Harvard iGEM 2005: Team BioWire and BioLoserz!!! LOL 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

Project Overview Our Approach – Signal: acyl-homoserine lactones (AHL) used by V. fischeri in quorum sensing – Transmission: pulse is controlled by a genetic incoherent feed-forward loop – Wire: engineered E. coli placed in wire form with agarose stamps

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 Animated line following flow of wire

Circuit Design (This page should include a more macroscopic design of our system, with stuff like sender cells, receiver cells, propagation cells, and how they’re placed in relation to each other, how circuits were cotransformed, etc)

Circuit Construction Constructed with BioBricks using parallel assembly 6 parts assembled in 3 stages (image from parts.mit.edu)

Final Constructs Sender Construct (LVA+): aTc -> AHL J06001 Receiver Test Construct: AHL -> YFP J06000 Receiver Repressor Component: AHL -> cI J06004 Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA- J06008

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

Photolithography Stamping process1mm perimeter lines Want picture of agarose stamp, possible master too and PDMS mold CAD design->Master mold using photolithography in cleanroom->Agarose stamp

Key Experiments Senders and Receivers – Testing signal reception in cells laid down with the stamp Propagation Constructs – Testing induction of propagation constructs with AHL

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

Senders and Receivers Results – Receiver cells fluoresced when laid down with sender cells. – [MOVIE? PICTURE? COOKIE?] Conclusions – Stamping is a viable method of laying down cells in a predetermined pattern for chemical signaling

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 – PICTURE Conclusions – Noise and leakiness are inherent in any biological system; synthetic constructs must be able to adjust for this

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 WICKED COOL database to organize the assembly process

Challenges

Achievements Made a helluva lotta parts Learned basic molecular biology techniques Pioneered the up and coming cutting edge field of SYNTHETIC BIOLOGY!!!!!1111one Did some computer modeling… oh wait, we didn’t. Obtained Alain’s credit card number. Heh heh heh.

Future Work Circle Oscillator – Las lactone shares similar pathway with Lux – Lux signal (A): moves both ways – Lux, Las signals (A,B): move unidirectionally BABA ABAB AA BB AA

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

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

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