Experiments

Phase 1: Protein Production & Isolation

  1. Two E. coli strains:
  2. Strain A: Expressing barnacle adhesive protein (BBa_K4854005, e.g., with a His-tag for purification).
  3. Strain B: Expressing protease (e.g., subtilisin or another candidate).
  4. Transformation using relevant parts in the distribution kit. Help:Protocols/Transformation - parts.igem.org
  5. Miniprep relevant parts https://www.brainvta.tech/html/Support/a/1111.html
  6. 3A assembly Help:Protocols/3A Assembly - parts.igem.org

Phase 2: Testing Protease Efficacy

If Strain B was not effective we could create multiple strains until we find a good candidate

  1. Protein extraction and isolation – 8 possible methods, narrowed down to either salting out or using glycine tags
  2. Testing efficacy of protease or similar enzyme

SDS-PAGE: Successful degradation: Bands for adhesive protein disappear/reduce in protease-treated sample vs. control.

If SDS-PAGE is unavailable, use a turbidity assay:

Aggregation Test:

  1. Barnacle adhesive proteins often aggregate. Mix protein + protease and monitor clarity over time.
  2. Degradation reduces turbidity (measure absorbance at 600 nm).

Adhesion Test:

  1. Coat microscope slides with adhesive protein (dry overnight).

Hazardous chemicals

Agarose gel components (performing gel electrophoresis):

We will avoid using Ethidium Bromide and instead use safer stains such as methylene blue.

  • Boric acid (irritant)
  • Isopropanol (flammable, irritant) – used in DNA precipitation
  • Ethanol (70-100%) (flammable, irritant)
  • SDS (Sodium Dodecyl Sulfate) (irritant) – in lysis buffers

3A Assembly (Restriction Digestion & Ligation)

Restriction enzymes (non-hazardous, but buffers may contain:

  • MgCl₂ (low hazard)

Antibiotics (depending on resistance markers used):

  • Ampicillin (can cause allergic reactions, handle with care)
  • Kanamycin (toxic, irritant)
  • Chloramphenicol (toxic, carcinogenic)
  • Tetracycline (light-sensitive, irritant)

3. Protein Expression & Purification

  • IPTG (Isopropyl β-D-1-thiogalactopyranoside) (irritant) – for induction
  • Lysozyme (low hazard, but powder can be irritant) – for cell lysis
  • Urea (irritant, harmful if inhaled) – in denaturing buffers
  • Imidazole (irritant) – for His-tag purification
  • β-Mercaptoethanol (BME) (toxic, strong odor) – reducing agent in SDS-PAGE
  • Ni-NTA resin (low hazard, but may contain nickel, which can be allergenic)

4. Protein Extraction & Isolation

  • Ammonium sulfate (irritant) – for salting out
  • Glycine (low hazard, but high concentrations may be irritant)
  • Tris-HCl (irritant) – buffer component
  • SDS (Sodium Dodecyl Sulfate) (irritant) – for denaturing proteins

5. SDS-PAGE & Turbidity Assay

  • Acrylamide/Bis-acrylamide (neurotoxic, carcinogenic) – for gel preparation
  • TEMED (Tetramethylethylenediamine) (corrosive, flammable) – gel polymerization
  • APS (Ammonium Persulfate) (oxidizer, irritant) – gel polymerization
  • Coomassie Blue stain (contains methanol & acetic acid – irritants)
  • Coomassie destain solution (methanol + acetic acid – flammable, corrosive)

6. Aggregation & Adhesion Tests

  • HCl/NaOH (corrosive) – for pH adjustments

General Hazardous Chemicals in Lab Work

  • Bleach (Sodium Hypochlorite) (corrosive) – for decontamination

AI

We will only be using it for suggestions and we will be verifying all information via other means to ensure that the information is accurate. We will not provide any personal information to the LLM and believe with these mitigations there is very low risk.

Hazards Presented by Organisms, Parts, Chemicals, or Experiments

1.Organisms & Parts:

  1. E. coli strains are Risk Group 1 (non-pathogenic) and will be handled at Biosafety Level 1 (BSL-1).
  2. The barnacle adhesive protein (BBa_K4854005) and protease (e.g., subtilisin) are not inherently hazardous, but standard lab precautions will be followed to avoid accidental exposure.

4.Hazardous Chemicals:

  1. Toxic/Corrosive Chemicals: β-Mercaptoethanol, acrylamide, TEMED, ethidium bromide/SYBR Safe, antibiotics (e.g., ampicillin, kanamycin).
  2. Irritants: Imidazole, SDS, IPTG, urea, ammonium sulfate.
  3. Flammables: Ethanol, isopropanol.
  4. Carcinogens/Mutagens: Chloramphenicol (low risk with proper handling), formaldehyde (if used).

9.Experimental Hazards:

  1. SDS-PAGE: Neurotoxic acrylamide, corrosive TEMED.
  2. Protein Purification: Nickel-NTA resin (potential allergen).
  3. Aggregation/Adhesion Tests: Potential aerosol generation during mixing (minimized via gentle pipetting).

13.Mitigation Measures:

  1. PPE: Gloves, lab coats, and goggles always required.
  2. Engineering Controls: Use of fume hoods for volatile chemicals (e.g., β-mercaptoethanol, phenol).
  3. Waste Disposal: Segregation of chemical/biological waste per institutional guidelines.
  4. Training: All team members trained in BSL-1 practices and chemical safety.

1. Organisms & Parts

  1. Accidental ingestion/inhalation of E. coli: If cultures are spilled or aerosols are generated during pipetting, lab members could ingest/inhale bacteria, though the risk of infection is minimal (although E.coli DH5α is non-pathogenic).
  2. Improper disposal of engineered strains: If strains are not autoclaved before disposal, they could persist in the environment, though they lack survival advantages.

2. Hazardous Chemicals

  1. Skin/eye contact with irritants (SDS, imidazole, β-mercaptoethanol): Splashes during pipetting or gel pouring could cause irritation or burns.
  2. Inhalation of toxic vapors (TEMED, phenol): Working outside a fume hood with volatile chemicals could lead to respiratory irritation or dizziness.
  3. Fire/explosion risk (ethanol, isopropanol): Improper storage near ignition sources (e.g., Bunsen burners) could cause fires.

3. Experimental Procedures

  1. Neurotoxin exposure (acrylamide): Unpolymerized acrylamide gel solution contacting skin could cause neurological harm with chronic exposure.
  2. Cuts from broken glass (SDS-PAGE gels): Handling glass plates or staining trays without care could lead to injuries.
  3. Allergic reaction to nickel (Ni-NTA resin): Repeated skin contact with resin during protein purification could trigger nickel allergies.

4. Dual-Use Considerations

  1. Protease misuse: If the engineered protease is highly efficient, it could theoretically be misused for unintended protein degradation (unlikely in this context).

5. Environmental Release

  1. Chemical contamination: Improper disposal of antibiotics could harm wastewater systems or microbial ecosystems.

Environmental Considerations

The plan is that the protein produced would be isolated and used in a formula for cleaning the boats more easily. However there is the possibility that bacteria could be developed as a biofilm on the underside of shipping vessels to prevent the adhesion of larger macrofouling fauna. This second application could represent a risk if the bacteria had some selective advantage it could be released and outcompete and endanger the species that it is modified to repel. There would need to be safeguards put in place if this was the case such as kill switches and experimental verification to minimise the risks of environmental harm.

Why are the strains used unable to spread?

1. Host Strain Limitations

DH5α and BL21(DE3) are Lab-Adapted Strains:

  • These strains have undergone extensive genetic modifications for optimal growth in laboratory conditions but are severely impaired in natural environments.
  • They lack essential survival mechanisms (e.g., robust stress responses, competitive fitness) needed to outcompete wild-type microbes in soil, water, or host-associated environments.

BL21(DE3) is an Auxotrophic Strain:

  • BL21(DE3) has mutations (e.g., ompT, lon, hsdSB) that reduce its ability to survive outside controlled lab conditions.
  • It lacks key virulence factors and metabolic pathways required for environmental persistence.

2. Plasmid-Based Containment (pET21a-balcp19k)

Dependence on Antibiotic Selection:

  • The plasmid carries an antibiotic resistance marker (ampicillin)
  • In the absence of selective pressure (antibiotics in the environment), the plasmid is likely to be lost over generations due to the metabolic burden it imposes.

No Known Horizontal Gene Transfer Mechanisms:

  • The pET21a vector lacks conjugation genes (tra genes) or mobilizable elements that would facilitate plasmid transfer to other bacteria.
  • The likelihood of environmental gene transfer is negligible under standard conditions.

3. Physical and Procedural Containment

Controlled Laboratory Conditions:

  • All work is conducted under Biosafety Level 1 (BSL-1) containment, following standard microbiological practices (e.g., sterilization of waste, use of disinfectants).
  • Engineered bacteria are not released into the environment and are destroyed via autoclaving after experiments.

No Pathogenicity or Toxin Production:

  • The modified strains do not produce toxins, virulence factors, or any known harmful metabolites that could pose a risk if accidentally released.

4. Additional Safety Measures

Inducible System Control:

  • Gene expression is tightly regulated and requires artificial inducer (IPTG) not found in nature.

Conclusion

Due to the host strain's lab-adapted weaknesses, lack of environmental survival mechanisms, absence of horizontal gene transfer risks, and strict lab containment protocols, our engineered E. coli strains are highly unlikely to establish or spread in natural environments.

Other support in managing risks:

We are members of CLEAPSS so we can enquire with them if unsure of a safety issue. Our senior laboratory technician Kelly.zhang@wellingtoncollege.cn is also experienced particularly with Chinese law and compliance with standards.

I would also seek the advice of iGEM safety help.

We have collaboration with technicians at NYU Shanghai and have visited their laboratory to seek help on managing risks.

PI Mentor - Lisa Rainer previous iGEM judge has been assigned by iGEM and is a useful sounding board.

UK CLEAPSS Resources

UK CLEAPSS (Consortium of Local Education Authorities for the provision of Science Services) https://dt.cleapss.org.uk/resources

  • CLEAPSS Guide L214b Examining Autoclaves, PressureCookers, Model Steam Engines: Written Scheme of Examination
  • CLEAPSS, Hazcards, current edition
  • CLEAPSS, Laboratory Handbook, current edition
  • CLEAPSS, Recipe Book, current edition
  • CLEAPSS, Managing Risk Assessment in Science url: https://science.cleapss.org.uk/login.aspx

UK Regulations

  • UK COSHH (Control Of Substances Hazardous to Health guidelines) downloaded 02-10-23 https://www.hse.gov.uk/coshh/[TE1]
  • UK Management of Health & Safety at Work Act downloaded 03-10-23 https://www.hse.gov.uk/legislation/hswa.htm
  • UK Dangerous Substances and Explosive Atmospheres Regulations (DSEAR) downloaded 03-10-23 https://www.hse.gov.uk/fireandexplosion/dsear.htm

RELEVANT LOCAL & NATIONAL REGULATIONS for Science Lab equipment in Primary & Secondary Schools JYT 0385-2006

  • Equipment standard of education equipment for science faculty in junior middle school JYT 0386-2006;
  • Equipment standard of education equipment for science in junior middle school JYT 0387- 2006 and JYT 0388-2006
  • Laboratory furniture – Fume Hood QB/T 5589-2021
  • Guidance to management of laboratory equipment GB/Z 27427-2022
  • Good research laboratory practice GB/T 27425-2020
  • Regulations for the safe use of chemicals in the workplace
  • Rule for the storage of dangerous chemicals GB 15603-1995
  • Regulations on the Administration of Precursor Chemicals
  • Measures for public security administration of explosive & dangerous chemicals
  • Public security requirements for storage sites with potentially explosive chemical synthesis
  • National Catalogue of Hazardous Wastes
  • Regulations on Labour protection in workplaces where toxic substances are used
  • General requirements of safety & control devices for gas burners and gas burning appliances GB/T 30597-2014
  • Technical specification for urban gas alarm control system CJJ/T 146

As discussed we will refer to CLEAPSS and comply with Chinese standards on control of substances hazardous to health.

Safety and security rules that govern our work:

UK CLEAPSS (Consortium of Local Education Authorities for the provision of Science Services) https://dt.cleapss.org.uk/resources

  • CLEAPSS Guide L214b Examining Autoclaves, PressureCookers, Model Steam Engines: Written Scheme of Examination
  • CLEAPSS, Hazcards, current edition
  • CLEAPSS, Laboratory Handbook, current edition
  • CLEAPSS, Recipe Book, current edition
  • CLEAPSS, Managing Risk Assessment in Science url: https://science.cleapss.org.uk/login.aspx
  • UK COSHH (Control Of Substances Hazardous to Health guidelines) downloaded 02-10-23 https://www.hse.gov.uk/coshh/[TE1]
  • UK Management of Health & Safety at Work Act downloaded 03-10-23 https://www.hse.gov.uk/legislation/hswa.htm
  • UK Dangerous Substances and Explosive Atmospheres Regulations (DSEAR) downloaded 03-10-23 https://www.hse.gov.uk/fireandexplosion/dsear.htm

RELEVANT LOCAL & NATIONAL REGULATIONS for Science Lab equipment in Primary & Secondary Schools JYT 0385-2006

  • Equipment standard of education equipment for science faculty in junior middle school JYT 0386-2006;
  • Equipment standard of education equipment for science in junior middle school JYT 0387- 2006 and JYT 0388-2006
  • Laboratory furniture – Fume Hood QB/T 5589-2021
  • Guidance to management of laboratory equipment GB/Z 27427-2022
  • Good research laboratory practice GB/T 27425-2020
  • Regulations for the safe use of chemicals in the workplace
  • Rule for the storage of dangerous chemicals GB 15603-1995
  • Regulations on the Administration of Precursor Chemicals
  • Measures for public security administration of explosive & dangerous chemicals
  • Public security requirements for storage sites with potentially explosive chemical synthesis
  • National Catalogue of Hazardous Wastes
  • Regulations on Labour protection in workplaces where toxic substances are used
  • General requirements of safety & control devices for gas burners and gas burning appliances GB/T 30597-2014
  • Technical specification for urban gas alarm control system CJJ/T 146

As discussed we will refer to CLEAPSS and comply with Chinese standards on control of substances hazardous to health.

Safety Protocols

Accident reporting: near miss and incident forms are completed and shared with health and safety committee. Personal protective equipment: Trained in use of gloves and goggles, ethanol sterilisation. Inventory controls: Science technicians check in and out all chemicals and we have video surveillance on the chemical store cupboard door which is locked with two person access required. Physical access controls: All laboratories are accessed by swipe cards which are only operable by science teachers, students don't have free access. Data access controls: passwords are used and ICT updates are regular. Waste management: managed by the technicians - autoclave and bleach is used - all biological waste is disposed of according to local regulations and is clearly labelled. Risk assessments are completed for each activity. Students are high school so will never be working unsupervised as an adult member of staff who is trained in safety protocols will always be present during practical activities.

All students are trained in aseptic technique and safe procedures for handling E.coli. Technicians are trained on safe disposal and management of tracking of chemicals. Thomas Edwards attended the iGEM online high school safety meeting . The team have decided to use E.coli as I it's level 1 and safer for our experiment, we will not be using any other organisms which helps mitigate risk. The other risk management actions were mentioned before, ensuring that a risk assessment is completed for each activity and that all students are supervised at all times by a member of trained staff.

The rules and guidance that identified help us to determine the hazard level and risk mitigation protocols which are appropriate for each activity. As a high school head of science, Mr Edwards has had health and safety training throughout his career, most recently in 2024 with a senior member of CLEAPSS who came and gave training at our school. Mr Edwarsd implemented his advice and updated our Science health and safety policy appropriately and is responsible for the health and safety in science. The school is a secure environment with card access only for staff, chemical storage and handling procedures are well established with experienced laboratory technicians helping to manage the space. The technicians are trained in waste treatment and we have collaborated with NYU laboratory in Shanghai to ensure our procedures are safe and effective. All students visited NYU laboratory and had safety training. The protocols which we will follow are well established and risk assessed before completion.