Team:LCG-UNAM-Mexico/Description

From 2009.igem.org

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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>
 
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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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==='''Phage detection'''===
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When phage T7 or T3 transduce their DNA into the host cell, the phage's polymerase will be able to bind the promoter of the system, which will activate two subsequent actions: production of toxins to inhibit further phage propagation, and a neighborhood alarm. The first thing translated is GFP
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the part contents, in order of appearance, are as follows.-
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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>

Revision as of 18:39, 20 October 2009


Description

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.


        • Creo que esta parte es la que muchos van a ver, estamos regresando al concepto de que es util en la industria y eso no es nuestro fuerte. Creo que nos conviene hablar de un ssitema que nosrmalmente es da;ino para una celula logramos hackearlo y usarlo a su beneficio o almenos defensa. Adem[as el sistema delivery y resaltar el modelo y las dinamicas que logramos reproducir. Creo que si seria bueno checar esto y en ultimo caso dejar el abstract de siempre


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).

    • Aqui estaria bien poner una animaci[on o imagen con los elementos logicos del sistema, o los pasos, como modificar p4, produccion, entrega, deteccion d fagos, respuesta anti fago,....

Design


En esta parte podemos justificar el sistema o almenos detallar las partes que usaremos. puede ser igual en una aimacion


Delivery


Main objective

The main goal of the delvery device is the construction of a new iGEM vector with the peculiarity of being part of a system for transduction of biobricks and synthetic devices in bacteria.

The system starts with a modified bacteriophage P4 genome. This viral vector will be modified to be compatible with iGEM standards for biobrick assembly. Also, because of the removal of the non-essential region from its genome and the use of natural mutant P4sid1, the capsid will be able to contain up to 25 kb of synthetic DNA. Remarkable characteristics expected in our system according to literature is the ability to function in an unusual host range which includes E.Coli, Klebsiella, Serratia and Rhizobium.

The second part of the system involves controlled production of P4 bacteriophages modified with the synthetic constructions. The idea is to create an E. Coli strain capable of producing phages under a certain stimulus.

In brief, we propose a complete, standardized and controllable system for production of phage vectors for delivery of

over 25 kb of synthetic constructs to a wide range of bacterial hosts. The relationships between bacteria and phages
is quite rich and dynamic, so hacking this system for our control will be an amusing challenge!

As can be imagined, this promises an important and powerful tool in Synthetic Biology with a great potential for expansions and applications.


Background


P2-P4 bacteriophages

Bacteriophage P2 and P4 are double stranded DNA enterobacteria viruses. Phage P4 is a satellite phage because it is dependent on the machinery of P2. Sometimes, it is called a "parasite phage", since it takes over the elements of P2

and leaves its "host phage" practically neutralized. 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.


P4 genome structure

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.


P4 sid mutation

As P4 thoroughly depends on P2 for capsid, tail and lysis functions, the difference in size between both genomes (+-

33kb for P2 whereas +-11kb for P4) came 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 1, 2 or 3 copies of its genome. The extra genome copies could be “something else”; this means 

P4 can transport over 20 kbs of extra foreign DNA attached to its genome.


Cos sites

Another important point is that the signal for encapsidation is located in the “cos” sites. It means that you only need this region (about 20 pb, but you increase the efficence of transduction with a region of more that 100 pb) to

encapsidate a double DNA strand disregarding the sequence in addition to the cos sites.



P4 Modifications


The main objectives in modifing P4 genome are:

  • Standardize P4 by removing forbidden restriction sites (EcoRI, XbaI, SpeI, PstI).
  • Add preffix and suffix at the ends so you can clone biobricks in it like any other standard plasmid.
  • Remove the integrase from the genome so P4 will be maintained as a plasmid in the bacterial host.
  • Remove P4 non-essential region. Coupled with the usage of P4sid1 variant, this raises cloning capacity inside the genome up to 25 kb.
  • Add an antibiotic resistance.
  • Add transcriptional terminators surrounding the cloning region.
  • Add annealing sequences for universal primers.


P4sid1-standardized production


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. Then we have our biobrick assembled inside ready-to-use phages that can deliver their genome to wildtype bacteria.

We also have biobricked the cos sites of P4. This biobrick should be coloned in any vector with your construction.

If you transform the productor strain of P4 with this vector then you infect with P4 you will have as a result some 

P4 phages and some of your vectors with the P4cos sites inside a capsid. It means you can encapsidate up to 33 kbs with this system. Until your production is not pure you can add a marker in the plasmid like an antibiotic resistance so after infection you select the colonies with the plasmid and not the natural P4.


System delivery: benefits and perspectives

One of the main motivations for the construction of this delivery system using P4 as the vector is to achieve insertion of devices into cells by transduction as an alternative way from traditional transformation. This extends the panorama of synthetic biology to the whole P4 host range, which involves especies of genera such as Rhizobium, Klebsiella, and Serratia besides Enterobacteria like E. coli. The delivery of parts into wildtype bacteria could be a pool for innovative applications and properties, such as the following:

1) A defense system against another phages for E. Coli delivered by P4 phage.

The idea we love is hacking one system that is harmful (if you are a bacterium), and using it then for your own protection against similar systems.

2) Refined phage therapy.

In addition to bacterial protection, we propose this system to protect humans. This could be using phage therapy to insert pathogenic bacteria. It would be an advantage in cases where extra control is needed, as in degrading toxins before killing the pathogen and so avoiding further immune response. Another benefit would be response specificity in hacking pathogen-specific regulators while the system is bypassed in non-hazardous strains.

As a first step in this area, we have adapted the kamikaze system to detect pathogens instead of phages. The target pathogen is EHEC and EPEC (Enterohemorragic and Enteropathogenic Escherichia coli). P4 will introduce a specific binding site for a pathogenicity-specific regulator LER, which in turn will activate the [[https://2009.igem.org/wiki/index.php?title=Team:LCG-UNAM-Mexico/Description#kamikaze system 3) Storage of information. We like the analogy of this modifiedP4 with an USB *** memoria, wehere you can strorage information or a complete program and then just plug it in another machine that will read the information or execute the program. We are sure it is a fascinating concept with many applications.

another usage could be to "train" the bacterial population by P4 infection so that it is sensitive to a future 

stimulus, like indirect activation of medicine producing devices inside bacteria through phage contact. 4) …



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.




Defense

Phage detection

When phage T7 or T3 transduce their DNA into the host cell, the phage's polymerase will be able to bind the promoter of the system, which will activate two subsequent actions: production of toxins to inhibit further phage propagation, and a neighborhood alarm. The first thing translated is GFP

the part contents, in order of appearance, are as follows.-





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.



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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