engineering a virus to select against antibiotic resistance
DESCRIPTION
Houston, TX. Engineering a virus to select against antibiotic resistance. Drug-Resistant Staph Germ's Toll Is Higher Than Thought Wednesday, October 17, 2007; Page A01. Tuesday, 10 October 2006. CDC concerned about drug-resistant TB. Friday, March 24, 2006. - PowerPoint PPT PresentationTRANSCRIPT
Engineering a virus to select against antibiotic resistance
Houston, TX
Drug-Resistant Staph Germ's Toll Is Higher Than Thought
Wednesday, October 17, 2007; Page A01
Tuesday, 10 October 2006
CDC concerned about drug-resistant TBFriday, March 24, 2006
Nearly 19,000 deaths in the U.S. in 2005 due to infections by a new virulent and rampant drug-resistant strain of Staphylococcus aureus
“If the mortality estimates are correct, the number of deaths associated with the germ, methicillin-resistant Staphylococcus aureus, or MRSA, would exceed those attributed to H.I.V.-AIDS, Parkinson’s disease, emphysema or homicide each year.”
Mechanisms of antibiotic resistance
•Efflux Pumps•Hydrolysis•Reduced Uptake•Sequestering•Enzymatic Modification
The Science Creative Quarterly 2: Jan-March 2007.
Existing strategies to reduce microbial antibiotic resistance all have significant drawbacks
Strategies Drawbacks Use large amounts of
antibioticsMerely slows the development of resistance, $$$
$$$, time consuming, unknown side effects
Develop new antibiotics
Select against specific mechanisms of resistance
Too specific, chemical side effects
Utilize mutually antagonistic antibiotics
Only targets resistance to specific antibiotics, hard to control
Project Roadmap
Computational ModelingCircuit Design
& Construction
Circuit Characterization
Project Roadmap
Computational Modeling
Circuit Characterization
Circuit Design & Construction•general circuit design
•create constructs with reporters
Circuit design principles: a stable method for programming phenotype-dependent fitness
Overarching goals:
1. use existing BioBricks
2. create a modular design
3. identify a robust architecture
4. circuit function selects regardless of resistance mechanism
5. self-propagating circuit
fitness
Pantibiotic
CellS > CellR
Nature has developed ways of detecting low [antibiotic] within cells
No Tetracycline (Tc):
No TetA no resistance
expressed
Low [Tc]
TetA resistance expressed
tetR tetA
tetR tetA
TetATetA
Fitness increased
Smaller effect on fitness
tetR fitness
tetR fitness
TcS bacteria
TcR
bacteria
Differentiating between sensitive (TcS) and resistant (TcR) cells affects fitness
LOW [Tc]
Bacteriophage lambda can stably alter fitness
Lysogeny Lysis Molecular Biology of the Cell. New York: Garland; 4th Edition, March 2002.
Bacteriophage lambda can stably alter fitness
Robust and stable
fitness alteration Lysogeny Lysis Molecular Biology of the Cell. New York:
Garland; 4th Edition, March 2002.
Bacteriophage lambda can stably alter fitness
Controllable through
BioBricks
Lysogeny Lysis Molecular Biology of the Cell. New York: Garland; 4th Edition, March 2002.
Bacteriophage lambda can stably alter fitness
Self-Propagation
Lysogeny Lysis Molecular Biology of the Cell. New York: Garland; 4th Edition, March 2002.
A circuit producing [CI] will increase phage immunity and fitness
lysogeny
lysis
Increased [CI] Reduced [CI]
Phage-encoded cI
Phage-encoded cI
Circuit-encoded cI
Increased [CI] Reduced [CI]
lysogeny
lysis
A circuit producing [CI] will increase phage immunity and fitness
PTet
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
Intr
ace
llula
r [te
tra
cycl
ine]
Imm
unity to
pha
ge
cI
PX
tetR
cI
PX
tetR
cI
PX
tetR
Our circuit design couples phage immunity to antibiotics
PTet
PTet
PTet
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
C
N
Our circuit design couples phage immunity to antibiotics
Intr
ace
llula
r [te
tra
cycl
ine]
Imm
unity to
pha
ge
cI
PX
tetR
cI
PX
tetR
cI
PX
tetR
TcR bacteria
TcS bacteria
PTet
PTet
Our circuit consists of three interconnected pieces
tetR
Px
Tc Sensor
eyfp
PTet
Reporter
PTetcI generator
cI
C
N
C
N
BBa_I744103
tetR
Px
Tc Sensor
eyfp
PTet
Reporter
Characterization constructs include the sensor and reporter
J23113
J23114
J23106
BBa_I744101
BBa_I744102
tetR
PTet
Tc Sensor
eyfp
PTet
Reporter
Characterization constructs include the sensor and reporter
TetRR0040
BBa_I744104
PTet
cI
C
N
C
N
The lambda cI generator construct confers immunity to phage
BBa_I744121
B0014
One full circuit has been constructed that uses feedback regulation
BBa_I744204
tetR
PTet
Tc Sensor
eyfp
PTet
Reporter
PTetcI generator
cI
C
N
C
N
TetRR0040 B0014
Project roadmap
Circuit Design & Construction
Circuit characterization
Computational Modeling• evaluate plasmid-based circuit
Using the Gillespie algorithm for modeling the effect of cell type on circuit output
Journal of Physical Chemistry. Vol. 81:25 p.2340-61.
Tc
tetR
PX
cI
PTet
TetR2
TetA
PNAS. Vol. 102:10 p.3581-3586.
CI2
Constitutive promoters produce too much TetR
Steady-state levels (copies/cell)
tetR
PxTc Sensor PTet ImmunitycI
The model predicts that the the circuit can differentiate cell types
tetR
PTetTc Sensor PTet ImmunitycI
Resistant
Sensitive
Mol
ecul
es
Minutes 100Time (minutes)
Molecules of cI/cell
Project Roadmap
Computational ModelingCircuit Design & Construction
Circuit Characterization• test circuit’s ability to
differentiate between cell type
tetR
PTet
Tc Sensor
eyfp
PTet
Reporter
PTetcI generator
cI
C
N
C
N
Registry Part # I744204
TetRR0040
EYFP output was used to characterize the full circuit
B0014
Cellular chassis for characterization
Journal of Bacteriology. Vol. 177:3 p.810-814.
Cell strains were graciously provided by G. N. Bennett
GNB8385K=TcS
GNB824=TcR
Tn10
TcS+circuit
TcS-circuit
TcR+circuit
TcR-circuit
Circuit: I744204
Control B0011
Excitation = 514 nm, Emission = 527 nm
No detectable EYFP output in TcR E. coli
[aTc] = 1 µMKD = 0.01nMBiochemistry. Vol. 35:23 p.7439-7446
0
1 105
2 105
3 105
4 105
5 105
6 105
7 105
0 20 40 60 80 100
Flu
ores
cenc
e/A
600
Time (min)
TcR+circuit
TcR-circuit
Time (min)
Excitation = 514 nm, Emission = 527 nm
In contrast, EYFP is produced under similar conditions in TcS E. coli
TcS+circuit
TcS-circuit
[aTc] = 1 µMKD = 0.01nMBiochemistry. Vol. 35:23 p.7439-7446
0
1 105
2 105
3 105
4 105
5 105
6 105
7 105
0 20 40 60 80 100
Flu
ores
cenc
e/A
600
Effects of increasing Tc on cell growth
[Tc] ≤ 2 µM can be used to drive our circuit with large adverse effects on cell growth
However, the effects of Tc on EYFP (and CI) production have not yet been characterized.
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
0.1 1 10
Tetracycline (uM)
OD
600
(AU
)
TcR +circuit
TcR
TcS +circuit
TcS
TcR-circuit
TcR+circuit
TcS-circuit
TcS+circuit
T = 240min
Conclusions
Computational Modeling
• created effective model and selected circuit for characterization
Circuit Design & Construction• built a variety of selection circuits
Circuit Characterization• demonstrated circuit’s ability to differentiate between cell types
Future Roadmap
Computational Modeling
• model circuit in phageCircuit Design & Construction• construct phage optimized circuit
Circuit Characterization• test circuit function within phage
COMPUTATION AND APPLIED MATH Steve Cox Jay Raol
CHEMICAL AND BIOMOLECULAR ENGINEERING Ken Cox Alec Walker
Baylor College of Medicine Bibhash Mukhopadhyay
BIOCHEMISTRY AND CELL BIOLOGY Beth Beason George Bennett Tina Chen David Kim Joff Silberg Taylor Stevenson Arielle Layman
BIOENGINEERING Christie Peebles Ka-Yiu San Thomas-Segall Shapiro
Linking our cell detection to phage lysis
0
1 105
2 105
3 105
4 105
5 105
6 105
7 105
0 20 40 60 80
Flu
ores
cenc
e/A
600
Time (min) Time (min)
0 20 40 60 80
Create TcR and TcS temperature sensitive lysogens and determine if circuit provides a relative fitness gain for TcS over TcR cells
TcR+/-circuit TcS
+/-circuit
Ptet Controlled Production of TetR Creates Resistance Phenotype
TcS+circuit
tetRPtet
TcS
I744204 B0011
Tetracycline Affects Growth Rates
t = 30min
1744204
B0011
(empty)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0.001 0.01 0.1 1 10 100
Tetracycline (uM)
OD
600
(AU
)
TcR +circuit
TcR
TcS +circuit
TcS
Tetracycline Affects on Growth Rates
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0.001 0.01 0.1 1 10 100
Tetracycline (uM)
OD
600
(AU
)
TcR +circuit
TcR
TcS +circuit
TcS
t = 240min
TcR
TcR+circuit
TcS
TcS+circuit
I744204 B0011
Phage Circuit Incorporation Relies Upon Recombination Plasmid
bleoR
lambda homologous
Biobrick restriction site
bleomycin resistance
λ1 λ2