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

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 & LuxIAHL

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 process 1mm 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

BA

AB

AA

AA

AABB

BB

BB

AA

AA

AA

AA

AAAA

AA

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 ENDINT INT INT INT 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.

AB BAAA AA AA AA AABB BB BB BB BB

A

B