Harvard iGEM 2005: Team BioWire

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Harvard iGEM 2005Team BioWire

• Orr Ashenberg, Patrick Bradley, Connie Cheng,
  Kang-Xing Jin, Danny Popper, Sasha Rush

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Project Overview

• Goal
• To engineer a biological wire capable of
  propagating a chemical signal down its length

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Our Approach

• Signal acyl-homoserine lactones (AHL) used in
  bacterial quorum sensing
• Lux system 3OC6HSL
• Las system 3OC12HSL
• Transmission pulse controlled by a genetic
  incoherent feed-forward loop
• Wire engineered E. coli placed in wire form with
  agarose stamps

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Transmission Circuit Design

• Incoherent feed-forward loop combined with
  positive feedback
• AHL upregulates production of cI, YFP, and LuxI
• LuxI produces more AHL molecules
• cI represses YFP and LuxI production

cI
YFP LuxI
AHL
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Transmission Circuit Design
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Constructs
Final Construct (cotransformed)
Test Constructs (separate cells)
Parts shown are for Lux system. Las analogues
were built as well.
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Wire Stamping

• Place lines of bacteria down on agar using
  micropatterned agarose stamps

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Wire Stamping
Stamping process
1mm perimeter lines
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Key Experiments

• All experiments were done on Lux system
• Senders and Receivers
• Testing signal reception in cells laid down with
  the stamp
• Propagation Constructs
• Testing induction of propagation constructs with
  AHL
• Testing intercellular propagation

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Senders and Receivers

• AHL producing sender cells were combined with
  receivers that fluoresced in response to AHL.
• Cells were laid down using agarose stamps

Senders
Receivers
1mm
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Senders and Receivers

• Results
• Receiver cells fluoresced when laid down with
  sender cells.
• Conclusions
• Test constructs work stamping is a viable method
  of laying down cells in a predetermined pattern

Receivers (near senders)
Receivers (far from senders)
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Propagation Constructs

• Propagation cells included the entire
  incoherent feed-forward loop/positive feedback
  system
• RBS and degradation tags on proteins were varied
• AHL was added to propagation cells in liquid
  media to test for induction

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Propagation Constructs

• Results
• Issues with noise - cells were either
  constitutively on or off regardless of AHL
  addition
• Conclusions
• Degradation tags, RBS/promoter strength may need
  fine-tuning
• Because of positive feedback, noise is amplified
• Further experiments necessary

AHL
AHL
-AHL
-AHL
YFP w/o degradation tag
YFP w/ degradation tag
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Propagation Constructs Take 2

• Combined propagation cells with reporter cells
  that respond to AHL
• Propagator reporter fluorescence should extend
  farther than reporter fluorescence alone
• Stamped with sender cells
• Well have results for you soon.

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Challenges

• Construction
• Time consuming nature of circuit construction
• Need for rapid and accurate verification of parts
  with sequencing
• Organization
• Difficulty in keeping track of large numbers of
  subparts involved in construction
• Sasha created a database to organize and automate
  the assembly process

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Challenges
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Achievements

• Constructed all parts for propagating signals for
  both the Lux and Las systems and routers
• Tested parts of the Lux system
• Successful induction of receivers via sender
  cells
• Preliminary tests on propagation systems
• Designed a protocol for stamping bacterial cells
  on agarose in any desired pattern with 500 micron
  resolution

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Future Work

• Debug Lux propagation system
• Test and characterize Las system
• Make dual-system oscillators

• 2 propagating wires using different signaling
  molecules (Lux, Las)
• Wires connected using routers that convert one
  signal to the other

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