Pr oj ect Logi - col [ i ] Pr oj ect Logi - col [ i ] Adventures - - PowerPoint PPT Presentation

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

Pr oj ect Logi - col [ i ] Pr oj ect Logi - col [ i ]

Adventures in genetic logic

The 2008 NINT iGEM Team

Pr oj ect Logi - col [ i ] Pr oj ect Logi - col [ i ]

2008 NINT iGEM Team

Sum 1 2 Carry 2 1

XOR1 XOR OR AND AND

Cin 3 Input 2 2 Input 1 1

Carry 2 2 Sum 1 1

XOR AND

Input 2 2 Input 1 1

Out 1 1

AND

Input 2 2 Input 1 1

Input 1 Input 2 Output 1 1 1 1 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/Attenuator anti-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 gate Anti-sense RNA Disrupted T/A gate, transcription proceeds Output RNA is input to the next T/A system

• Predictable structure allows simple design
• f 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/mol E: -11.6 kJ/mol E: -7.2 kJ/mol E: -11.7kJ/mol

Design of T/AasL gates:

A B Out 1 1 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 T aT anti-Terminator

Requires aaT > aT > T

OR Gate

aaT T

A

aaT T

B A

aT aaT

B

A B Out 1 1 1 1 1 1 1

Design of T/AasL gates:

NAND Gate

(The Universal gate)

Cryptic terminator sequence

aT aT

A B

aT aT

A B A B

T aT

A

aT

B

A B Out 1 1 1 1 1 1 1

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 TA3In LacZα BamHI NcoI EcoRI 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

10 20 30 40 50 60 70 80 90 100

N

• input

Input

Expected results (single input gates):

Relative LacZ Activity TRUE his Synthetic anti-Terminator

LacZ assay - terminator efficiency

Miller Units

Construct

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) Relative terminator efficiency

No correlation

• 0.25

0.00 0.25 0.50 0.75 1.00

• 80
• 70
• 60
• 50
• 40
• 30
• 20
• 10

LacZ assay - anti-sense activation

No activation – but we have some more ideas

Construct Activation ratio

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 1 2 Carry 2 1

XOR1 XOR OR AND AND

Cin 3 Input 2 2 Input 1 1

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/AasL Electrical 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