LCG-UNAM-MEXICO 2009 MOTIVATION Industry processes involving - - PowerPoint PPT Presentation

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LCG-UNAM-MEXICO 2009 MOTIVATION Industry processes involving - - PowerPoint PPT Presentation

Jul 12, 2023 29 likes •580 views

LCG-UNAM-MEXICO 2009 MOTIVATION 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

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LCG-UNAM-MEXICO 2009

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  • 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

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Engineer and standardize a bacteriophage so it delivers a synthetic defense construction, thus leaving bacteria

  • f interest protected against other phages.

SOLUTION

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  • 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

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Delivery / Defense

1) Delivery system 2) Defense system

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Delivery system

a) Production b) Delivery

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Defense system

a) DETECTION b) KAMIKAZE c) GOSSIP d) PARANOIA

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Defense system

DETECTION: A specific element of the phage (Bad guy) triggers a transcriptional response.

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Defense system

a) DETECTION b) KAMIKAZE c) GOSSIP d) PARANOIA

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Defense system

KAMIKAZE: Kills the cell as fast as possible to avoid the formation of new viral particles.

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Defense system

a) DETECTION b) KAMIKAZE c) GOSSIP d) PARANOIA

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Defense system

GOSSIP: Like an alarm, bacteria start spreading the rumor that a phage (bad guy) is near.

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Defense system

a) DETECTION b) KAMIKAZE c) GOSSIP d) PARANOIA

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Defense system

As an extention….. PARANOIA: Anticipates a response against phages before the infection happens.

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The complete system

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Main Goal

  • Engineer a device which can transduce

synthetic DNA constructions into different bacterial hosts.

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Overview

  • Select, standardize viral plasmid
  • Control the production of phages
  • Transduce bacteria with viruses
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It’s a non lytic phage It complements P4 reproduction!!

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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

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P4 Production

Goals.- Engineer a strain with P2 particle formation genes Control P4 particle formation with P2 regulators (control construction)

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  • IPTG inducible
  • Cox and ogr regulators (global for P2)

Control construction

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Mass production

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Applications

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Applications

Insert pathogen-only gene traps into

  • bacteria. (refined phage therapy)

Insert genes to fight other phage’s infections (coming up next!!)

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System dynamics

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System dynamics

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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.

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Multipromoter functionality

T7 RNA polymerase show specific transcriptional activity.

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Multiscale System

  • Individual dynamics: biochemical reactions

inside the cell.

  • Population dynamics: infection spread.
  • We need to unify both scales.
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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

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Molecular Model diagram

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Simulated burst size

  • distribution. Experimentally

reported values are shown in vertical red bars.

Our Burst Size Distribution

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Objectives

  • Simulate:
  • Bacterial behaviour
  • Phage spread
  • AHL diffusion
  • Infection
  • The Defense System
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. 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.

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Simulation of WT infection

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Simulation of WT infection

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Molecular Model diagram

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Simulation of infection with the KAMIKAZE system

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Simulation of infection with the KAMIKAZE system

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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

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Sensitivity Analysis for Ribosome 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

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  • 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.

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  • 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

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  • 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.

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  • They helped us with relevant aspects of
  • ur project model.
  • We supported them with transformations

and plasmid extractions for the biobricks they needed.

IPN-UNAM-MEXICO

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Acknowledgments

  • We want to thank everybody

who made possible this project, specially Universidad Nacional Autonoma de Mexico for beliving in our work.