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 release of engineered genes into the environment. Physical containment with proper management can be enforced in laboratory setting. Nevertheless, 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. This so called kill switches have a wide variety of applications.

Application of Kill switches

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 requires numerous iterations before the design comes to realization. Currently, there is no harmonized framework for kill switch design and construction. Team NUSgem aims to develop a methodology (which includes the use of computer aided design and modelling tools, development of toolkit and customised chassis) to make engineering of customized kill switch easier. As a proof of concept, we designed and developed a kill switch layered with phosphate and temperature sensors for biocontainment of engineered probiotics.

Our idea is to have an engineered E. coli chassis in which the killing module has been integrated in the chromosome. As such, users can just focus on the designing the sensors and logic required to trigger the immunity protein. Due to time constraint, we collaborated with the Swedish Team which created the E. coli chassis with killing module integrated in the chromosomes while we focus on developing the toolkit and the methodology for designing the sensors and logic. The toolkit consists of three major categories: sensor for designed inputs, logic gates to be implemented, and the killing mechanism.

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

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

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. TlpA promoter is temperature sensitive (Piraner, Abedi, Moser, Lee-Gosselin & Shapiro, 2016). It is activated when the temperature is above 36 degrees 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 are generally high except in the colon (Metcalf et al., 1987). Temperature does not matter in the transportation stage, and will be above 36 degrees Celsius after the probiotics are consumed (human body temperature). 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.