Project Description

Recent advancements in engineered probiotics have enabled a host of novel medical therapeutics and diagnostics. One such example is scientists using engineered microbes to successfully target tumors in the gastrointestinal tract with minimal collateral damage and invasiveness [1, 2, 3, 4].Yet, despite these developments in engineered therapeutics, the risk of engineered microbes leaking into a non-designated environment remains an unresolved threat: a threat that could disrupt ecosystems and increase antibiotic resistance of natural bacteria. Therefore, critical to the feasibility of a functional engineered probiotic are control systems called killswitches that allow for spatial and temporal killing of the engineered probiotic once removed from its specific target site. However, the function of many existing killswitches is limited because they rely on: (1) Single input biosensors which decreases specificity [5]; (2) artificial amino acids which are difficult and unsuitable for implementation in the human body [6]; and (3) active control based on controlled expression of a toxin which in turn can lead to microbe survival upon mutagenesis of the killswitch [7, 8, 9].

To improve current switches, Team NUSgem proposes a novel passive biocontainment system for the human gut with enhanced specificity, safeguard and with a newly added function of verification . Our cascaded dual-biosensor killswitch is configured to achieve an OR gate logic upon detection of a physical (temperature) and chemical (phosphate) input to enhance specificity. Upon detection of a wastewater environment outside of the human body, the system will inhibit the expression of anti-toxin IM2, thereby allowing the constitutively produced toxin-endonuclease E2, to destroy the engineered probiotic. Our design purposefully places the E2 toxin gene in the probiotic’s genome to not only to create plasmid addiction of an IM2 plasmid, but also to safeguard against mutagenesis. Moreover, we use the bidirectional properties of the pTlpA36 temperature sensitive promoter to express the verification of engineered probiotic death. Added verifcation provides monitoring of the killswitch's effectivness which in turn, has the potential to used as a stimulus for a feedback-loop driven killswitch.