Bacteria functionality model

Because of the high degree of randomness and limited capabilities of a particular bacterium, communication between two individual bacteria can be unreliable. In addition, the delay in the communication process can be fairly large due to biological actions such as transcription and translation. Also, the effects of gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with cellular context currently make it a challenge to engineer single cells. Most applications or tasks in present synthetic biological systems are generally completed by a population of cells, not any single cell [179]. Hence, to achieve reliability of the communication system, here, the communication model between two populations of bacteria which is proposed in [180] is taken into consideration. In this model, a cluster of bacteria trapped in a chamber is considered as a node. These clusters of biological nodes, which are able to transport information from one point to another, are considered to be the basic building blocks of the communication system. In short, an individual bacterium is very primitive and unreliable and hence incapable of transferring information by itself. Instead, a cluster of bacteria has been applied, which are collectively capable for reliable transmission and reception of molecular information, to form a biological node [180].

The model consists of the transmitter node, the receiver node and the communication channel. Both the transmitter and receiver nodes are considered to be genetically modified bacteria, which can sense specific types of signals and respond accordingly, generally by means of readily isolating plasmids from bacterial cells and altering in

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vitro by inserting or deleting specific sequences of DNA [181] [182]. Molecular communication between two bio-nodes can be made up of three procedures. The transmitter node produces the signalling molecules by adequate stimulation, then these molecules propagate through the medium undergoing Brownian motion and finally the receiver node senses the concentration of the local signalling molecules and takes appropriate actions. The communication system is assumed to be in a theoretically infinite space. The transmitted information is encoded via the concentration of signalling molecules, i.e. the embedding of the information is by alteration of the concentration of the molecules and its transmission relies on diffusion. The output of the receiver node, in the form of luminescence, is measured in steady-state to estimate the concentration of signalling molecules at the vicinity of the node, and hence decode the transmitted information [180].

In this proposed model, both the transmitter and receiver nodes contain instances of the bacterium, V. fischeri, which was introduced in Chapter 3 and is the most commonly studied QS system in gram-negative bacteria. These bacteria are motile, gram-negative rods, 0.8-1.3 in diameter and 1.8-2.4 in length [183]. As a marine bacterium, V. fischeri exists at low cell densities when free living and at high cell densities when colonising the light organ. The luminescence is governed by the expression of certain genes, called the lux operon, in the cell, which in turn is controlled by the density of cells in a population. The regulation of the luminescence genes, named luxCDABE, depends on the production and detection of the signal (Type-I autoinducer), which is synthesised by the protein LuxI and sensed by the protein LuxR. Figure 4.1 illustrates the process with the structure of a bacterium used in such a node shown in Figure 4.1 (a). The features of the production of Green fluorescent protein (GFP), which is the protein responsible for detection or

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production of molecules, is stored in the plasmid which can be added to a bacterium who does not naturally emit GFP through genetic engineering techniques. The receiver node senses the surrounding concentration of Type-I autoinducers, which will trigger the production of GFP and the process is shown in Figure 4.1(b).

Plasmid

DNA AHL

AHL molecule AHL binds to the

receptor (LuxR+AHL)

Molecule Receptor

(LuxR) LuxR+AHL binds to DNA and activates GFP gene

GFP

(a) (b)

Figure 4.1 Bacterial communication: (a) bacterium structure; (b) GFP production.

In V. fischeri, bioluminescence is controlled by the QS system, which is composed by two regulatory genes, luxI and luxR, coding for proteins LuxI and LuxR, respectively. At low cell densities when only a small number of bacteria are present, the signal AHL, which is synthesized by the protein LuxI, is produced by the bacteria at a low level. Then the molecules diffuse out of the bacteria cells and propagate into the surrounding environment. When the bacteria population increases, the concentration of AHLs around the node will grow. If the concentration of the signal reaches a critical threshold, it is able to interact with the LuxR protein, which acts as the ligand receptor for AHL. The LuxR/AHL complex binds to a region of DNA called the lux box, activating the transcription of the bioluminescence operon, which is made up of the luxCDABE genes. In addition, the LuxR/AHL complex also triggers the AHL (via LuxI) to be produced at a higher level. Thus the AHL is said to auto-induce its own synthesis [180].

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Figure 4.2 shows the schematic for the communication between two populations of bacteria. The specifics of the nodes will be discussed in the next section. In this work, the number of bacteria in each node is assumed to be constant. The bacteria inside the node can grow, divide and die to maintain the constant population through the process of gene regulation [10]. It is assumed that each bacterium can sense and produce two different types of AHL molecules, specifically Type-I (3-oxo-C6-HSL) and Type-II (C8-HSL) autoinducers mentioned above [184]. Hence, each bacterium must be equipped in general with two distinct receptor types (for Type-I and Type-II molecules) to perform its functionality as a transmitter or receiver. The transmitted information which will be transmitted at the transmitter is encoded into the concentration of signals, denoted by . The bacteria inside the transmitter node can produce various concentrations of Type-I molecules to be transmitted through the channel by the stimulation of different levels of concentration of specific stimulants. The emitted signalling molecules would then diffuse through the channel to the receiver which is at the distance from the transmitter. At the receiver, each bacterium senses the concentration of Type-I molecules through the corresponding Type-I receptors (LuxR), followed by the production of GFP by bacteria, which is used to decode the input signal concentration . However, it should be noted that depending on the different functionalities, as a transmitter or receiver, only one type of receptors is activated. For the bacteria in the transmitter node, only Type-II receptors (AinR) are activated and the gene expression of AinS is repressed, while for bacteria in the receiver node, only Type-I receptors (LuxR) are enabled and the gene expression of luxI is repressed, a process which can be controlled by proper enzymes. The corresponding reasons will be discussed later.

79 Transmitter Node Receiver Node Bacteria Diffusion Channel Transmitter Node Receiver Node

Bacteria Signalling Molecules

Diffusion Channel

R d R

Figure 4.2 Bacterial communication setup consisting of the transmitter node, the diffusion channel and the receiver node.