Self-differentiated Bacterial Assembly Line Peking 2007 IGEM

1. Self-differentiated Bacterial Assembly LinePeking 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 LexArepresssPku • 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

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

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

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. oriT GFP oriT Experimental Result • 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 • http://parts.mit.edu/igem07/index.php/Presentations • Peking 2007 Wiki Project Notes • http://parts.mit.edu/igem07/index.php/Peking_The_Projects