A Genetic Differential Amplifier: Design, Simulation, Construction

and Testing

Seema Nagaraj and Stephen Davies

University of Toronto

Edward S. Rogers Sr. Department of Electrical and Computer Engineering

Institute of Biomaterials and Biomedical Engineering

Engineered Genetic Circuits

• artificial networks of interacting DNA, mRNA and proteins

• behavior is analogous to electrical devices

Genetic Differential Amplifier

• differential amplifiers are fundamental components in many electronic circuits

• a genetic differential amplifier could be a building block for more complex genetic circuits

Electronic Differential Amplifier

Amplifier Behaviour

Gene Expression

geneDNA

Promoter

Gene Expression

gene

RNA Polymerase

DNAPromoter

Gene Expression

gene

RNA Polymerase

DNA

mRNA

Promoter

Gene Expression

gene

RNA Polymerase

Ribosome

DNA

mRNA

Promoter

Gene Expression

gene

RNA Polymerase

Ribosome

DNA

mRNA

Protein

Promoter

Gene Networks

Gene Networks

transcription factor

(repressor)

X

Gene Networks

transcription factor

(activator)

Genetic Amplifier Topology

cI

cro

gfp

Plac

Ptet

PRM -

(+) activator

(-) repressor

Wild Type Promoter

gfp

PRM

Double Mutation

gfp

PRM

Implementation

p15A CAM

PRM-

egfp

PtetPlac

cIcro

pSC101 AMP

Input Amplifier

Stochastic Modeling

• includes inputs and output• system behaviour is described through

reaction probabilities• system behaviour is simulated using the

Gillespie algorithm

Simulation Results(Low Copy Number)

Simulation Results(High Copy Number)

• Construct HIGH and LOW copy number variants of the circuit

• Compare performance to simulated results

Experimental Results

Experimental ResultsHigh Copy Number

15000

16000

17000

18000

19000

20000

21000

22000

23000

0uM IPTG 2uM IPTG 20uM IPTG 200uM IPTG

10 ng/mL aTc

5 ng/mL aTc

0 ng/mL aTc

Experimental ResultsLow Copy Number

7000

7500

8000

8500

9000

9500

0 uM 2 uM 20 uM 200 uM

10 ng/mL

0 ng/mL

Conclusions

Simulation:• low copy number amplifier based on double-

mutant PRM yields “ideal” behaviour

Experimental:• high copy number amplifier displays

differential amplifier behaviour• low copy number amplifier displays positive

results

Acknowledgements

• Anja Lowrance• Members of Davies Lab

• Rogers Scholarship