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 & LuxI AHL

Transmission: Circuit Design

Constructs Parts shown are for Lux system. Las analogues were built as well. Final Construct (cotransformed) Test Constructs (separate cells)

Wire: Stamping Place lines of bacteria down on agar using micropatterned agarose stamps

Wire: Stamping Stamping process1mm 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 1mm

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 YFP w/o degradation tag YFP 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 BABA ABAB AA BB – – 2 propagating wires using different signaling molecules (Lux, Las) – – Wires connected using routers that convert one signal to the other