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