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

2. Project Overview Goal: to engineer a synthetic, macroscopic biological “wire,” capable of propagating a chemical pulse down its length – 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

3. BioWire: “Pulse” Propagation Initial Signal

4. Circuit Design: Pulse Generator Incoherent feed-forward loop plus positive feedback – AHL stimulates production of cI, YFP, and LuxI – cI represses YFP and LuxI – Result: a pulse of fluorescence, followed by a refractory period cI YFP & LuxI AHL Animated line following flow of wire

5. Circuit Design: BioWire Incoherent feed-forward loop plus positive feedback (GET RID OF THIS SLIDE, CONSOLIDATE INTO PREVIOUS) – LuxI produces more AHL, which triggers neighboring cells cIYFP & LuxIAHL

6. Construction Part pictures (propagators,cross-linkers etc), number of ligations, Sasha’s database

7. Photolithography Place lines of bacteria down on agar using micropatterned agarose stamps CAD of stamp

8. Photolithography Stamping process1mm perimeter lines Want picture of agarose stamp, possible master too and PDMS mold ->Mask->Master->PDMS molds->agarose stamps

9. Experiments AHL sender-receiver

10. Future Work Circle Oscillator – Signal moves in a circle – 1 signaling molecule A: signal goes both ways – 2 signaling molecules A, B: signal is unidirectional BABA ABAB AA BB AA

11. Sender / Receiver Phase Sender / Receiver GFP Receiver Phase Receiver GFP

12. Senders, 100X phase Senders, 100X GFP Receivers, 100X phase Receivers, 100X GFP

13. Receivers near senders, 100X phase Receivers near senders, 100X GFP Receivers far from senders, 100X phase Receivers far from senders, 100X GFP

14. Timing Diagram for Pulse YFP cI AHL

15. New Ideas: Toward Oscillators Line Oscillator – “Bounce” a signal back and forth along a line Initial Signal

16. New Ideas: Toward Oscillators Line Oscillator (design 1) – 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

17. New Ideas: Toward Oscillators Line Oscillator (design 2) – Two signaling molecules A and B Lux with either Las or Rh1 (respond to different variants HSL) – 4 cell types: AB, BA, AA, BB. Cell XY takes input X and pulses Y in response. 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. Issues: Crosstalk and BioBricks. ABABBABAAA BB A B