Introduction

One of our main goals is to engineer CATE such that the release of payload is controlled on different levels. 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 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 if they have reached tumor tissue. Only then the third checkpoint, thermoactivation by focused ultrasound, comes into play.

Choosing Inputs

To realize the second level of control, namely avoid activation of our engineered system outside of tumors, we devised a strategy for CATE to be able to differentiate between healthy and tumor tissue. This is achieved by AND-logic integration of two inputs: AHL and Lactate. AHL is produced by the bacteria themselves and reaches high concentrations only in case the population is dense. 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, only in tumor tissue AHL should be present in sufficient amounts. The second input is 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 such 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-homoacylserine lactone (AHL) plays a central role. AHL 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 the 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. Only in the case where both inducers are present, no loop is formed and Plux is accessible to dimerized LuxR, which lead to transcription activation. See the illustration for a graphical representation of the action of our synthetic AND-gate promoter.