Harvard iGEM 2005: Team BioWire Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush.

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

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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