Description

The Project

Bacteriophage infection represents an interesting process in science and industry. The idea of being able to contend at a population level with such infections is the main motivation for the development of our project.

We propose a population level approach relaying on a defense system delivered by an engineered version of the enterobacteria phage P4. The purpose of the defense construction is to make a bacteria to hold back the process of infection by triggering a cellular death response when a cell encounters a specific component of the infective phage. Such response will be fast enough to stop the formation process of viral particles, thus preventing the phage proliferation and population decline.

Delivery of the defense system take advantage of the satellite properties of P4 phage. This means that a P4 phage engineered with the defense construction will be able to infect an E.coli strain which harbors some genes from the helper phage P2 that are used for complementing and completing P4 life cycle, hence creating a production line of our version of P4.
On the other hand the defense system will consist of DNA and RNA degradation by toxins which will be transcribed by T3 or T7 RNA-Polymerases fast enough to stop phage assembly and scattering in the environment. Simultaneously, a quorum sensing signal will be difunding to the non-infected bacterias acting as a transcriptional activator of an antisense RNA against bacteriophage's transcriptional machinery , hence "warning" the population to prepare against further T3 or T7 infection.

Furthermore, we will implement a stochastic multi-scale model. The model will simulate the behaviour at the intracellular scale using stochastic molecular simulations and at the populations scale using a Cellular Automata and a system of ODE's.

Design

Delivery

Defence

The Defence System We designed a kamikaze system that will prevent the spreading of phage infection. We fused T7’s promoter with the rRNAse domain of colicin E3 and GFP gene as a reporter. Colicin E3 is a toxin that cleaves 16s rRNAs in active ribosomes of E. Coli.
Naive T7 will infect protected E. Coli which will start producing toxins that deactivate ribosomes. The result: No translation Machinery, no phages production and a heroic bacterium’s death. We expect the burst size to be significantly reduced when our system is working.

A virus infection is a process that takes place inside and individual but the real consequences of the infection become important at the population scale. In order to efficiently and accurately simulate the behaviour of The Defence System we need to implement two different kinds of approaches: an individual-based simulation and a population simulation, and then integrate them in a Multi-Scale Model.

Our construction for the Defence System also integrates LuxI in order to create an Alarm Response. Once a bacterium gets infected T7 promoter will activate the transcription of E3, GFP and LuxI so AHL will be produced and diffused to the extracellular environment.

In order to simulate the spatial dynamics of the Defence System we designed and implemented a Cellular Automata (CA). Using the CA we can approach several problems at the same time: E. Coli movement and duplication, AHL and phage diffusion and the infection process. Parameters for the bacteria in the CA are random variables so we sample the distributions created by the Stochastic Kinetic Simulations:

Finally we create the multi-scale model sampling the distributions created by the Stochastic Kinetic Simulations and use those values as parameters for the cells in the CA.

System Specifications

Construction:
E Coli Strain:
Toxins:
Bacteriophages:

Model Validation

We expect the Burst-Size to be significantly reduced. An optimal result would be a Burst-Size of 0; we see in our results that this is not the case. The BSD has mean ___ and variance___. We can calculate the likelihood of the model (BSD) given the observed burst size for both the wild type and modified E.Coli. The CA and the ODE’s generate growth curves that can be compared with those obtained experimentally.

Relevance of the project

Application areas

Bacteria play a fundamental role in human life. They are still the preferred models in science for the study of the molecular dynamics of organisms; probiotics are of vital importance in industry and food manufacturing. Infection by phages represents a relevant and expensive problem. That is the reason why we decided to construct a system to contend bacteriophage infection.

Portability

The project is designed in such a way that contributes on molecular biology as well as on industry. Our aim is to achieve this by making the defense system portable enough to be used as a tool for protection of profitable bacteria. With portability we mean that we will be able to work with the device in a wide range of bacterial species. Because of the system activation relies on the presence of the replication machinery of the infectious phage not depending on the identity of the protected bacteria, thus leaving the possibility to modify the multi promoter that controls the device to be triggered against specific phages. In the other hand, phage P4 seems to be able to infect a wide range of bacteria, which would contribute to the portability of the system.

Defence approach

An important artefact concerning with the defense system is the use of toxins as the main element in the disruption of phage’s assembly and scattering. Even though the contention of the infection implies that some bacteria will die, the use of a RNAse and a DNAse induces a delay of the phages production by beating host machinery. This in turn, avoids the possibility of the phage to getting resistance against toxins.

Using a population approach makes feasible to achieve a faster and wider protection response by amplifying the infection signal of the delivery phage in order to increase the number of "immune" bacteria at every lytic cycle.

Standarization and delivery

Standardization of biobricks has become an alternative in the development of easier and more profitable tools for genetic engineering. In this context, our project takes advantage of the phages property for infecting and transducing genetic material into bacteria. We will modify P4 bacteriophage in such a way that facilitates gene cloning into phage’s genome for its subsequent transduction into bacteria harboring the P2 genes for completing lytic cycle of the carrier phage and its exponential release.