Lab Safety Instruction

Prelab Training

We received online lab training on a lab model website. There are multiple periods, and we got to operate the equipment ourselves after listening to lessons. After receiving training online, we went to the lab. The tutor helped us to recall the use of each apparatus and showed us the procedure of each experiment we were about to carry. And other teachers reminded us of safety repeatedly.

The safety training includes the following topics:

• Lab access and rules (e.g. appropriate clothing, eating and drinking)
• Responsible individuals (e.g. lab or departmental specialist or institutional biosafety officer)
• Differences between biosafety levels
• Biosafety equipment (e.g. biosafety cabinets)
• Good microbial technique
• Disinfection and sterilization
• Emergency procedures
• Rules for transporting samples between labs or shipping between institutions
• Physical biosecurity (e.g. tracking materials, access controls)
• Chemical, fire and electrical safety

Other actions we have taken to manage the risks in our project are the following:

• Project-specific safety or security training (e.g. training on handling certain organisms)
• Consulting with other experts about managing risks (e.g. an institutional biosafety officer)
• Consulting with stakeholders about managing risks
• Evaluating countermeasures against your organism, parts, or other products (e.g. efficacy of therapeutics, detection in case of environmental release)
• Crafting a responsible communication plan (e.g. redacting specific information, highlighting the biosafety measures used)

Lab Location

Zhejiang University Biology National Experimental Teaching Centre

Work Areas

• Open bench
• Biosafety cabinet
• Chemical fume hood

Rules

We follow the rules set by Chinese government to conduct our experiment.

• Accident reporting (system to record any lab accidents)
• Personal Protective Equipment / PPE (wearing lab coats, gloves, eye protection, etc.)
• Inventory controls (tracking who has what physical materials and where the materials are)
• Physical access controls (controlling who can access your lab or storage spaces)
• Data access controls (controlling who can access computers or databases)
• Lone Worker or Out of Hours policy (procedures for working alone or at times when normal support is unavailable)
• Medical surveillance (finding out if you get sick because of an organism or chemical you used)
• Waste management system (such as decontaminating waste before it leaves your institution)

Experiment Design

We plan to construct an engineered bacteria that can express fusion proteins of spider silk and PET enzymes. The fusion proteins could be used to capture and degrade microplastics in the environment. We are interested in PETase expressed in bacterium, and spider silk proteins expressed in spider. We get the protein sequences form NCBI and literature. PCR technology will be used to amplify the target to obtain DNA fragments, and nucleic acid electrophoresis will be used for confirmation. The constructed plasmid will be transformed into Escherichia coli for expression. SDS-PAGE gel electrophoresis will be used to check the protein expression, and protein purification technology will be used to express and purify large-scale recombinant protein.

None of our parts could be hazardous to the Registry.

During the experiment, the controlled chemical reagents we will use include:

• 1) Flammable includes anhydrous ethanol, which is used to prepare other reagents, alcohol lamp fuel, diluted disinfection, etc.
• 2) Acrylamide with neurotoxicity and potential cancer risk is used to prepare polyacrylamide gel for SDS-PAGE protein electrophoresis

However, it is at low risk that this would cause actual harm. Firstly, we need to undergo formal lab training, everyone has participated in the training program for safe operation. Secondly, regarding the volatile toxicity, we will operate in that ventilated area.

These are the experiments we did: PCR, electrophoresis, restrictive endonuclease digestion, double enzyme cleavage connection, resuscitation of engineering bacteria, preparation of receptive cells, transformation of E.coli, screening of recombinant bacteria. Considering the safety of the experiment, we disinfect and sterilize our engineering bacteria after usage. Also, we would use non-toxic and harmless stain in electrophoresis.

We predict that the future development of our project would not require release beyond containment.

Conversation about possible bad outcomes of our project:

• Our team had a conversation on how bad outcomes might be caused in our project. We conclude that our project could be harmful to materials, equipment, and infrastructure if there is accidental release of an engineered organism or part into the environment.
• As Engineered Escherichia coli may survive in the wild. In order to avoid their potential harm to the environment we will treat engineering bacteria in strict accordance with the disinfection and sterilization procedures of biological wastes in the laboratory to ensure that engineering bacteria will not be released into the environment.

Managing Risks

• Dr. Dong Shan, Dr. Jing Shi, Professor Zhou Tan, and the manager of the lab are our safety administrators and responsible for daily safety-check.