Team:NYMU-Taipei/Notebook-Biosafety
Overview
In our project we used E. coli, Metarhizium anisopliae, and Bactrocera dorsalis, which are all classified as biosafety level 1 organisms. All of the organisms were cultured and used under well-designed protocol and by students equipped with full personal protection gear. Also, all lab members went through wet lab training under the tutelage and supervision of our team instructors and their counterparts from NYMU 2015 iGEM team. The training included lab emergency protocols, operation protocols for various machines, and biological waste decontamination and disposal protocols.
Escherichia coli DH5alpha:
We are well aware of the skin, eye, and respiratory tract irritation E. coli may cause, therefore, all members of our wet-lab team are required to wear full personal protection gear when operating with E. coli competent cells and cultures. All materials that came in contact with bacterial cultures or competent cells were autoclaved and disposed properly.
Metarhizium anisopliae:
Our experimentation with Metarhizium anisopliae is split between two locations. We were able to culture, transform, and perform genomic DNA extraction on M. anisopliaein the fungal lab of Dr. Ying-Lien Chen, thanks to his kindness. We also designated a M. anisopliae-experiment-only area.
Bactrocera dorsalis:
Bactrocera dorsalis, or oriental fruit fly, serves as the experimental host insect for transformed and wildtyep M. anisopliae. The infection rate of B. dorsalis by wildtype M. anisopliae will be tested live individuals. B. dorsalis is a major fruit pest around the world. To minimize the possibility of escape and property damage by B. dorsalis specimen, we have constructed plastic and cardboard containers with netted entrance. These containers allow us to feed and clean excrement while the flies are contained.
Killswitch Biosafety
The main focus of our project is to create a biosafety mechanism, a killswitch in this case, for M. ansiopliae. Our kill switch will include a KillerRed production circuit controlled by a hemolymph-induced promoter, PMcl1. This circuit will be activated when M. anisopliae enters the hemolymph after it penetrates the cuticle of its insect host. The KillerRed protein with NLS that was produced, due to the need of yellow spectrum light to activate, its will stay inert as long as the fungus is within the host insect. However, when M. anisopliaeemerges from its host for conidiation, the yellow spectrum light in sunlight will allow KillerRed proteins to produce highly reactive oxygen species (O2•-) in nucleus, which in turn will kill the fungus. The transformed fungus will pose no additional threat to humans.
Future Outlook and Risk Evaluations
Large-scale spraying, the current deployment method of Metarhizium anisopliae, may pose several potential threats to environment if the payload consists of genetically modified M. anisopliae conidia. That is why, we develop a bait trap that could minimize these threats by attracting male oriental fruit flies, infecting them with spore solution and spreading the fungus to other members of its species, including the pheromone-trap-resistant females. When genetically modified Metarhizium anisopliae is deployed via our oriental fruit fly bait trap, it comes in direct contact with the surrounding environment. This is why its development needs careful evaluation if further application is desired. As for this subject, we will continue to consult the Food Drug Administration and the Council of Agriculture in Taiwan and will continue advocating the legislation on related issues. We would follow the standards provided by WHO, which compels us to continue the evaluation of the stability, horizontal gene transfer rate, and toxicity of our inserted genes if further field test and application are conducted.
