E3

E9

GFP

Terminator

T7Promoter

LuxI

LuxRTerminator

TetPromoter

Lux Box

RFP

Terminator

Lux Box

T3Antisense

T7 Antisense

KanamycineResistance

Su�x

Pre�x

P4 genome T3Promoter

Ori

Possible bacteria states during phage infection Growth Curve. E. coli WT

AHL concentration in enviroment Growth Curve. E.coli infected with T7

Multi-Scale integration using Cellular AutomataThe main goal of the Cellular Automata (CA) was to integrate the information contained in the Molecu-lar Distributions (particularly the BSD) with a popu-lation simulation in order to observe the behaviour of the whole system under di�erent conditions. The experimental work with T7 and the CA show the same overall behaviour.

Defense

The device was designed as a tool for delivering biobrick assemblies of up to 26 kb. We took advantage of the phages' natural ability for transducing DNA. The bacteriophage of choice was P4 along with basic elements of phage P2 that complete its cycle. In order to gain room for the insertion of constructs, the non essential region for the replication of P4 was eliminated. The targets could be several bacteria belonging to the P4 host range such as Klebsiella, Salmonella, Shigella, Serratia and even Rhizobium. (referencia)

The previously described delivery device will be used for the distribution of a defense system, whose main elements are two colicins employed to destroyDNA and RNA. Such toxins will be transcribed by the (infectious) phage RNA-polymerase, fast enough to stop phage assembly and scattering in the enviro-ment. Symultaneously, a quorum sensing signal will spread out to the non-infected bacteriA warning and preparing them against future T3 or T7 infec-tion. We propose the use of an antisense RNA that could be expressed in order to target phage's replication.

Delivery

Devices

Fight Fire with FirePhage-mediated bacterial bite back

LCG-UNAM-Mexico Team

Abraham Avelar, Willebaldo García, Laura Gómez, Adrián A. Granados, Luis F. Montaño, Libertad Pantoja, Enrique Paz, Jorge Quintana, Eduardo Soto, Minerva S. Trejo, Uriel Urquiza, Carlos Vargas, Arturo Velarde, Miguel A. Ramírez, Osbaldo Resendis.

Abraham Avelar, Willebaldo García, Laura Gómez, Adrián A. Granados, Luis F. Montaño, Libertad Pantoja, Enrique Paz, Jorge Quintana, Eduardo Soto, Minerva S. Trejo, Uriel Urquiza, Carlos Vargas, Arturo Velarde, Miguel A. Ramírez, Osbaldo Resendis.

Abraham Avelar, Willebaldo García, Laura Gómez, Adrián A. Granados, Luis F. Montaño, Libertad Pantoja, Enrique Paz, Jorge Quintana, Eduardo Soto, Minerva S. Trejo, Uriel Urquiza, Carlos Vargas, Arturo Velarde, Miguel A. Ramírez, Osbaldo Resendis.

Abraham Avelar, Willebaldo García, Laura Gómez, Adrián A. Granados, Luis F. Montaño, Libertad Pantoja, Enrique Paz, Jorge Quintana, Eduardo Soto, Minerva S. Trejo, Uriel Urquiza, Carlos Vargas, Arturo Velarde, Miguel A. Ramírez, Osbaldo Resendis.

Abraham Avelar, Willebaldo García, Laura Gómez, Adrián A. Granados, Luis F. Montaño, Libertad Pantoja, Enrique Paz, Jorge Quintana, Eduardo Soto, Minerva S. Trejo, Uriel Urquiza, Carlos Vargas, Arturo Velarde, Miguel A. Ramírez, Osbaldo Resendis.

The ProjectBacteriophage infection represents an interesting process in microbiology and in-dustry. The idea of being able to contend at a population level with such infec-tions is the main motivation for the development of our project.

We propose a population approach based on a defense system delivered by an engineered version of the enterobacteria phage P4. The purpose of the defense construction is to provide bacteria with a system that holds back the infection process by triggering cellular death response when a cell encounters a speci�c component of the infective phage. Such response must be fast enough to stop the formation of viral particles, thus preventing phage proliferation and popula-tion decline.

We also propose the use of the delivery phage as a standardized method for clo-ning of synthetic biobricks based on the natural properties of phages such as P4 and P2, which transduce into a range of novel hosts.

Plasmid

ogr

cox terminator

lysis tail capsid

P2E3

E9

GFP

Terminator

T7Promoter

LuxI

LuxRTerminator

TetPromoter

Lux Box

RFP

Terminator

Lux Box

T3Antisense

T7 Antisense

KanamycineResistance

Su�x

Pre�x

P4 genome T3Promoter

Ori

Induction

T7 RNA polymerase

T3 RNA polymerase

DNA

E3

E9

GFP

Terminator

T7Promoter

LuxI

LuxRTerminator

TetPromoter

Lux Box

RFP

Terminator

Lux Box

T3Antisense

T7 Antisense

KanamycineResistance

Su�x

Pre�x

P4 genome T3Promoter

Ori

GFP

DNALuxI

AHL

LuxR+ AHL

T7genome T3genome

Ribosomes

Modelling Approaches

Population Scale

Molecular ScaleBased on previous works we simulated chemical-kinetic systems of the life cycle of phage T7 using a stochastic framework. One of the systems corresponds to the Wild-Type Model (WTM) of the life cycle of phage T7 in E Coli.A second model, the KamikaZe Model (KZM) simulates the interplay and performance between the kamikaze system and the phage T7 infection. At a molecular level, KZM integrates contribution of ribosomes to the translation rates and attack of toxins over bacterial translation machinery.

Ensembles of runs of WTM and KZM will provide data to build Burst Size Distri-butions (BSDs)* for each model. BSDs are further used in the population models to recreate the impact of our synthetic circuit on phage infection at a population level. *Burst size: Typical number of phage released by an infected bacterium.

We used three di�erent approaches: * Multi-Scale integration using Cellular Automata * Mathematical Model using Delay Di�erential Equations * Agent Based Simulation Applet

Stochastic molecular simulations for essential reactions of T7 life cycle with kamikaze system.

To predict whether our defense system will function inside the cell andwhether our population will survive a T7 phage infection.

Agent based simulation using Net Logo.

Results

T3/T7 multipromoter functional-ity was tested qualitatively with GFP using an IPTG-inducible T7 RNA polymerase.

T3/T7 multipromoter functional-ity was tested qualitatively with GFP using an IPTG-inducible T7 RNA polymerase.

Fig 2. Optical Density measurements for T3 and T7 infections on E.coli C-1a strain. Blue line show growth without phages.

Multipromoter

40X micrography of BL21 strain carry-ing multipromoter. Induced by 0.1mM IPTG.

Concluding remarks

Both, delivery and defense systems represent a promising introduction of bacteriophages as rich elements in synthetic biology.

We designed a transduction system based on the natural properties of phages such as P4 and P2, which could deliver synthetic biobricks into a wide range of hosts.

A possible clinical application is the sabotage of pathogenicity regulators in bacteria for manipulating disease development.

We have seen molecular and population models working separately, but we have never seen a multi-scale model that integrates molecular and population dynamics in such a delightful and realistic way as we did.

Our model successfully predicted the experimentally calculated burst sizes for T7.

The model is a reliable tool for prediction of infected population behaviors. As well as in the analysis of the sensitivity of infection to pa-rameter perturbations.

References1.Propagation of satellite phage P4 as a plas-

mid. Goldstein R, Sedivy J, Ljungquist E.

2.Mechanisms of Genome Propagation and

Helper Exploitation by Satellite Phage P4.

Lindqvist BH, Deh˜ G, Calendar R. (1993)

3.Evolution of Bacteriophage T7 in a growing

plate. Yin. (1992)

4.Intracellular Kinetics of a Growing Virus: A

Genetically Structured Simulation for Bacte-

riophage T7. Endy D., Kong D., Yin J. (1996)

5. E�ects of Escherichia coli Physiology on

Growth of Phage T7 In Vivo and In Silico. Yin J.

(2002)

6.Stochastic Models in Biology S. Goel.

(2003)

AcknowledgmentsDavid Romero Camarena, PhD

Guillermo Dávila Ramos, PhD

Ian Molineux, PhD

José Luis Reyes Taboada, PhD

Julio Collado Vides, PhD

Karla Cedano Villavicencio, M.S.

Luis Kameyama, PhD

Otto Geiger, PhD & his research group

Rafael Palacios, PhD & his research group

Richard Calendar, PhD

Sabino Pacheco, M.S.

State Government of Morelos, Mexico

Undergraduate Program in

Genomic Sciences(LCG)

Fig 1. Simulated wild type Burst Size Distribution for T7. Experimentally reported values are shown as vertical red bars. +- 1 standard deviation from the mean is shown as a horizontal red bar.

Fig 3. Simulation using the Cellular Automaton for the experiment described in Fig 2.

Fig 4. Theoretically predicted behaviour for infection process with the Defense System.

Burst Size Distributions were simulated for both wild type and Kamikaze E.Coli using WTM and KZM respectively. WT BSD is supported by experimentally reported data (�gure 1).Cellular Automaton simulations sample BSDs in real time.

Burst Size Distributions were simulated for both wild type and Kamikaze E.Coli using WTM and KZM respectively. WT BSD is supported by experimentally reported data (�gure 1).Cellular Automaton simulations sample BSDs in real time.

Burst Size Distribution

Simulations using the CA accurately reproduced the behav-iour of the T7 infection experiments. The simulations and the experiments were made under the same initial conditions. This results show the reliability of our multiscale model predic-tions.We observe that the bacteria population successfully con-tended the whole infection. Our results suggest that the De-fense System will work as expected.

Simulations using the CA accurately reproduced the behav-iour of the T7 infection experiments. The simulations and the experiments were made under the same initial conditions. This results show the reliability of our multiscale model predic-tions.We observe that the bacteria population successfully con-tended the whole infection. Our results suggest that the De-fense System will work as expected.

Bacteriophage Infection.