Team:FJNU-China/Safety
Safety
Our team members have organized a laboratory safety test. Everyone have read and learned the safety protocols carefully and apply them in our experiments, and prepared our work surroundings to ensure the biosafety of our experimental environments.
Safe lab work
Which organisms we use?
Our starting strain is Escherichia coli that is biosafety classification of 1 based on U.S. Public Health Service Guidelines. The odorless host (the tnaA gene deletion strain of Escherichia coli ) is introduced in our experiments. These organisms are commonly used with safety in our lab. And the Staphylococcus epidermidis used in the experiment is also a biosafety level 1 strain which is one of the normal skin microorganisms and not pathogenic. We also sterilized the equipment before and after the experiment , by which we minimize the risk of infection. We process our experimental samples reasonably and our experiments will comply with laboratory safety guidelines.
What we do with our organisms?
Our bacteria will be engineered to express a series of enzymes. Such as Escherichia coli atoDA and Clostridium acetobutylicum adhE2 were co-expressed in the host strain to transform E-3M2H into alcohols. Additionally, the broad-spectrum antibacterial phenyllactic acid (PLA) and the pleasant rose-like fragrance 2-phenylethanol (2-PE,) biosynthesis pathways were co-constructed in the host with circuit to achieve antibacterial and natural deodorizing effects.
What do we refer to?
We all complied with the regulations below: 1. The general requirements for Laboratory biosafety, and other regulations about pathogenic microorganisms, medical wastes and experimental animals 2. Fujian Normal University's Regulations on Strengthening Laboratory Safety Management 3. Fujian Normal University Laboratories Safety Management Measures for Dangerous Chemicals 4. Fujian Normal University Laboratory Hazardous Waste Management Measures Our experiments will follow the above guidelines and proceed reasonably. We received training before entering the lab, we received training from our Second PI. • decontamination of materials prior to disposal, using an autoclave. • personal protective equipment (lab coats, eye protection, and gloves) • mechanical pipetting only • the prohibition of food, drinks, and any smokable materials, etc.
Safe project design
In addition to uncontrollable factor in science, the public and government also have their concern， due to limited application scenarios and biosafety concern, it’s often difficult to put these genetically engineered bacteria into practical use. The mazEF is a toxin-antitoxin module located on the Escherichia coli chromosome and some of other bacteria, including pathogens. mazF specifies for a stable toxin protein, and mazE specifies for a labile antitoxin, which antagonizes toxin protein[1]. mazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and mazE inhibits the RNase activity of mazF by forming a complex[2]. In our project We used mazF toxin protein as our killer to ensure biosafety.
The suicide switch we use in project
In the garbage can application , the light control promoter to expressed a toxic protein mazF. When our engineering bacteria were revealed to natural environments, they will faded away soon.
In the bromidrosis application , with this circuit below, we can realize the switch of our engineering bacteria metabolic state switching from the cell growth mode to suicide mode. AraC repressor inhibits transcription from the PBAD promoter. When transcription by the PBAD promoter is induced arabinose addition, expression of toxin protein （mazF） is promoted and the expression of 2-PE under the PLtetO1 promoter is inhibited by the TetR repressor.
Bioremediation - Three Plus Net
The use of genetically engineered bacteria for environmental bioremediation is currently drawing more attentions for its economical applications.On top of due to limited application scenarios and biosafety issues, it is often difficult to put these genetically engineered bacteria into practical use. In addition to public and government acceptance is also a concern in our project.
In order to solve these problems, 3+net is a new concept of the environment track, which was jointly proposed by East China University of Science and Technology (ECUST) and many universities in China. The purpose is to provide guidance and reference for the bioremediation field. 3+net was separately elaborated from the three perspectives of application, biosecurity and society, and explored the relationship among the three aspects. In order to provide more feasible suggestions for people, it is better to apply laboratory results into technologies, resulting in enormous social values.
Base on the collaboration with East China University of Science and Technology (ECUST) , we try our best to apply the results into practical applications and produce greater social values. In scientific methods, we cite some simple methods, such as designing suicide switches, knocking out resistance genes, genome integration, visualization of genetically engineered bacteria, hardware design, network monitoring, and so on. We summarize the current common suicide switches for different types of environmental conditions.