Palo Alto High School UC Berkeley

1. Univ. of Calif. San Francisco Lauren Jann Eric Meltzer Jimmy Huang Alex Ng Eric Chou Robert Ovadia Michael Chen Lincoln High School, S.F., CA Palo Alto High School UC Berkeley

2. LOCATIONLOCATIONLOCATION: Directing Biology through Synthetic Assemblies and Organelles

3. How does a cell carry out so many different processes?

4. How does a cell carry out so many different processes? 2. compartments 1. Protein complexes One Simple Solution: Spatial Organization “Location, Location, Location”

5. How does a cell carry out so many different processes? 2. compartments 1. Protein complexes • Molecular machines • Can be organized by scaffold proteins • Often organize signaling pathways One Simple Solution: Spatial Organization “Location, Location, Location”

6. How does a cell carry out so many different processes? 2. compartments 1. Protein complexes • organelles (e.g. nucleus, mitochondria) • concentration - efficiency • isolation - limit toxicity One Simple Solution: Spatial Organization “Location, Location, Location”

7. Common SynBio Problem: How do we get parts to function together as specific system? Can we apply this strategy of spatial organization to synthetic biology?

8. Our Goal: Manipulate Spatial Organization 1. Rewire a kinase signaling pathway using a scaffold scaffold Trying to use scaffold as “molecular breadboard” to build new cellular circuits

9. Our Goal: Manipulate Spatial Organization Even more ambitious . . . 2. Build a new organelle “Synthesome” - a synthetic organelle A place to house: • Drug Factory • Biofuel Factory Potentially useful for any SynBio System!

10. Now more on the two projects . . .

11. PROJECT 1: Using a protein scaffold to rewire a MAP kinase signaling pathway MODEL SYSTEM: Yeast mating pathway - example of conserved MAP kinase cascade found in all eukaryotes This pathway requires scaffold protein (Ste5) that binds & organizes all three kinases Pheromone Pheromone Receptor Receptor MAP3K MAP3K Scaffold MAP2K MAP2K Scaffold MAPK MAPK mating response mating response Scaffold is like “molecular breadboard” OUTPUT NO OUTPUT

12. GOAL: Alter pathway output by recruiting new negative effector proteins to scaffold HOW: Add new interaction site to scaffold - leucine zipper Pheromone Receptor MAP3K synthetic recruitment site (leucine zipper) Scaffold MAP2K MAPK negative effector (+zipper) Repression mating response

13. P WHAT EFFECTORS? Use bacterial enzymes that suppress MAPK pathways in the human immune system Toolkit: “Borrow” bacterial enzymes that are known to act on human MAPK signaling: • OspF • MAPK Phosphothreonine Lyase Pheromone Receptor MAP3K Scaffold MAP2K MAPK MAPK MAPK Irreversibly removes phosphorylated side chain mating response

14. How important is recruitment of effectors to scaffold? Predictions: • Experimental Setup: Make 3 circuit variants • No effector • Effector recruited to scaffold (via zipper) • Effector - Unrecruited (defective zipper) Induce with alpha-factor Measure output by GFP reporter

15. RESULT: As predicted, recruitment of negative effectors to scaffold strongly represses pathway output OspF irreversible No Effector Unrecruited Pathway Output (GFP Fluorescence) Recruited Time Bottom-line: Recruited-- strong repression Unrecruited -- weak or no repression

16. MORE COMPLEX REWIRING: Can we build negative feedback loop ? DESIGN: Express negative effector from promoter activated by pathway RESULTS: OspF Wild-type Unrecruited Pathway Output (GFP Fluorescence) Recruited GFP expression stops Time NEGATIVE FEEDBACK LOOP YIELDS ADAPTATION: Initial response like wild-type, BUT then at ~40 min automatically stops expressing more GFP

17. Conclusions: Project 1 • Bacterial effectors are powerful new toolkit for engineering MAPK pathways • The artificial recruitment of negative effectors to scaffold can dramatically repress MAPK pathway output • Synthetically recruited effectors can be used to build feedback loops and create adaptation response.

18. PROJECT 2:BUILDING A NEW ORGANELLEFOR SYNTHETIC BIOLOGY A quick recap wild-type cell create “synthesome” use “synthesome” STEP 2: Recruit proteins to carryout any synbio process of choice (e.g. drug or biofuel factory) STEP 1: Create spatially distinct membrane compartment that has unique molecular identity OUR FOCUS (Ask about in questions)

19. BUILDING A NEW ORGANELLESTEP 1: create compartment with novel molecular identity code Introducing phosphoinositides PI = Endoplasmic Reticulum

20. BUILDING A NEW ORGANELLESTEP 1: create compartment with novel molecular identity code Introducing phosphoinositides P = Endoplasmic Reticulum

21. P P BUILDING A NEW ORGANELLESTEP 1: create compartment with novel molecular identity code P P PI[3,5]P Late Endosome PI[4,5]P Plasma Membrane PI[3]P PI[3,5]P PI[4]P PI[4,5]P P PI[3]P Early Endosome

22. BUILDING A NEW ORGANELLESTEP 1: create compartment with novel molecular identity code P PI[5]P = ???

23. BUILDING A NEW ORGANELLESTEP 1: create compartment with novel molecular identity code P PI[5]P = Synthesome Can we create a synthetic membrane compartment containing this novel phospholipid?

24. Taking advantage of nature:The Ste2 Receptor Endocytosis Pathway Stimulation with Mating Factor The Ste2 Receptor Endocytosis 3 P X ! X ? 3 P 5 P Late Endosome Early Endosome 3 3’ phosphoinositide phosphatase (MTM) PI[3,5]P2 P PI[5]P 5 P 5 P Vacuole (Lysosome) desired species Found in “higher” eukaryotes

25. Stimulation with Mating Factor V 3 Endocytosis P 3 P 5 Late Endosome P V Early Endosome V 5 P X Vacuole (Lysosome) V NEW LIPID => NEW COMPARTMENT Strategy: Recruit lipid phosphatase to receptor (via zipper) to convert late endosomes to new organelle Ste2 Receptor + MTM Phosphatase (via zipper recruitment) V

26. How can we detect the synthesome? Specific lipid recognition domain (PH domain) - tagged with RFP RFP P GFP V Receptor - tagged with GFP and lipid phosphatase V V

27. Project 2 -- Milestones for creating compartment with new lipid identity • Fuse Ste2 receptor to GFP and zipper • Create and tether lipid phosphatase to Ste2 via zipper • Confirm receptor assembly is functional • Observe blocking of endosome / vacuole fusion • Use RFP-tagged PI[5]P binding domain to detect new lipid • Use biochemical assays to detect new lipid

28. GFP Tagged receptor is properly localized and functional  V Undergoes efficient endocytosis GFP Before Alpha-factor stimulation After Did we actually make a new compartment?

29. Tagged receptor is properly localized and functional We’re not sure  V V Before Alpha-factor stimulation After

30. Project 2 -- Milestones for creating compartment with new lipid identity • Fuse Ste2 receptor to GFP and zipper • Tether lipid phosphatase to Ste2 via zipper • Confirm receptor assembly is targeted to endosomes • Observe blocking of endosome / vacuole fusion 5. Use RFP-tagged PI5P binding domain to detect new lipid 6. Use biochemical assays to detect new lipid Positive controls fail; switching to GFP

31. UCSF iGEM 2007 - Overall Summary • Cellular microenvironments are convenient platforms for controlling the flow of cellular information in diverse processes. • Project 1: Recruitment of pathway modulators to protein scaffolds allows us to flexibly engineer cell signaling. • Project 2: Targeting lipid modifying enzymes (lipid kinases/phosphatases) may allow the creation of novel membrane bound compartments with unique molecular identities - all-purpose chassis for housing whatever synthetic system you could imagine!

32. Special Thanks to: Julie Reis George Cachianes

33. Step 2: Using the SynthesomeSome Thoughts Simple “Scaffold” (3D -> 2D) 2. Fusion to extracellular Tail of receptor 3. Import System Level of Complexity