Team:LCG-UNAM-Mexico/Description

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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.

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).

Design

Delivery

Background

  • P2-P4 bacteriophages
  • P4 genome structure
  • P4 sid mutation
  • Cos sites

Bacteriophage P2 and P4 are double stranded DNA enterobacteria virus. 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. About 100 P4 are produced for each P2 particle **** Esto dependeria de la cepa no ¿?? ***** . 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 (+- 33kb 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 1, 2 or 3 copies of its genome. The extra genome copies could be “something eles”, it means P4 can transport over 20 kbs of extra foreign DNA if atached to its genome.

Another important point is that the signal for the 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 without regarding of the sequence in adition to the cos sites.


Main objective and relevance

This part of the project consists in a system for transduction of synthetic constructions.

The system starts with a modified bacteriophage P4 genome. This viral vector will be modified to be compatible with iGEM standard for biobrick assembly. Also, because the remotion of non-essential regio non its genome and the use of a natural mutant of this phage P4sid1, the capsid will be able to suport up to 25 kbs of synthetic DNA. Another remarkable characteristic that we expect in our system according to litterature, is the ability to function in an inusual host range than includes E.Coli, Klebsiella, Serratia and Rhizobium.

Second part of the system is the production under our regulation of these P4 bacteriophages with the synthetic constructions. The idea is to create an E. Coli as a productor strain of this phages that becomes production of P4 only under a stimulus. So you only will need to transform this strain with the viral vector + your construction and the production of the P4 phages may become only when an activation of two main regulators in the system.

In conclusion we propose a complete, standardized and controllable system for production of P4 bacteriophages than can carry over 25 kbs of synthetic DNA and can transduce an inusual host range of bacteria. Another interesting point is that the relation between bacteria and their bacteriophages is quite rich and dynamic so hacking this system to now control it has been really entertaining.

As you can imagin it mean san important and powerfull tool in Synthetic Biology with a great potencial for expansions and applications.


P4 Modifications

The main objectives of modifing P4 genome are:

  • Standardize P4 by removing forbiden restriction sites (EcoRI, XbaI, SspeI, PstI).
  • Add preffix and suffix so you can clone biobricks like in any other standard plasmid.
  • Remove the integrase from the genome so P4 will be maintained as a plasmid in the bacteria host.
  • Remotion of the non-essential region. Together the use of P4sid1 variant means the ability to carry about 25 kbs of *synthetic DNA.
  • Adding an antibiotic resistance.
  • Adding transcripcional terminators bordering the cloning region.
  • Adding sequences for primering with universal primers.

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


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. Now 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.

P4 infection – transduction – delivery

The process of infection requires a protocol to increase efficence. The protocol we have used are:

For transduction, an important characteristic of P4 bacteriophage is its unusual range of infection. In litterature is reported that this unusal range includes Klebsiella, E. Coli, Serratia and Rhizobium.


Applications

Applications and extensions are many. Two of them we propose for this year are:

1) A defense system agains another phages for E. Coli delivered by P4phage. The idea we love is the hacking of one system that is harmful (if you are a bacteria), and use it then for your own protection agains similar systems.

2) Refinated phage therapy. In adition of protection of bacteria we propose this system for protecting humans. This by creating an important tool for a refinated phage teraphy were you not only kill bacterias with phages *** but you insert a program inside the pathogen. Its an advantage in cases were you need extra control, in time for example or a program to degrade toxins befote killing the pathogen so toxins are not Spreads in the organism avoiding an increased immune response.

As a first step in this area we have adapted the kamikazee system to detect phatogens instead of phages. The paghes to detect is EHEC, by detecting its molecule for quorum sensing, the kamikazee construction will kill quickly this cell.

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.

4) …


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 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.


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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