LCG-UNAM-MEXICO - 2009.igem.org2009.igem.org/files/presentation/LCG-UNAM-Mexico.pdf ·...
Transcript of LCG-UNAM-MEXICO - 2009.igem.org2009.igem.org/files/presentation/LCG-UNAM-Mexico.pdf ·...
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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
of 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 systemAs 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 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
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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 our 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.