Introduction

One of our main goals is to engineer CATE such that the release of payload is controlled via multiple checkpoints. The first checkpoint is the preferential accumulation of bacteria in solid tumors upon intravenous administration. According to literature, four days after administration E. coli Nissle 1917 is found exclusively in tumor tissues. Up to this point however, small numbers of the bacteria are also present in liver and spleen [1]. Therefore, the second checkpoint is evaluated by the bacteria themselves - they should be able to autonomously decide whether they are located in a tumor or healthy tissue. Only then the third checkpoint, thermoactivation by focused ultrasound, comes into play.

Choosing Inputs

To pass the second checkpoint, namely activating our circuit only in tumorous tissue, we implemented a AND gate of two inputs: acyl-homoserine lactone (AHL) and L-lactate. AHL is produced by the bacteria themselves and reaches high concentrations only in dense populations. This system, termed quorum sensing, is used by a range of bacteria in nature to sense their population density and change bevaviour accordingly [5]. Thus, AHL should only be present in tumor tissue in sufficient amounts. The second input is L-lactate, a chemical produced in high amounts by cancer cells as a consequence of defects in cellular respiration and other aberrations, even under normoxic conditions [2]. Hence, a combination of lactate and AHL is a unique molecular pattern that would only occur in tumor tissue.

Defining the Strategy

In order to achieve an AND-gate behaviour, we relied on the previously characterized parts LldP/LldR and Plldr as well as Plux and LuxR . For lactate sensing, the two main components are LldP and LldR. LldP is a transmembrane protein that imports L-lactate from the environment into the cell, while LldR is an intracellular protein that binds to two sites on the DNA, termed O1 and O2. Binding of LldR to these sites leads to formation of a DNA-loop that “hides” the region in between O1 and O2 from the transcription activation complex. On the other hand, once L-lactate is present, LldR binds it and undergoes a conformational change as a consequence. This results in the opening of the loop whereby intervening regions are exposed again [3].

For quorum sensing the molecule N-acyl homoserine lactone (AHL) plays a central role. AHL - or more precisely 3-oxohexanoyl-homoserine lactone - binds to LuxR whereby LuxR undergoes a conformational change which in turn leads to formation of homo-dimers. This complex will then bind to the Plux promoter sequence and recruit RNA polymerase, thus activate transctription of downstream genes [4].

Final Design

We reasoned that flanking the Plux promoter with O1 and O2 should lead to AND-gate behaviour. In absence of lactate, pLux should be looped and therefore inaccessible to LuxR, independent of AHL being present or not. On the other hand, if lactate is present but AHL is not, Plux is exposed but will not be activated. Once the lactate-part is triggered, pLux is accessible for the dimerized transcription factor LuxR, leading to transcriptional activation of the downstream gene.