Harvard iGEM 2005

1. Harvard iGEM 2005 Kang-Xing Jin Yin Li Thomas Noriega Daniel Popper Alexander Rush Yves Wang Orr Ashenberg Patrick Bradley Connie Cheng Chris Doucette Hing Eng Jennifer Gao

2. Our Projects • Biowire • The Wire Made of Biology • Biosketch • The Bacterial Sketchpad

3. BioWireThe Wire Made of Biology Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Daniel Popper, Alexander Rush

4. Initial Signal Project Overview • Goal • To engineer a biological “wire” capable of propagating a chemical signal down its length

5. Our Approach • Signal: acyl-homoserine lactones (AHL) used in bacterial quorum sensing • Lux system: 3OC6HSL • Builds on work by Ron Weiss • Las system: 3OC12HSL • Transmission: pulse controlled by a genetic incoherent feed-forward loop • Wire: engineered E. coli placed in wire form with agarose stamps • Based on technique by George Whitesides

6. Transmission: 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 AHL cI YFP& LuxI

7. Constructs Final Construct (cotransformed) Propagation Component:AHL->{YFP, AHL}, cI repressible Repressor Component: AHL->cI Sender Construct: Constitutive AHL Test Constructs (separate cells) Receiver Construct: AHL->YFP YFP luxR luxPR luxPL Parts shown are for Lux system. Las analogues were built as well.

8. Wire: Stamping • Place lines of bacteria down on agar using micropatterned agarose stamps

9. Wire: Stamping

10. Wire: Stamping • Stamping process • 1mm perimeter lines

11. Key Experiments • All experiments were done on Lux system • Senders and Receivers • Testing signal reception in cells laid down with the stamp • Propagation Constructs • Testing induction of propagation constructs with AHL

12. 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 Sender Construct: Constitutive AHL Receiver Construct: AHL->YFP luxR luxPR YFP luxPL 1mm

13. Senders and Receivers • Results • Receiver cells fluoresced when laid down with sender cells. • Conclusions • Test constructs work; stamping is a viable method of laying down cells in a predetermined pattern Receivers (near senders) Receivers (far from senders)

14. 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 Component:AHL->{YFP, AHL}, cI repressible Repressor Component: AHL->cI Two components cotransformed to form propagation construct

15. Propagation Constructs • Results • Issues with noise - cells were either constitutively “on” or “off” regardless of AHL addition +AHL +AHL -AHL -AHL YFP w/o degradation tag YFP w/ degradation tag

16. Propagation Constructs • Conclusions • Degradation tags, RBS/promoter strength may need fine-tuning • Because of positive feedback, noise is amplified • Further experiments necessary

17. Achievements: Construction • Constructed all parts for propagating signals for both the Lux and Las systems and routers • Approximately 120 parts sent to Registry • Everything assembled using BioBricks; no DNA synthesis • Needed to keep track of large numbers of subparts involved in construction • Sasha created a database to organize and automate the assembly process • Database determined pairwise ligations, insert/vector, plasmid resistances necessary to construct each final part

19. Achievements: Experiments • Tested parts of the Lux system • Successful induction of receivers via sender cells • Preliminary tests on propagation systems • Designed a protocol for stamping bacterial cells on agarose in any desired pattern with 500 micron resolution

20. Future Work • Debug Lux propagation system • Test and characterize Las system • Make dual-system oscillators • 2 propagating wires using different signaling molecules (Lux, Las) • Wires connected using routers that convert one signal to the other BA AA BB AA BB BB AA AB

21. BioSketchThe Bacterial Sketch Pad Chris Doucette, Hing Eng, Jenny Gao, Yin Li, Thomas Noreiga, Yves Wang,

22. Goal • To make a sketch pad with bacteria • Write with a UV pen • Erase with heat BACTERIA HEAT UV

23. Project Concept

24. Circuit Design • A and B repress each other • Writing: UV cleaves A, allowing expression of B and reporter • Erasing: Heat denatures B, allowing expression of A UV A B REPORTER HEAT

25. Circuit Construction UV A B REPORTER HEAT

26. Testing the Circuit • Two l CI-LacIts switches built • Can we turn switches OFF with heat? • Can we turn switches ON with UV? • Can we maintain the states? • Assays: • Fluorescence microscopy • FACS analysis

27. Day 1 30°C o.n. Day 2 30, 32, 35, 37, or 40°C o.n. Day 3 Assay Testing: Turning Switch OFF • Experimental Design • Strains (LacI-) • Switch • GFP only • No construct

28. Switch 30°C  30°C 30°C  32°C 30°C  37°C 30°C  40°C GFP alone No construct Heat represses fluorescence • Temperatures from 37°C to 40°C almost completely abolishes fluorescence. • Cells transformed with only GFP are always fluorescent. • Cells with no introduced constructs were never fluorescent.

29. Day 1 40°C o.n. Day 2 0, 6, 12, 24, 48 J/m2 4h @ 30°C Assay Testing: Turning Switch ON • Experimental Design • Strains (LacI-) • Switch • GFP only • No construct

30. Switch 0 J/m2 6 J/m2 24 J/m2 48 J/m2 UV increases fluorescence • Increasing intensity of UV correlates with increasing intensity of cell fluorescence. • UV irradiation did not alter the fluorescence profiles of cells with only GFP construct or no construct.

31. State Maintenance • Cell populations have 3 states • Mixed (some ON, some OFF) • ON (UV) • OFF (heat) • Cells should “remember” their state • If turned on, should stay on indefinitely • If turned off, should stay off indefinitely • Experiments show state is unstable • Observed expected state after 4 hours • After 24 hours, returns to mixed state

32. BioBricks Implementation • Collins switch equivalent (λ CI and LacIts) • Made 2 LacIts QPIs by mutating normal LacI QPI • Designed alternative switch • 434 CI as UV sensitive component • λ CIts as heat sensitive component UV sensitivepromoter Heat sensitive QPI UV sensitive QPI Reporter

33. Current Work • Improve on results from modified Collins l CI-LacIts switch • Get better induction with UV • Achieve more robust state-maintenance • Draw/erase on a lawn • BioBrick switches • Rebuild l CI-LacIts switches • Assemble 434 CI-l CIts from synthesized parts • Other reporters • Build both switches using “mCherry” reporter which is visible without microscopy

34. Accomplishments • Constructed switches that can be turned ON by UV, and turned OFF by heat. • Added 29 new parts to the Registry including: • Bacterial mCherry reporter • Made into BioBricks with and without LVA degradation tags • Also synthesized codon-optimized version • 2 temperature sensitive LacI coding sequences and LacI QPIs • Synthesized l CIts QPI • In progress: Constructing improved switches using mCherry

35. Acknowledgments • Alain Viel, Ira Phillips, Sasha Wait • Pam Silver, George Church, Kit Parker, Radhika Nagpal • Harvard Provost Office, HHMI, DEAS, Division of Life Sciences • Registry of Standard Biological Parts at MIT • Ron Weiss Lab (Princeton) • George Whitesides Lab (Harvard) • Kit Parker Lab (Harvard) • Jim Collins Lab (Boston University) • Center for Nanoscale Systems at Harvard • Dana Farber Flow Cytometry Facility • Bauer Center Flow Cytometry Facility • The Chinese Kitchen food truck