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

2. Project Overview Initial Signal cIGFP AHL m & LUXI Pulse through incoherent feed-forward loop Propagation through feedback loop

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

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

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. ABABBABAAA BB A B

11. New Ideas: Toward Oscillators Circle Oscillator – Move signal around in a circle – 1 signaling molecule: signal goes both ways… – 2 signaling molecules BABA ABAB AA BB 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): “