Safety concerns about our engineered bacteria

Our engineered bacteria are able to capture heavy metals due to the additional metal binding domains, which are MBP3 and SUMO-SmtA, located downstream CsgA in plasmid. We are concerned about safety issues related to our bacteria in our project. Our safety considerations regarding how to keep the bacteria under control will be discussed in this section. The bacteria related risks can be assigned into two stages: inside the heavy metal treating module and beyond the module. The following evaluation will imply that risks and possible deleterious substance leakage harming the ecosystem can be neglected.

Inside the module

We make use of biofilm formed by our engineered bacteria to capture heavy metals like silver, copper and cadmium. This, however, introduce one endogenous risk factor derived from the nature of biofilm, which is its spontaneous detachment and dispersal.¹ With slight differences, these two processes both contribute to the falling off of the bacteria and enabling it to go with the effluent flow, all the way to the outlet. This may cause the leakage of genetically-modified organisms (GMOs). We will employ high-density membrane filtration system, which contains semipermeable membrane, to hinder the GMOs from escaping the system. The filtration facility was designed modularly, facilitating the replacement of filter module.

Beyond the module

Though utilizing semipermeable membrane to preventing bacteria from escaping the filtration module, small amounts of GMOs may make their way out of containment. Considering that this may happen anyway, we would minimize the influence of escaped GMOs on ecosystem with the help of the industry itself. Our heavy metal capturing module will be installed in a wastewater treatment facility. One of the steps during wastewater treatment is to adjust pH value of wastewater. Mixing the wastewater with escaped GMOs and low pH wastewater will eventually kill all GMOs, which are susceptible to pH value.

Some factories may use other sterilizing methods like adding chemicals or using ultra violet (UV) light to kill or make organisms have defect in reproduction. Our filtration module can be installed before such treatment steps. The efflux entering such sterilizing facility will be sterilized, ensuring no living or fertile GMOs escaping the whole wastewater treatment facility.

Lab safety

We conduct our expeeriments in laboratories of Biological Science Department and Environment Department due to lack of equipment. We follow the Health and Safety Handbook of Biological Sciences Department and Environment Department of Xi’an Jiaotong-Liverpool University (XJTLU). All of the students who get involved in experiments have received adequate safety training, including using equipment properly, experiment operated safely, waste disposal method and action in case of emergency. We also reported all harmful and potentially harmful substances to the Departments before we use them by filling in safety data sheets (SDS).

Biological experiment

Biological laboratories

We conduct our experiments in Biosafety Level 1 (BSL-1) laboratory, which only use nonpathogenic organisms like E. coli K12 strains.²

Conduct experiments safely

Our experiments are under the supervision of the Department staffs in charge of laboratory safety. Personal protection equipment (PPE) is also a mandatory requirement for entering laboratories. Disposable rubber gloves and lab coats are worn during experiments. All experiments related to GMOs are conducted inside the biological safety cabinet (BSC) to prevent potential biohazards leakage.

All experiments regarding GMOs are conducted inside the BSCs

All consumables like used micro pipette tips, centrifuge tubes and microcentrifuge tubes contaminated by GMOs are preserved converged in a closed container. Students wearing PPE are not allowed to leave the laboratory unless removing any potentially contaminated PPE.

Number of students who work in laboratory is required to be more than two. Students are also not recommended to work after 5 p.m. in laboratory. If students have to work in laboratory alone or after 5 p.m., he/she shall report his/her working schedule and experiment protocol to the Department staff and inform other students in experiment group.

After experiments - waste disposal

All contaminated consumables and some PPE like disposable rubber gloves are centralized and autoclaved at 121 degree Celsius for 15 minutes for sterilization. Students who use autoclave are supervised by qualified staff from the Department of Biological Science. A usage record should be filled in before using the autoclave. Students are required to wear autoclave gloves when dealing with autoclave. All vessels with lids are loosen before entering autoclave process.

An autoclave that employed to sterilize waste contaminated by bacteria

Other used but not contaminated consumables are discarded in dust bin, which will be dealt with accordingly.

In case of emergency

All laboratories are equiped with emergency shower and emergency eyewasher. A spill kit is also prepared in case of any chemical spillage.

Collaboration with environment laboratories

Due to lack of equipment, we have to seek help from the Department of Environment to finish our experiment. The equipment we use are Inductively coupled plasma mass spectrometry (ICP-MS) instruments and microwave digestion system. Students have to fill in usage records before using any equipments including balance and oven.

A usage record of oven

ICP-MS

Students are not allowed to conduct ICP-MS without supervision of qualified staff from Department of Environment. Students must follow the instruction from supervisor. Working after 5 p.m is not allowed.

Microwave digestion system

Before conducting digestion process, all samples should first be digested by aggressive reagents. What we use are nitric acid (70%) and hydrogen peroxide (30%). These chemicals are under strict supervision of the Department of Environment and Suzhou Industrial Park Police Department. Students who use these chemicals should wear PPE with higher protection level, for example, vinyl gloves and goggles. All chemicals involved in experiments should be kept in cabinet with higher safety level and handled in fume hood.

Students are not allowed to work with these chemicals alone. A minimum number of working students is two.

Before using any of the aggressive reagents, students are required to submit the work schedule and protocol to the Department of Environment in advance. A registy form should be filled in and filed in the Department. A registed staff will take the chemicals out and supervise the students conduction experiments. All chemicals left after use will be collected by the staff and returned to the cabinet. Striking visual signs and instructions are hung on the entrance of the storage area.

Safety signs and emergency procedure hung on the door of chemical storage room

Considering that pressure vessels are used during microwave digestion, students are required to conduct experiments under the surveillance of qualified staff from the Department of Environment. Students are not allowed to work in laboratory after 5 p.m.

After the experiments, all inorganic chemical waste including digested bacteria and waste nitric acid and will be discarded centralized in a bucket with lid, which will be collected by qualified chemical waste disposal companies.

Different waste are sorted according to their chemical properties

Reference