BioWire Progress Report Week Two

1. BioWire Progress ReportWeek Two Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Paula Nunes, Danny Popper, Sasha Rush

2. & LUXI Initial Signal Project Overview • Pulse through incoherent feed-forward loop • Propagation through feedback loop AHLm cI GFP

3. Last Week • Received BioBricks, Lab Work • Some key parts missing • Miniprepped the BioBricks • Ordered More BioBricks • Modeling • Photolithography • Weiss • New Ideas

4. Modeling • Located parameters for all AHL rate constants • Updated the discrete model to use a faster algorithm • Expanded the continuous model to model both transcription and translation (should yield better timing data) • Issues: Major discrepancies for constants in different papers.

5. Photolithography • Went through microprinting process for mammalian cells in Kit’s lab. • In contact with member of Prof. Whitesides’ lab; we should be able to get a 50 μm line. • Also working on CAD design for our own masks and stamps.

6. Update on Weiss • Weiss is working on the same PPG (propagating pulse generator) design as us. • They have simulations done • Potential for back-propagation and noise causing random pulses. • They have constructed the circuit but have not been able to get pulse behavior experimentally (yet)

7. New Ideas: Refinements and Extensions • PPG Refinements: • Use an intracellular lactonase • Allow for faster AHL degradation to reduce noise and potential back-propagation. • Extensions (“cool patterns”) • Assume that we have a working pulse • Oscillators • line and circle

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

9. 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 INT INT INT INT END

10. 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. A AB AA AA AA AA AA BA BB BB BB BB BB B

11. New Ideas: Toward Oscillators • Circle Oscillator • Move signal around in a circle • 1 signaling molecule: signal goes both ways… • 2 signaling molecules BA AA AA AA BB AA AA AA BB AA BB AA AA AA AB AA

12. This Week • Make test constructs and begin experiments that were scheduled for last week (as described in last week’s presentation) • Test receiver cells’ response to AHL • Test sender cells’ ability to produce AHL • Begin testing crosstalk • Today: • Miniprep 28 BioBricks (grown in culture over the weekend) • Ligate to make our receiver and tester constructs • Order some more parts (in particular, aiiA lactonase)

13. Schedule (old) • Week 1 (6/6): Project Choice and Design • Week 2 (6/13): Test AHL receiver/sender • Week 3 (6/20): Test sender+receiver and repressor • Week 4 (6/27): More tests • Week 5 (7/4): Finish testing • Week 6 (7/11): Test cell signaling density; photolithography done • Week 7 (7/18): Construct IFFL (incoherent feed-forward loop) • Week 8 (7/25): Test it • Week 9 (8/1): “ • Week 10 (8/8): “ • Week 11 (8/15): “ • Week 12 (8/22): “

14. Schedule (new) • Week 1 (6/6): Project Choice and Design • Week 2 (6/13): Got parts and set up tests • Week 3 (6/20): Get remaining parts; Test all • Week 4 (6/27): Tests we couldn’t get to last week • Week 5 (7/4): Finish testing • Week 6 (7/11): Test cell signaling density; photolithography done • Week 7 (7/18): Construct IFFL • Week 8 (7/25): Test it • Week 9 (8/1): “ • Week 10 (8/8): “ • Week 11 (8/15): “ • Week 12 (8/22): “