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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