2008 NINT iGEM Team:J. Bolstad, J. D'Amico, S. Deane, H. Gaber, N. Glass, Q. Li, E. Nguyen, J. Robinson-Anagor, J. Rodway, R. van den Hurk, Z. Wiltshire

Team Instructor:W. Materi

Project Logi-col[i]Adventures in genetic logic

The 2008 NINT iGEM Team

Project Logi-col[i]2008 NINT iGEM Team

Sum 12

Carry 21

XOR1

XOR

OR

AND

AND

Cin3

Input 22

Input 11

Carry 22

Sum 11

XOR

AND

Input 22

Input 11

Out 11

AND

Input 22

Input 11

Input 1 Input 2 Output 10 0 01 0 00 1 01 1 1

Electronic logic

What could we build with genetic logic?

Anderson et al. 2006

Bacteria that target metastatic tumors

New organisms with controlled structure

Inexpensive houses from engineered plants

Genetic equivalence to electronic logic?

Connectivity

Electrical systems

Extensibility

Biological systems

The problem with current approaches

Controls transcription through activator/repressor proteins

Requires specific protein - DNA interaction

Protein engineering is still in its infancy

Connectivity

Biological systems Electrical systems

Extensibility

The problem with current approaches

Terminator/Attenuatoranti-sense Logic (T/AasL)

Transcription is a complex, regulated process

Transcription

promoter sequence

3’

3’

5’

5’

copied DNA strand

RNA polymerase

Transcription Termination

3’

5’ RNA polymerase

Stem-loop structure

mRNA

RNA regulation of transcription

Terminator at start of transcript = Attenuator

3’ U-rich region

Principles of T/AasL

T/A gateAnti-sense RNA

Disrupted T/A gate, transcription proceeds

Output RNA is input to the next T/A system

•Predictable structure allows simple design of multiple logic gates that are– Connectable – output and input are same– Extensible – rational design algorithms based on

thermodynamic principles– A complete implementation of PoPs!

Principles of T/AasL

Logic Gate Design

Prediction of T/A stability

• Based on M-fold algorithm• Can process 4 interacting strands of RNA

Stability of stem loop structures:

• # nucleotides in loop• type of nucleotides in

loop• length of stem• %GC content in stem

E: -10.5kJ/molE: -11.6 kJ/mol

E: -7.2 kJ/mol E: -11.7kJ/mol

Design of T/AasL gates: A B Out0 0 01 0 00 1 0

1 1 1

AND Gate

A

A B

T T T

T T

T/aT/aaT sequences

• aaT & aTs not followed by UA rich region• Relative stabilities affect equilibrium

anti-anti-Terminator Terminator

aaT TaT

anti-Terminator

Requires aaT > aT > T

OR Gate

aaT T

A

aaT T

B

A

aT

aaT

B

A B Out0 0 01 0 10 1 1

1 1 1

Design of T/AasL gates:

NAND Gate(The Universal gate)

Cryptic terminator sequence

aTaT

A B

aT aT

A B

A

BT

aT

A

aT

B

A B Out0 0 11 0 10 1 1

1 1 0

Requires aT > T

Design of T/AasL gates:

From logic gates to plasmids…

Connecting devices

Connected plasmids

EcoRI

XbaI

SpeI

PstI

Device 1

TA1

TA2In

EcoRI

XbaI

SpeI

PstI

Device 2

TA2

TA3In… …

Input test harness

ara

Input test harness plasmid

EcoRI

XbaI

SpeI

PstI

Device 1 TA2In

PBAD

araC TA1

XhoI

HindIII

Output test harness

LacZα

Output test harness plasmid

EcoRI

XbaI

SpeI

PstI

Device 2

TA2

TA3InLacZα

BamHI

NcoIEcoRI

XbaI

SpeI

PstI

Device 1 TA2In

PBAD

araC

XhoI

HindIII

Experimental Results

What is the best way to disrupt a T/A?

Reporter Ratio

What is the best way to disrupt a T/A?

2.70

2.38

2.46

0

10

20

30

40

50

60

70

80

90

100

No input

Input

Expected results (single input gates):

Rela

tive

Lac

Z Ac

tivi

ty

TRUE his Synthetic anti-Terminator

LacZ assay - terminator efficiencyM

iller

Uni

ts

Construct

0

100

200

300

400

500

600

700

800

900

2021 2031 2041 2051 2061 2071 2111 2121

TRUE (always ON)

His term(THRU)

“Improved” synthetic gates

2081 2091 2101

His term + pause

Synthetic + pause

with pause

no pause

anti-terminators

Terminator efficiency and stability

Terminator stability (kcal/mol)

Rela

tive

ter

min

ator

eff

icie

ncy

No correlation

-0.25

0.00

0.25

0.50

0.75

1.00

-80 -70 -60 -50 -40 -30 -20 -10 0

LacZ assay - anti-sense activationNo activation – but we have some more ideas

Construct

Acti

vati

on r

atio

0

0.5

1

1.5

2

2021 2031 2041 2051 2061 2071 2081 2091 2101 2111 2121

Future Directions and Final Thoughts

T/A gate

2º structure

anti-sense input

Version 1.2 (based on Isaacs et al., 2004)

Bulges in stem

YUNR sequence

Future research•Version 1.2 of gates and anti-sense input•Design and characterize dual input devices• Implement OR, AND and NAND gates •Design and implement XOR gate•Connect devices into more complex circuits • Full adder circuit

Sum 12

Carry 21

XOR1

XOR

OR

AND

AND

Cin3

Input 22

Input 11

Summary•Modeling

•stem-loop stability•multi-input T/A gates

•Experimental Research•Natural and synthetic terminator efficiency•Anti-sense disruption efficiency•26 BioBrick parts submitted•Designed a viable PoPs implementation

Connectivity

Extensibility

T/AasLElectrical systems

Conclusion - a viable PoPs design

Acknowledgements: Our sponsors:

And a special thanks to our team instructor, Dr. Wayne Materi

Thank you!

Input test harness plasmid(with hammerhead ribozymes)

EcoRI

XbaI

SpeI

PstI

Device 1 TA2In

PBAD

araC

XhoI

HindIII

HH2

HH1

Taira et al., 1991

RNA Cleavage

Bayer and Smolke, 2005

RNA aptamer regulation of translation