LCG-UNAM-MEXICO

2009

• Industry processes involving microorganisms could be

severely affected by viral infections.

• Manipulate and model the infection process is an

interesting challenge.

• Constructing a transduction iGEM standard system,

portable wide host range.

MOTIVATION

Engineer and standardize a bacteriophage so it delivers

a synthetic defense construction, thus leaving bacteria

of interest protected against other phages.

SOLUTION

• Design and standardize a biobrick delivery system

based on transduction.

• Construct a population-scale phage defense system.

• Develop a multiscale model to simulate phage

infections and performance of our defense system.

MAIN OBJECTIVES

Delivery / Defense

1) Delivery system 2) Defense system

Delivery system

a) Production b) Delivery

Defense system

a) DETECTION

b) KAMIKAZE

c) GOSSIP

d) PARANOIA

Defense system

DETECTION:

A specific element of the phage (Bad guy)

triggers a transcriptional response.

Defense system

a) DETECTION

b) KAMIKAZE

c) GOSSIP

d) PARANOIA

Defense system

KAMIKAZE:

Kills the cell as fast as possible to avoid

the formation of new viral particles.

Defense system

a) DETECTION

b) KAMIKAZE

c) GOSSIP

d) PARANOIA

Defense system

GOSSIP:

Like an alarm, bacteria start spreading the

rumor that a phage (bad guy) is near.

Defense system

a) DETECTION

b) KAMIKAZE

c) GOSSIP

d) PARANOIA

Defense systemAs an extention…..

PARANOIA:

Anticipates a response against phages

before the infection happens.

The complete system

Main Goal

• Engineer a device which can transduce

synthetic DNA constructions into different

bacterial hosts.

Overview

• Select, standardize viral plasmid

• Control the production of phages

• Transduce bacteria with viruses

It’s a non lytic phage

It complements P4 reproduction!!

Nonessential region+ integrase

Bjorn H. Lind Qvist, Gianni Deho and Richard Calendar, Mechanisms of genome propagation and

helper explotation by satellite phage P4. Microbiological Reviews, Sep. 1993, p. 683.702

P4 Production

Goals.-

Engineer a strain with P2 particle formation

genes

Control P4 particle formation with P2

regulators (control construction)

• IPTG inducible

• Cox and ogr regulators (global for P2)

Control construction

Mass production

Applications

Applications

Insert pathogen-only gene traps into

bacteria. (refined phage therapy)

Insert genes to fight other phage’s infections

(coming up next!!)

System dynamics

System dynamics

Expected Behaviour

• No translation, no phage production and a

heroic bacterial suicide.

• Because of the lack of protein production

we expect a reduction in the number of

newly synthesized phages.

• The population will survive the infection

process.

Multipromoter functionality

T7 RNA polymerase show specific

transcriptional activity.

Multiscale System

• Individual dynamics: biochemical reactions

inside the cell.

• Population dynamics: infection spread.

• We need to unify both scales.

Simulating the burst size

• In 1945 Delbrück obtained values which

indicated an exceedingly wide variation.

All reported values we found for T7 are between 100 and 300

Delbrück, 1945

Molecular Model diagram

Simulated burst size distribution. Experimentally reported values are shown in vertical red bars.

Our Burst Size Distribution

Objectives

• Simulate:

-Bacterial behaviour

-Phage spread

-AHL diffusion

-Infection

-The Defense System

. We can think of the cells in the grid as biological cells.

- We define the rules for the evolution of the system and we can simulate

biological behaviour.

Simulation of WT infection

Simulation of WT infection

Molecular Model diagram

Simulation of infection with the

KAMIKAZE system

Simulation of infection with the

KAMIKAZE system

Drew Endy, Deyu Kong, John Yin. 1996.

Intracellular Kinetics of a Growing Virus: A

Genetically Structured Simulation for

Bacteriophage T7

Lingchong You, Patrick F. Suthers, and John Yin.

2002. Effects of Escherichia coli Physiology on

Growth of Phage T7 In Vivo and In Silico

Sensitivity Analysis forRibosome inactivation rate.

-Burst size reduced to zero for inactivation rates greater than 10e-5

-Mean burst size reduced to ~6 for inactivation rate 10e-5. Bacteria die!

Automata simulation for mean BS ~6

Sensitivity analysis

• We designed a transduction system based on the

natural properties of P4 and P2, which could deliver

synthetic biobricks into a wide range of hosts.

• Both, delivery and defense systems represent a

promising use of bacteriophages as rich elements in

synthetic biology.

• The delivery system is still under construction but

expectatives for the wide host range are high.

• Our simulations are consistent with the experimental wild type

behavior and suggest that the defense device will work as

expected.

• Although there have been attempts of integrating individual

and population models, we think that ours is an innovative

approach for the study of complex behaviour arising in

biological systems

• Furthermore, the integration of this kind of multiscale

approaches with the experimental work will indeed be crucial

for the future design and study of biological systems

• Our model successfully reproduced the

experimentally calculated burst sizes for T7.

• The model is a reliable sampling tool of the

diverse molecular species involved in the

process of bacteriophage infection.

-They helped us with relevant aspects of our project model.

-We supported them with transformations and plasmid extractions for the biobricks

they needed.

IPN-UNAM-MEXICO

Acknowledgments

• We want to thank everybody

who made possible this project,

specially Universidad Nacional

Autonoma de Mexico for

beliving in our work.