Download BioWire Progress Report Week Eight

Harvard iGEM 2005:
Team BioWire and
BioLoserz!!! LOL
Orr Ashenberg, Patrick
Bradley, Connie Cheng,
Kang-Xing Jin, Danny
Popper, Sasha Rush
Project Overview
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Goal: to engineer a synthetic, macroscopic
biological “wire,” capable of propagating a
chemical pulse down its length
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Chemical signaling uses homoserine lactones
(Lux, Las) (quorum sensing pathway in V. fischeri)
picture?
Pulse is controlled via transcriptional regulation
Bacteria placed in wire form with photolithography
BioWire: “Pulse” Propagation
Initial Signal
Circuit Design: Pulse Generator

Incoherent feed-forward loop plus positive
feedback
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AHL stimulates production of cI, YFP, and LuxI
cI represses YFP and LuxI
Result: a pulse of fluorescence, followed by a
refractory period
AHL
cI
Animated line following flow of wire
YFP & LuxI
Circuit Design: BioWire

Incoherent feed-forward loop plus positive
feedback (GET RID OF THIS SLIDE,
CONSOLIDATE INTO PREVIOUS)
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LuxI produces more AHL, which triggers
neighboring cells
AHL
cI
YFP & LuxI
Construction
Part pictures (propagators,cross-linkers etc), number of ligations,
Sasha’s database
Photolithography

Place lines of bacteria down on agar using
micropatterned agarose stamps
CAD of stamp
->Mask->Master->PDMS molds->agarose stamps
Photolithography
Want picture of agarose stamp, possible master too and PDMS mold
Stamping process
1mm perimeter lines
Experiments
AHL sender-receiver
Future Work
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Circle Oscillator
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Signal moves in a circle
1 signaling molecule A: signal goes both ways
2 signaling molecules A, B: signal is unidirectional
BA
AA
AA
AA
AA
AA
AA
AA
AA
AA
BB
AA
BB
BB
AA
AB
Sender / Receiver
Sender / Receiver
Phase
GFP
Receiver
Phase
Receiver GFP
Senders, 100X phase
Receivers, 100X phase
Senders, 100X GFP
Receivers, 100X GFP
Receivers far from senders, 100X phase
Receivers far from senders, 100X GFP
Receivers near senders, 100X phase
Receivers near senders, 100X GFP
Timing Diagram for Pulse
YFP
cI
AHL
New Ideas: Toward Oscillators
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Line Oscillator
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“Bounce” a signal back and forth along a line
Initial Signal
New Ideas: Toward Oscillators
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Line Oscillator (design 1)
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1 signaling molecule, 2 types of cells: “End” cells
and “Interior” cells
End cells have a longer delay (> 1 interior cell
refractory period) between receiving input and
generating pulse output
END
INT
INT
INT
INT
INT
END
New Ideas: Toward Oscillators

Line Oscillator (design 2)
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Two signaling molecules A and B
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4 cell types: AB, BA, AA, BB. Cell XY takes input X and
pulses Y in response.
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Lux with either Las or Rh1 (respond to different variants HSL)
e.g. AB takes input A and pulses B in response.
Requires 2 signaling molecules but does not require
different time scales on end cells.
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Issues: Crosstalk and BioBricks.
A
AB
AA
BB
B
AA
BB
AA
BB
AA
BB
AA
BB
BA