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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