This year, team SKLMT-China proposed a promoter library for pseudomonas fluorescence and use it to heterologously express the nicotine degradation gene cluster. The biosafety level is well recognized and the protocol for experimental manipulation is well established. Although nicotine we used is toxic, we made sure that every step is under rigid control so that no hazardous chemical will be let out. We consulted the PI from labs that have been studying nicotine degradation for a relatively long time for safety protocol when working with it.

Safe though it may seem, there still could have been unsolicited consequences if no attention for safe experiments is heeded. In order to minimize any unwanted accident, we received proper training and adopted several safety regulations in the laboratory. Now we’d like to share our experiences with safety control.

Safe Project Design

Choosing a non-pathogenic chassis

In our project, non-pathogenic strains of E. coli were the chassis used to perform molecular cloning. The other chassis used to perform protein expression is Pseudomonas fluorescence pf-5. This strain belongs to the Risk Group 1 microorganisms according to iGEM white list. It is not pathogenic to mammals and has passed the acute toxicity test of the Ministry of Agriculture of China.

Choosing parts that will be environmentally friendly

Harmful parts are not allowed in our project. The parts in our project are most commonly used recombinases and expression control units (such as terminators and promoters widely used in research) so that none of our parts would raise any safety issues according to current professional knowledge.

Safe Lab Work

Before we carried out our experiments, all of our team members had received strict lab safety training, including standard experimental protocols, proper disposal of biological and chemical waste, information on hazardous chemicals and use of biosafety cabinets given by PI in the lab. Such guidance was documented on the online portal in detail. Team members involved in bench works had all went through orientations with mentors or managers in charge of specific equipment used before gaining access to the lab. According to the requirements of iGEM policy, in our daily bench work, we never performed any dangerous experiments or faced any unusual safety issues. Our bench work followed some basic regulations as follows:

1. Duplicating a key to the laboratory without permission is strictly prohibited.

2. Participants need to understand the experiment completely.

3. All experiments are done with rubber gloves.

4. Steps with any probability of contamination should be performed in a biosafety cabinet.

5. Fire, electric heaters, and microwave ovens should never be left unattended.

6. All wastes should go through a rigid disposal process before discarding.

7. Sanitize the lab using UV-light every week.

8. The last person to leave the lab should make sure that water, electricity, gas, and air conditioners have been turned off, and doors and windows have been locked before leaving.

Safe Project Design

As mentioned above, our DNA parts are absolutely safe because of they are common-used recombinases and expression control units(promoter) or those encode safe proteins. The DNA parts are safely contained within 96 well plates as the Parts registry requires.