Our project, Mangroves PE&CO2 Hunters, is focused on using modified mangrove bacteria, Pseudomonas aeruginosa PAO1, to degrade microplastics, and using Rhodopseudomonas palustris CGA009 to fix carbon dioxide and maintain the positioning of the blue carbon system in the mangrove forest. Our ultimate goal is to apply the entire system to mangroves, so when we designed the project, we took into account the hazards that the system releases into mangroves. We designed the corresponding safety module to prevent plasmid loss and target the suicide of engineered bacteria when they escape the mangroves or when harmless chemical is artificially applied into soil. In addition, we also pay attention to the safety of the people who come to the experiment and the safety of HP activities. On this page we will give a comprehensive presentation of the safety and reliability of our project.
Pseudomonas aeruginosa PAO1 is a bacterium found in mangrove ecosystems that possesses the strongest ability to degrade polyethylene (PE) plastics. The PAO1 strain is a common reference strain of P.aeruginosa, originating from Bruce Holloway's laboratory, and has become the standard strain for studying the genetics, physiology, and metabolic functions of this bacterium. With proper safety precautions, Pseudomonas aeruginosa can hardly pose a hazard to people in laboratory environment. Pseudomonas aeruginosa is an opportunistic pathogen with little pathogenicity in healthy humans. To prevent P. aeruginosa and the plasmids introduced into engineered bacteria from doing harm in open environment, we designed our suicide system so that the bacteria would kill themselves under certain circumstances or if we needed.
Rhodopseudomonas palustris CGA009 is also a common chassis, and is a Biosafety Level 1 strain, so it is relatively safe to work with and release it into the open environment.
We introduce the hok/sok system (TA) from the plasmid R1 of Escherichia coli to prevent plasmid loss. Considering that resistance gene may transfer from engineered bacteria to non-engineered bacteria and produce more resistance pathogens, we decided to use hok/sok system to help our engineered bacteria keep their plasmids themselves. Assuming that if the recombinant plasmid in engineered bacteria is accidentally lost, both of hok and sok will not transcribe in the bacteria. However, since the degradation rate of sok antisense RNA is much faster than that of hok RNA, the remaining pool of hok RNA in the cell can express after all the sok antisense RNAs are degraded. Then the hok toxin proteins will be encoded, leading to the death of the engineered bacteria that have lost the plasmid, which is able to keep the plasmid stable in the engineered bacteria. Moreover, we replace the promoters of hok with a common weak promoter PcW, as well as the one of sok by popdH which is able to sense concentration of citrate, to conduct bacteria suicide after leaving the environment with citrate. When the engineered bacteria are in the environment with citrate, the strength of PcW is much weaker than PopdH so that the sok antisense RNA can completely repress the effect of hok RNA to keep cells alive; when engineered bacteria escape from the environment with citrate, the strength of PopdH will be weaken and the amount of sok antisense RNA will decrease, and the released hok RNA is now able to encode toxin and kill the engineered bacteria. In addition, we designed an inducible promoter PQJ to artificially control bacterial suicide. When we delivere cumate to the target region, PQJ is able to sense cumate and activate the transcriptional translation of the hok toxin protein. This will prevent sok from fully inhibiting the effect of hok RNA. The hok RNA is able to kill the engineered bacteria in the target region as we needed.
When working on synthetic biology projects, we need to focus on safety issues at every step. Our team takes the safety of our members and the environment as an important prerequisite. Consequently, we are dedicated to taking all necessary precautions to avoid personal or environmental injury. Our team has improved laboratory safety guidelines, standardized experimental operations, and conducted systematic experimental operation training to jointly ensure experimental safety from various aspects.
Figure 1 Laboratory Safety Information Label
Figure 2(left) Micro Fire Station Fire War Cabinet
Figure 3(right) Emergency Showers
First, we clarified the rules regarding personal protection when entering the laboratory. Team members need to wear lab coats, gloves, masks, and wear goggle when necessary. Second, we isolated laboratories and rest areas. It is strictly forbidden to bring food into the laboratory and take things away from the laboratory at the same time. Third, we provided explicit guidance on chemical safety. It is necessary to achieve classified and standardized storage. Irritating volatile drugs must be operated in a fume hood. Our laboratory is equipped with perfect fire prevention and emergency equipment, such as fire blankets, fire extinguishers, smoke alarms, emergency showers, eyewashes and so on.
Figure 4 Team members working with safety covering
Our project is aim to use P.aeruginosa to degrade microplastic. P.aeruginosa is a Biosafety level 2 strain, we required all team members to wear gloves and masks when performing relevant operations to ensure safety. At the same time, we performed operations such as bacterial inoculation in specific biosafety cabinets and culture in different incubators to avoid contamination and infection.
Microplastics are plastics with a diameter less than 5mm which may have adverse effects on the human body. When operating the experiment with microplastic we require team members to wear gloves, N95 respirator and goggle. Besides, we manipulate the microplastics in fume hoods so that they cannot accidentally fall into the environment and enter our respiratory system.
Additionally, we benefited from the guidance and expertise of experienced instructors. These instructors provided comprehensive instructions for experimental procedures and safety protocols. This concerted effort guaranteed not only a secure experimental environment but also a smoother workflow for all team members.
Figure 5(left) Goggles and N95 masks for 3-micron microplastics
Figure 6(right) The fume hood in our laboratory used to handle microplastics and volatiles reagents
Before all experiments are conducted, we conducted laboratory training for all team members, including experimental skills, safety training, and the operation of related experimental equipment. In the early stage of the experiment, we made sure that two people work in pairs to supervise each other and ensure the safety of the experiment. Teachers regularly check the safety of the laboratory and regulate the behavior of the laboratory team members. Prominent and comprehensive safety signs are prominently displayed on large instruments.
Our project required an electroporator to transfer the constructed plasmid into P.aeruginosa. We trained team members on the use of the electroporator and precautions, and printed and pasted the relevant operation steps next to the electroporator.
Figure 7 Guidance on using electroporator
We required team members to perform experiments related on attenuated Salmonella in a biosafety cabinet. Before the start of the experiment, we conducted training on the use of biological safety cabinets, emphasizing the differences between biological safety cabinets and ultra-clean workbenches. The latter mainly protect samples in the work area from external environmental pollution, but do not effectively protect operators. When using a biosafety cabinet, avoid placing too many items in the cabinet, so as not to affect the airflow and safety performance of the safety cabinet. Before and after the use of the safety cabinet, we need to follow the procedure to sterilize the surface of the safety cabinet.
Figure 8 A biosecurity cabinet in our lab
The training content includes the use of large instruments such as high-speed centrifuges, autoclaves, inverted fluorescence microscopes and so on. The training details the principles of action of these large instruments and highlights the precautions. The use of high-speed centrifuges requires high attention to strict trim. We should check the liquid level before each use of the autoclave and promptly remove items post-use. Inverted fluorescence microscope needs to be observed in a dark space. We should pay attention to wait for the eyes to adapt to the dark environment before observing and at the same time we need to wear protective glasses when adjusting the light source to avoid ultraviolet rays damage to the eyes.
Before the formal experiments, we systematically conducted online group training using the MOOC and virtual simulation laboratory platform of Beijing Normal University, focusing mainly on molecular experiments and microbiology experiments, including experimental principles, experimental design and experimental operations. In particular, the virtual simulation project allows us to complete the experimental process by operating simulation, master experimental techniques and methods, and make up for the shortcomings caused by the inability of experimental training to enable us to perform experimental operations efficiently and autonomously.
Figure 9 Stimulation diagram of virtual simulation lab operations
Figure 10 Online training MOOC learning progress
Figure 11 Watching online video of molecular experiment operations
Prior to entering the laboratory and engaging in activities such as bacteria painting and lab tour, we took great care in educating visitors about laboratory safety rules. We emphasized the importance of wearing protective coverings, including masks and gloves, as a strict requirement. In order to make people more aware of the importance of mangroves and the significance of our project, we carried out HP activities in the mangroves, during which we were travelling in groups and guided by professionals to ensure safety.
In the integrated human practice, we ensured the personal privacy of every participant and their individual rights. Specifically, when collecting survey information, we employed an anonymous approach and committed to not using the gathered data for any other purposes. During interviews, if audio or video recording was required, we obtained prior consent from the interviewees. If we intended to publish the interview content online, we sought approval from the interviewees in advance for review. In essence, we made every effort to guarantee the personal privacy of our participants.
