Description

The Project

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.

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.

The idea for structuring our project on two subsystems emerges from this global protection vision. The coupled expression of the subsystems leads to a cascade dependent on the presence of an infectious phage. This property gives an extra versatility to our project because the defense turns on fast enough to hold back infection and remains enough to give immunity to the population.

Design

Delivery

Defense

The Defense 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 an individual; however, the real consequences of the infection become important at the population scale. In order to efficiently and accurately simulate the behaviour of The Defense 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 Defense 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 Defense 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.

Defense 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.