self-differentiated bacterial assembly line peking 2007 igem all ideas and images from peking 2007...
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SELF-DIFFERENTIATED BACTERIAL ASSEMBLY LINEPEKING 2007 IGEMAll ideas and images from Peking 2007 IGEM team unless otherwise noted
Allen Lin
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Issue
Proteins whose production interferes with each other Need for seperation
Seperation from homogenous conditions in division of labor
Solutions: Temporal Differentiation Spatial Differentiation
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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
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Binary Switch
Bistable switch (stabilizes over time) NOR gate to reset to initial conditions
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Binary Switch5
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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
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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
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Choosing the right parts8
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Confirmation of Bistable Switch
Top figure: Each colony has a stable color
Bottom figure: Two states exist
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How to Select Best Parts
Graphical display of effectiveness (NOR gate)
Blue graph – desired; others – not so much
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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?
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Hop Count
Spatial Seperation – need for different production systems that interfere with each other to happen in fixed proportions
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Plasmids
Main idea: Plasmid vector length as counter In conjugation, DNA between two OriC sites
deleted
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Conjugation
oriT traI TraI
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Isolating Components in Separate Plasmid
Signaling
First delete oriT and traI and place it signaling plasmid Counter will be in
signaling plasmid
Original Plasmid
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Theoretical Model
Cell 1
Helper
Signaling Plasmid
Cell 2
Helper
Signaling Plasmid
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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
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Experimental Result
GFPoriT oriT
After conjugationBefore conjugation
Before conjugation, promoter stops at terminators
Conjugation step removes two terminators between oriT sites
Thus, after conjugation, GFP expressed
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Experimental Result II
Shows that absence of TraI prevents conjugation completely
Plasmid F
with traI
deleted
Wild type
plasmid F
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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?
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Sources
Peking 2007 iGEM presentation and powerpoint http://parts.mit.edu/igem07/index.php/Pres
entations Peking 2007 Wiki Project Notes
http://parts.mit.edu/igem07/index.php/Peking_The_Projects
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