and future industrial scale-up. These considerations, along with our corresponding control strategies, form an integral part of our project's safety framework, ensuring comprehensive risk management throughout the development process.

Risk

Chemical Hazards

In the laboratory, some potentially hazardous chemicals are frequently used, such as

Flammable Chemicals:methanol and ethanol

Carcinogenic Chemicals: Safe Gel Red nucleic acid dye, polyacrylamide (PAGE), Tris, SDS, and Coomassie Brilliant Blue.

Toxic Chemicals:2,6-DMP，ABTS

Corrosive chemicals: NaOH solution，H2O2 solution

Sharp Object

Sharp objects such as scissors, needles, and broken glass may cause cuts or punctures

Biohazardous production

Yeast fermentation supernatant and E. coli lysate

Industrialization Risks

Strains with higher antibiotic resistance typically achieve greater expression yields, necessitating the use of high-expression, stable strains for industrial production. However, since multicopy integration in Pichia pastoris occurs with low probability, screening for such strains requires high concentrations of antibiotics at a large scale. This practice poses a significant risk of antibiotic misuse.

Safety measures

To reduce safety risks in the laboratory, we have implemented comprehensive safety measures, including

Training and Education

We have established a comprehensive laboratory safety training system to ensure all students pass a formal safety assessment before being authorized as full laboratory members. The assessment consists of two integral components: a written examination and a long-term practical evaluation.

The written exam, curated and reviewed by two experienced principal investigators, includes two versions (Form A and Form B) with identical content but varied question sequences and answer choices. It covers essential topics such as equipment and reagent safety, standard microbiological practices, and includes specialized questions—for instance, on the sporulation behavior of haploid Pichia pastoris under nitrogen-deficient conditions—to ensure thorough understanding of potential biological risks.

A minimum score of 90 out of 100 is required to pass the written test. Subsequently, candidates enter a long-term observational assessment phase, during which an experienced instructor monitors their laboratory work. Any unsafe practice is documented: the first instance results in a warning, the second in a symbolic monetary contribution to the team fund, and the third leads to temporary revocation of laboratory access, requiring re-examination.

This structured approach ensures that all student researchers are thoroughly prepared and consistently mindful of safety throughout their experimental work.

Strict Zoning Regulations

The laboratory space is divided into distinct zones: the Molecular Biology Area, Electrophoresis Contamination Area, Microbiology Laboratory, and Materials Science Laboratory. Personal items are strictly prohibited in all laboratory zones, and cross-use of reagents and consumables between different experimental areas is not permitted.

Electrophoresis Contamination Area: Experiments conducted in this area include agarose gel electrophoresis, SDS-PAGE gel electrophoresis, and Western blot. Personnel must strictly adhere to safety protocols to avoid any physical contact with surfaces, reagents, consumables, equipment, or the floor.

Microbiology Laboratory: This laboratory is used for microbial culture, fermentation, and cell disruption experiments, utilizing Pichia pastoris, Saccharomyces cerevisiae, and Escherichia coli. It is equipped with a biosafety cabinet for aseptic operations. Regular UV sterilization is performed, and all waste must be securely sealed in yellow sterilization bags and autoclaved before removal.

Molecular and Materials Science Laboratories: Experimental workstations in these labs are assigned to specific users. All benches must be wiped with alcohol before and after use to prevent potential exposure to hazardous substances.

Additionally, operations involving flammable or volatile reagents—such as glacial acetic acid, methanol, petroleum ether, and 2,6-DMP—as well as treatments of straw with sodium hydroxide, water, or sodium hypochlorite must be conducted inside a fume hood.

Meanwhile, we have established a strict waste disposal system to prevent environmental contamination and microbial infection. Specifically for the disposal of culture media, we adhere to the following protocols:

In response to the iGEM competition’s specific safety guidelines regarding yeast usage—particularly for high school teams—we have implemented rigorous rules to raise student awareness of biosafety when working with haploid yeast strains. These measures ensure that all team members fully recognize and adhere to safe experimental practices throughout the project.

Solid Media Handling:Cultures maintained on solid media for over 3 days will be transferred to 4°C storage to suppress metabolic activity. Any plates stored beyond 2 weeks will undergo autoclave sterilization. All solid plates are securely sealed with parafilm to prevent leakage.

Liquid Culture Management:YPD Liquid cultures will be discarded after 24 hours to ensure optimal conditions.

Induction Media (SD-CAA/BMMY): Cultures will not exceed 72 hours of use; any samples surpassing this duration will be immediately sterilized.

Waste Disposal: All terminated or expired cultures will be sterilized via autoclaving prior to disposal, ensuring full compliance with biosafety regulations.

For all potentially hazardous chemicals or mildly toxic substances involved in the experiment, we have conducted detailed research on their usage specifications and toxicity introductions to ensure the safety of team members during the experiment.

Biohazardous production: The E. coli lysate and P. pastoris fermentation broth must be aseptically filtered through a 0.22 μm sterile membrane under a laminar flow hood prior to subsequent steps—including protein lyophilization for spinning, enzyme activity assays, and straw pretreatment via cell surface display.

Pichia pastoris is renowned for its high-level secretory expression of functional proteins, including toxins, enzymes, and antibodies. In our project, we plan to rely on this yeast for the industrial-scale production of scaffold proteins and three lignin-degrading enzymes. Moreover, we may consider expressing additional enzyme combinations to further enhance lignin degradation, which would require a large number of high-expression strains. However, the transformation and screening of high-expression P. pastoris typically involve significant risk of antibiotic overuse.

If antibiotic resistance genes are retained in the resulting engineered strains, they may enter environmental microbial communities through horizontal gene transfer—via plasmid conjugation, transduction, or transformation—potentially leading to the emergence of "superbugs" and endangering public health. This risk of antibiotic contamination is particularly pronounced during the high-copy screening of Pichia pastoris. Since high expression in this yeast often depends on high gene copy numbers, and the probability of secondary genomic integration is only about 1%-10%, it is necessary to generate a large number of transformants to select for high-copy strains. Although high-copy screening was not performed in our project, we aimed to adopt an appropriate vector strategy to minimize antibiotic contamination risks in future product development. Therefore, we used the Cre/lox system to eliminate resistance genes.

Cre/lox Site-Specific Recombination System

The Cre/loxP system is a widely adopted site-specific recombination tool derived from bacteriophage P1. In this system, the Cre recombinase enzyme recognizes and catalyzes recombination between two specific 34-base-pair DNA sequences known as loxP sites. When these sites are arranged in the same orientation, Cre-mediated recombination efficiently excises the DNA segment located between them. This system has been successfully adapted for use in Pichia pastoris, enabling precise, programmed removal of selected genetic elements.

Building on this mechanism, a Cre/lox-mediated recombination module is integrated into our plasmid backbone. Upon successful incorporation of the exogenous gene, the expressed Cre recombinase excises the kanamycin resistance (kanR) and zeocin resistance (zeoR) cassettes by recognizing and cleaving the flanking lox71 and lox66 sites (plasmid design illustrated in Figure A). This process ensures complete removal of superfluous antibiotic resistance genes (ARGs) from the final engineered strain, thereby substantially reducing the probability of their environmental dissemination through horizontal gene transfer (HGT).

The use of Cre/lox system to remove resistance genes can bring two core benefits: firstly, completely removing resistance markers, achieving "scar free" modification, and making the final engineered strain comply with the mandatory requirements of genetically modified organism safety regulations for "no resistance residue"; Secondly, the resistance box itself is a repetitive sequence, which eliminates homologous recombination hotspots and allows subsequent vectors to be dispersed and integrated into different sites of the genome after excision, thereby significantly reducing genetic instability mediated by repetitive sequences.

However, we have chosen not to recommend the vector we used to the iGEM community at this time, due to an unexpected issue: leaky expression of Cre recombinase in E. coli. Although we incorporated a lacO sequence between the AOX1 promoter and the Cre gene to block potential AOX1-driven expression in E. coli—despite AOX1 being a tightly regulated methanol-inducible promoter specific to eukaryotic yeast—we were surprised to find that plasmid segments were still lost during the construction of yeast expression vectors with different signal peptides in DH5α. Even when our instructor repeated the experiment using Top10 cells, the same issue occurred, significantly delaying our experimental progress.

Since Pichia pastoris transformation requires 5–10 μg of linearized DNA, we had to retransform the initially successfully constructed plasmids into fresh E. coli and scale up the culture immediately after transformation to obtain sufficient plasmid quantities. We observed that the longer the plasmid resided in E. coli, the more severe the segment loss became. As a result, our plan for high-copy integration could not be accomplished within the iGEM competition period.

Nevertheless, we hope that future iGEM teams can refine and stabilize our vector design based on our findings. More importantly, we urge all teams to recognize that screening for P. pastoris and other chassis organisms requiring copy number selection can lead to increased risks of antibiotic misuse. In addition to adopting novel yeast expression vectors, we have also optimized the enzyme screening process to eliminate dependence on high-concentration antibiotic selection.

Throughout our human practices activities, we recognized 2 primary safety considerations beyond the laboratory: protecting participant privacy and ensuring the accuracy of information shared during public engagements.

To address privacy concerns, we implemented strict protocols during all interviews and educational events, obtaining explicit consent before capturing any photos or videos and clearly explaining how such materials would be used. This approach ensured that every participant felt respected and that their personal information remained secure.

Regarding information accuracy, we recognized that public outreach carries the responsibility of conveying scientifically reliable content. Before each educational activity, our team thoroughly researched the topics to be presented, cross-referencing multiple authoritative sources such as peer-reviewed journals and established scientific databases. This preparation ensured that all workshop materials, presentations, and answers to public questions reflected current scientific understanding and avoided potential misinformation.

By integrating these safety measures into our human practices framework, we maintained high standards of ethical engagement while building trust with the diverse communities we served—from farmers in Fengkai County to students in our synthetic biology workshops. This commitment to responsible outreach represents an essential dimension of safety in iGEM, where proper information and respectful interaction are as crucial as laboratory biosafety.

Our project implements a comprehensive safety framework spanning personnel management, laboratory operations, and environmental protection. All members must pass a dual assessment—written exam (score ≥90/100) and practical evaluation—before gaining lab access. We adopted the Cre/lox system to eliminate antibiotic resistance genes in P. pastoris, avoiding traditional high-copy screening methods that risk antibiotic misuse. Strict waste protocols require sterile filtration of all microbial cultures prior to further processing. In human practices, we maintained strict ethical standards for interviewee privacy and promoted biosafety awareness. While further vector optimization is needed, we urge future teams to develop safer, antibiotic-free selection systems. Through these measures, we ensure project safety while supporting sustainable straw-to-textile technology development.