Team:CSU Fort Collins/KillSwitch/

From 2014.igem.org

Kill Switch

Creating a Kill Switch

While other parts of our project were based around the idea of recycling waste material into useful products, some environmental concerns still remained, such as how to control the E. Coli in the event of a containment breach and minimize its impact to its surroundings. To alleviate these issues, we decided to insert a kill switch into our E. Coli as an added safety mechanism.

Utilizing the gene known as KillerRed (Bulina, 1), as well as a negative Tryptophan promoter (BBa_K588001), we were able to construct a basic kill switch. When the E. Coli is exposed to Tryptophan, the KillerRed gene is repressed, and the cells continue to grow and carry out normal function. However, when removed from a Tryptophan-rich system (ie. escaping containment and entering the general environment) the KillerRed gene is no longer repressed and quickly begins producing reactive oxygen species- ROS- when exposed to red spectrum light. These ROS cause rapid apoptosis, leading to mass cell death soon after the E. Coli enters an uncontrolled environment.

After constructing our Trp+KillerRed plasmid, we ran several experiments designed to test its effectiveness as a kill switch (see below). While our data did not show that all of our E. Coli was killed off via KillerRed, it did show that the gene had an affect, as plates grown from samples irradiated with steady white light for 30 minutes had significantly less colonies than those taken from samples not exposed to light.

Moving forward, we would like to study the Trp-KillerRed system in greater detail, by modelling out their function based on experimental data with the goal of optimizing the system. Reducing leakage and lag time of the kill switch would greatly increase its impact on the safety of the system.

1. Bulina, M., et. al. "A genetically encoded photosensitizer.” Nature Biotechnology, 95-99. 2005. October 2014.