SELF-DIFFERENTIATED BACTERIAL ASSEMBLY LINEPEKING 2007 IGEMAll 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 Switch5

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

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 parts8

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 2nd state

to 1st 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

oriT traI TraI

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 oriT

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 http://parts.mit.edu/igem07/index.php/Pres

entations Peking 2007 Wiki Project Notes

http://parts.mit.edu/igem07/index.php/Peking_The_Projects

21