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Harvard iGEM 2005:Team BioWire
Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush
Project Overview
Goal– To engineer a biological “wire” capable of
propagating a chemical signal down its length
Initial Signal
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
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 & LuxIAHL
Transmission: Circuit Design
QuickTime™ and aAnimation decompressor
are needed to see this picture.
Constructs
Parts shown are for Lux system. Las analogues were built as wellParts shown are for Lux system. Las analogues were built as well ..
Final ConstructFinal Construct(cotransformed)(cotransformed)
Test ConstructsTest Constructs(separate cells)(separate cells)
Wire: Stamping
Place lines of bacteria down on agar using micropatterned agarose stamps
Wire: Stamping
Stamping process 1mm perimeter lines
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
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
1mm1mm
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)
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
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 +AHL+AHL -AHL-AHL
YFP w/o degradation tagYFP w/o degradation tag YFP w/ degradation tagYFP w/ degradation tag
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
We’ll have results for you soon….
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
Achievements
Constructed all parts for propagating signals for both the Lux and Las systems and routers
– Approximately 150 parts
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
Future Work
Debug Lux propagation system Test and characterize Las system Make dual-system oscillators
BA
AB
AA
AA
AABB
BB
BB
– 2 propagating wires using different signaling molecules (Lux, Las)
– Wires connected using routers that convert one signal to the other