Introduction

Sensing the temperature is crucial for the lifecycle of many bacteria and virus. We adapt this function from a specific heat-inducible system and engineer it to fit our needs for activating the toxin release in CATE.

Various thermosensitive operator systems exist in nature and they differ massively in their way of function. Four general classes of thermosensors exist: DNA, RNA, protein or lipid-protein thermosensors. DNA thermosensors rely on the bending of DNA at lower temperatures, which enables cooperative binding of DNA-associated proteins. RNA based thermosensors form a stem-loop in the messenger RNA, which hides the Shine-Dalgarno sequence and the AUG translation initiation codon. At higher temperatures, the hydrogen bonds of the stem-loop break apart and the ribosomal subunits can associate with the RNA and initiate translation. While DNA-and RNA thermosensors act before translation, for protein mediated temperature sensing, a translated protein needs to be present in the cytoplasm. This comes with the potential to tune the translation initiation rate by changing the ribosome binding site affinity to the ribosome.

CATE's heat sensor

For CATE, we chose the TlpA thermosensor, which is derived from Salmonella and belongs to the protein thermosensors. The advantage of this system is the high on/off-ratio of up to 300 and induction temperature of 45 °C, a temperature not reached by fever, but still below levels that cause damage in tissues surrounding a tumor. We adapt this function so that we can use it to trigger the release of the Anti-Cancer Toxin.

The TlpA system consists of a constitutively expressed regulator protein called TlpA and an inducible TlpA operator-promotor called P_TlpA. TlpA contains an approximately 300-residue coiled-coil domain at the C-terminus that uncoils between 42 °C and 45 °C. In low temperatures, its N-terminal domain is in a dimeric state and can bind the 52-bp P_TlpA. Transcription of the downstream gene can therefore happen at temperatures above 42 °C but not below. [1][2]

Checkpoint 2

The Heat Sensor is used as Checkpoint 2 to ensure only bacteria in the tumor site release the Anti-Cancer Toxin.

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