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

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

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

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

Binary Switch

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

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

Choosing the right parts

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

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

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?

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

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

Conjugation oriT traI TraI

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

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

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

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

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

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?

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_Pr ojects