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 ·...
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.