SELF-DIFFERENTIATED BACTERIAL ASSEMBLY LINE PEKING 2007 IGEM All ideas and images from Peking 2007 IGEM team unless otherwise noted Allen Lin 1

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

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 3

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

Binary Switch 5

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 2 nd UV light, LexA degraded, Pku on -> Prm on, red color, back to start  cI(ind-) repressions Pr 6

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 7

Choosing the right parts 8

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

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

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 2 nd state to 1 st state  Use of computation beforehand  Identical input to produce varied output based on previous history -> counter? 11

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

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

Conjugation oriTtraITraI 14

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

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

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 17

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

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

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? 20

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