Team:LCG-UNAM-Mexico/Description

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(The Defense System)
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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.<br><br>
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.<br><br>
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Our multipromoter 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.<br><br>
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Our multipromoter 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.<br><br>
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:<br><br>
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:<br><br>
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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.<br>
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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.<br>
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==='''System Specifications'''===
==='''System Specifications'''===

Revision as of 02:49, 20 October 2009


Description

Design

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.

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 protecting 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.


Logic and justification of the project subsystems

The first level of the expression cascade consists on the delivery (Phage Delivey) of the protection system that will be immediately activated when a phage is detected (Defense system: Phage sabotage). At the time that formation of infectious viral particles is hold back, a diffusing signal alarms neighboring cells of the presence of infection. (Gossip). The reaction of alarmed cells consists on turning on a device, which allows a delay in case of turning on the defense system (Paranoia).



Delivery

Production

P4 sid1 genome structure and components

Phage P4 is a satellite phage because it is dependent on the machinery of another phage called P2. Sometimes, it is called a "parasite phage", since it takes over the elements of P2 and leaves its "host phage" practically neutralized. About 100 P4 are produced for each P2 particle. There are several interesting features of the P4 genome, including transactivation zones (the genes that respond to the presence of P2 along with P4 and vice versa) that function in domination of the late P2 genes. Important elements of this kind are gene P4 delta and P2 ogr, which work synergistically together in activating P2 genes. Given these interesting properties, P4 has been exhaustively studied.


We can divide P4 genome into two main regions: the essential and non-essential region. The essential region contains operons intended for replication and hijacking of P2, and the non-essential region contains accessory genes for special situations as lambda infections, as well as the integrase and attachment site. Removing the latter two would result in a permanent plasmid-state P4 with a unique multicopy replication system.


As P4 thoroughly depends on P2 for capsid, tail and lysis functions, the difference in size between both genomes (+-50kb for P2 whereas +-11kb for P4) came up to attention. P4 protein sid is able to scaffold a smaller capsid with the same structural proteins as P2. A sid mutant was found that made P4 pack its genome inside bigger-sized capsids, which can hold up to +-26 kb overall (more than 15 extra kb). In our project, we found this P4 mutation useful for the purposes of synthetic biology.


The main goal in P4 work will be to amplify the essential P4 region so it acquires the standard preffix and suffix. This will be readily useful to be ligated with the desired bioparts and further packed into sid1 capsids. Thus, we obtain the biobrick delivery system inside non-lytic viral particles.


P4 stock production: P2 helper bacteria

Of course after the ligation we don't have viral particles, but naked DNA alone. We thought of a way to overproduce our viral particles without being forced to infect with P2 or getting P2 particles as a byproduct. The solution planned was to construct an E. coli strain containing all the useful genes for P4 in P2 (capsid, tail and lysis operons). In addition to these genes, the helper would also contain the main P2 transactivators (cox and ogr) under a lac operator. This way, after we transform the helper cell with our desired P4 plasmid, we would decide when to promote lysis of the helper bacteria and P4 stock production by adding IPTG. Now we have our biobrick assembled inside ready-to-use phages that can deliver their genome to wildtype bacteria.


Delivery

The Delivery System

System Specifications


Construction:
E Coli Strain:
Toxins:
Bacteriophages:

Relevance of the project

Application areas

Portability

Approach

Standarization

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.

Our multipromoter 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 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.


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.


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