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Progress Report on LUXI Initial Signal Project - Week Two Overview

This report summarizes the progress of the LUXI Initial Signal Project during Week Two. Key activities included miniprepping BioBricks, ordering more parts, and advancements in modeling techniques. We encountered issues with discrepancies in AHL rate constants across different papers and worked on photolithography for designing microstructures in mammalian cells. New ideas for oscillators and a pulse generator design have been discussed, focusing on minimizing noise and back-propagation. Upcoming tasks involve testing receiver and sender cells’ responses to AHL.

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Progress Report on LUXI Initial Signal Project - Week Two Overview

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  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): “

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