As the field of synthetic biology advances, the use of engineered microbes beyond a contained laboratory is increasingly commonplace. Development of such genetically engineered organisms raise concerns of unknown proliferation or unintended widespread of engineered genes in the environment. Physical containment with proper management can be enforced in all the settings. However, accidental release is unavoidable. To enclose engineered microbes in their designated environment, built-in containment systems specific to the environment are used to enhance safety. Kill-switches have a wide variety of applications.

While kill-switches are important, their applications are limited to a specific environment. Different kill-switches have to be designed for different purposes. Different kill-switches would require different sensors and logic to achieve the desired outcome. The process of designing, constructing and testing is no easy work. The process repeats numerous times before the design comes to realisation. Team NUSgem aims to design a toolkit to reduce the iterations, and make engineering of a customised kill-switch easier.

The toolkit consists of three major categories: sensor needed, logic gates to be implemented, and kill mechanism.

The idea is to select the sensors specific to the environment, align the sensors such that it will generate the desired output and kill with the different logics, and eventually construct the plasmid as designed.

The toolkit works with our Design-Model-Build-Test framework.

As a proof of concept, we will design a kill-switch layered with phosphate and temperature sensors for biocontainment of probiotics.

Our kill-switch relies on a dual-input (temperature and phosphate) cascade to achieve an OR gate logic. Upon detection of a wastewater environment (low temperature and low phosphate), the expression of anti-toxin IM2 is inhibited, thereby allowing the constitutively produced toxin, endonuclease E2, to destroy the engineered probiotic. Our design integrates E2 gene into the genome to create plasmid addiction of an IM2 anti-toxin plasmid.

In the presence of phosphate, PhoR, a sensory histidine kinase, is inhibited. PhoR is activated in low phosphate concentration, phosphorylating PhoB and activating the phoB promoter. TlpA36 promoter is temperature sensitive (Piraner, Abedi, Moser, Lee-Gosselin & Shapiro, 2016). It is activated when the temperature is above 36 degree Celsius, upregulating the downstream IM2 production. E2-IM2 is a toxin-antitoxin system. IM2 is an immunity protein that binds to Colicin E2 and inhibit the endonuclease activity.

Probiotics are transported in a phosphate-containing medium and phosphate levels in the gastrointestinal tract is generally high except in the colon (Metcalf et al., 1987). Temperature does not matter in the transportation stage, and will be above 36 degree Celsius when probiotics are consumed. IM2 production is only inhibited when both phosphate and temperature levels are low, resulting in killing of the probiotic. Figure 5 represents the states at different timings.

To make sure the circuit will work as expected, we modeled a design and completed our experiments.