1. Self-differentiated Bacterial Assembly Line Peking 2007 IGEM
All ideas and images from Peking 2007 IGEM team unless otherwise noted
Allen Lin

2. Issue Proteins whose production interferes with each other
Need for seperation
Seperation from homogenous conditions in division of labor
Solutions:
Temporal Differentiation
Spatial Differentiation

3. Push-on-Push-off Switch
Temporal Seperation
Same input at different times -> different output
Simple finite state machine (has a current state and cyclic) – binary

4. Binary Switch Bistable switch (stabilizes over time)
NOR gate to reset to initial conditions

5. Binary Switch

6. Details of Binary Switch
At first, Prm on -> CI and lacI produced, red color
C1 represses Pr, lacI represses Pku
After UV light, CI degraded; Pr stronger promoter -> CI434 produced, green color
Pr represses Prm, lacI production falls (mRNA degraded), so only LexA represss Pku
After 2nd UV light, LexA degraded, Pku on -> Prm on, red color, back to start
cI(ind-) repressions Pr

7. Theoretical Computation (with ODE)
Allen’s comments:
NOR gates can be used to build AND, OR, INV gates
Note the time scale
GFP and RFP can be replaced by two different production systems

8. Choosing the right parts

9. Confirmation of Bistable Switch
Top figure: Each colony has a stable color
Bottom figure: Two states exist

10. How to Select Best Parts
Graphical display of effectiveness (NOR gate)
Blue graph – desired; others – not so much

11. Allen’s Thoughts How was the circuit designed?
The circuit is a 2-state, cyclic system – can it be expanded? What components are specific to this implementation?
Needed a NOR gate to reset from 2nd state to 1st state
Use of computation beforehand
Identical input to produce varied output based on previous history -> counter?

12. Hop Count
Spatial Seperation – need for different production systems that interfere with each other to happen in fixed proportions

13. Plasmids Main idea: Plasmid vector length as counter
In conjugation, DNA between two OriC sites deleted

14. Conjugation
oriT
traI
TraI

15. Isolating Components in Separate Plasmid
First delete oriT and traI and place it signaling plasmid
Counter will be in signaling plasmid
Signaling
Original Plasmid

16. Theoretical Model Cell 1 Cell 2 Signaling Plasmid Signaling Plasmid
Helper
Signaling Plasmid
Cell 2
Helper
Signaling Plasmid

17. Details
Important that the different oriT/traI systems do not interfere (crosstalk) with each other
Things to note:
One promoter – only traI nearest promoter is transcribed, terminator ends that transcription
DNA replication occurs in opposite direction of promoter
Thus lose traI + terminator + functional oriT every step

18. Experimental Result Before conjugation After conjugation GFP oriT
Before conjugation, promoter stops at terminators
Conjugation step removes two terminators between oriT sites
Thus, after conjugation, GFP expressed
Before conjugation
After conjugation

19. Experimental Result II
Shows that absence of TraI prevents conjugation completely
Plasmid F with traI deleted
Wild type plasmid F

20. Allen’s Thoughts
Shows two steps needed for hop count system to work once
Place assembly line specific genes in between oriT sites before terminator
Ensures that one cell does each step; other cells in assembly line not far away
What would happen if we combined temporal and seperation? Get bistable neighboring cells? Checkerboard configuration?

21. Sources Peking 2007 iGEM presentation and powerpoint
Peking 2007 Wiki Project Notes
ojects