Safe
Working Practices
General Laboratory Safety
Prior to starting lab work, the safety level of the laboratory was analysed and classified as Biosafety level 2. As a result, further precautionary action was taken to ensure team members were aware and trained on safe work practices. Team members received a PC2 Bio Safety Training session and completed relevant online safety modules. UNSW online safety modules included:
- Laboratory Safety Awareness
- Hazardous Substances, Gene Technology,
- Fume Cupboards and Safety
- Ergonomics and Manual Tasks,
- Work Health and Safety Awareness.
Laboratory team members read the Safe Work Procedure (SWP) and Risk Management Form (RMF) and received a PC2 induction, building induction, lab induction and training on relevant equipment prior to use.
General Laboratory Safety Protocols and Precautions
- Specific safety equipment: The location of and operating procedures of emergency eyewashes and showers, fire blanket, fire extinguisher as well as, spill cleanup kit.
- Personal Protective Equipment (PPE): Lab gown, safety goggles, gloves, enclosed foot-wear which greatly reduced risk of direct skin contact with harmful substances. Lab gowns are not removed from the laboratory to prevent contamination of the external environment.
- Waste disposal: Waste materials are sorted into hazard categories (biological, chemical, corrosives), labelled and disposed of according to waste disposal protocols. Biological waste was autoclaved or chemically disinfected before disposal as directed.
- Chemical safety: Locations, appropriate storage and handling methods for flammable and hazardous materials were introduced. Chemicals were kept in separate well-labelled storage cabinets, with all samples being fully labelled.
OGTR
To ensure our team's safety we followed OGTR guidelines for working in a PC2 laboratory4.
- Safety sheets are placed on the door containing information of emergency contacts, hazards present and expected safety practices to be maintained.
- Lab gowns are kept within the lab by the entrance to reduce the need to remove decontaminate clothing prior to leaving the lab. By removing the need to transport items in and out of the lab, thus contamination of the external environment.
- The first aid kit is kept near the entrance of the laboratory.
- There are three safety showers, eye wash stations, fire blankets and fire extinguishers distributed within the lab. Located by the two entrances of the lab and in the middle.
- Chemicals and materials are kept in well labelled glassware, containing information of its contents, hazard category, and safety information.
- Flammable materials are stored in its designated cabinet, away from heat and flames.
- Waste are classified as: non-hazardous, sharps, corrosives, flammables and harmful waste. Each material is contained within it's own designated waste container with proper labels attached.
- Waste container labels.
- Once waste containers are full, they are placed in the waste room for proper decontamination and/or neutralising before disposal. The waste room has designated shelves for different waste categories.
Safe Project Design
Chassis Organism and Parts
Our team used four strains of competent E.coli cells and S. cerevisiae as a chassis for our project:
| Strain Type | Chassis | Biosafety Level | Function |
|---|---|---|---|
| Cloning | NEB Dh5-alpha E. coli | 1 | Optimal for sufficient plasmid production and stability |
| Cloning | Promega HB101 E. coli | 1 | Optimal for sufficient plasmid production and stability |
| Expression | NEB T7 Express E. coli | 1 | Optimal for sufficient protein production |
| Expression | Novagen OrigamiTM 2 (DE3)pLysS E. coli | 1 | Optimal for sufficient protein production. Contains Origami plasmid that aids in protein folding for soluble proteins. |
| S. cerevisiae BY4741 | 1* | Protein production |
The chassis organisms pose minimal risk to team members in the laboratory and the environment as they are not classified as a dangerous substance or mixture according to the Globally Harmonized System (GHS). They are also not known to consistently cause disease in immunocompetent adult humans1 2 3. Moreover, DH5α does not mutate or pass on DNA that it hosts and has been engineered to lack the ability to produce thiamine and leucine, making it unable to survive in the environment without these two compounds supplemented.
All parts used in our project are classified in a risk group of 0-1 by DSMZ and iGEM Safety/Risk Groups, and do not pose any disease risk to humans or the environment6. Additional information on the parts we used can be found here (link to parts overview page).
| Part | Risk Group | Purpose |
|---|---|---|
| Alpha Prefoldin-SpyC | 1 | Subunit of Assemblase scaffold |
| Beta Prefoldin-SnoopC | 1 | Subunit of Assemblase scaffold |
| LXYL-P1-2-SpyT | 1 | Enzyme in Paclitaxel production pathway |
| DBAT-SnoopT | - | Enzyme in Paclitaxel production pathway |
| PAM-SnoopT | - | Enzyme in Paclitaxel semi-synthetic production pathway |
| TycA-SpyT | 1 | Enzyme in Paclitaxel semi-synthetic production pathway |
| mCerulean3-SnoopT | - | Fluorescent protein |
| mVenus-SpyT | - | Fluorescent protein |