Palo Alto High SchoolUC Berkeley

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Univ. of Calif. San Francisco

Lincoln High School, S.F., CA

Lauren JannEric MeltzerJimmy HuangAlex Ng

Eric ChouRobert OvadiaMichael Chen

LOCATIONLOCATIONLOCATION:

LOCATIONLOCATIONLOCATION:

Directing Biology through Synthetic Assemblies and

Organelles

How does a cell carry out so many different processes?

1. Proteincomplexes

One Simple Solution: Spatial Organization“Location, Location, Location”

2. compartments

How does a cell carry out so many different processes?

1. Proteincomplexes

One Simple Solution: Spatial Organization“Location, Location, Location”

2. compartments

•Molecular machines•Can be organized by scaffold proteins•Often organize signaling pathways

How does a cell carry out so many different processes?

1. Proteincomplexes

One Simple Solution: Spatial Organization“Location, Location, Location”

2. compartments

• organelles (e.g. nucleus, mitochondria)• concentration - efficiency• isolation - limit toxicity

How does a cell carry out so many different processes?

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?

Our Goal: Manipulate Spatial Organization

1. Rewire a kinase signaling pathway using a scaffold

Trying to use scaffold as “molecular breadboard” to build new cellular circuits

scaffold

2. Build a new organelle

• Drug Factory• Biofuel Factory

Even more ambitious . . .

“Synthesome” - a synthetic organelleA place to house:

Our Goal: Manipulate Spatial Organization

Potentially useful for any SynBio System!

Now more on the two projects . . .

PROJECT 1: Using a protein scaffold to rewire a MAP kinase

signaling pathway

MAP2K

Receptor

Pheromone

MODEL SYSTEM: Yeast mating pathway - example of conserved MAP kinase cascade found in all eukaryotes

Sc

affo

ld MAP3K

MAPK

mating response

MAP2K

Receptor

Pheromone

MAP3K

MAPK

mating response

NO OUTPUTOUTPUTScaffold is like “molecular breadboard”

This pathway requires scaffold protein (Ste5) that binds & organizes all three kinases

Sc

affo

ld

GOAL: Alter pathway output by recruiting new negative effector

proteins to scaffold

MAP2K

Receptor

Pheromone

Sca

ffo

ld

MAP3K

MAPK

mating response

synthetic recruitment site (leucine zipper)

negative effector(+zipper)

HOW: Add new interaction site to scaffold - leucine zipper

Repression

Toolkit: “Borrow” bacterial enzymes that are known to act on human MAPK signaling:

– OspF• MAPK Phosphothreonine

Lyase

WHAT EFFECTORS? Use bacterial enzymes that

suppress MAPK pathways in the human immune system

PP

MAPK MAPKMAP2K

Receptor

Pheromone

Sca

ffo

ld

MAP3K

MAPK

mating response

Irreversibly removesphosphorylated side chain

How important is recruitment of effectors to scaffold?

Predictions:Experimental Setup:

Make 3 circuit variants

1. No effector

2. Effector recruited to scaffold (via zipper)

3. Effector - Unrecruited (defective zipper)

Induce with alpha-factor

Measure output by GFP reporter

0

1000

2000

3000

4000

5000

6000

0 20 40 60 80 100 120 140

RESULT: As predicted, recruitment of negative effectors to scaffold

strongly represses pathway outputOspF

irreversible

Pat

hw

ay O

utp

ut

(GF

P F

luo

resc

ence

)

Time

Recruited

UnrecruitedNo Effector

Recruited -- strong repressionUnrecruited -- weak or no repression

Bottom-line:

500

700

900

1100

1300

1500

1700

1900

0 20 40 60 80 100 120 140

MORE COMPLEX REWIRING: Can we build negative feedback loop ?

Pat

hw

ay O

utp

ut

(GF

P F

luo

resc

ence

)

Time

OspF

RESULTS:

NEGATIVE FEEDBACK LOOP YIELDS ADAPTATION: Initial response like wild-type, BUT then at ~40 min automatically stops expressing more GFP

DESIGN: Express negative effector from promoter activated by pathway

Recruited

Unrecruited

Wild-type

GFP expression stops

• 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.

Conclusions: Project 1

PROJECT 2:BUILDING A NEW ORGANELLE

FOR SYNTHETIC BIOLOGY

STEP 1: Create spatially distinct membrane compartment that has unique molecular identity

wild-type cell create “synthesome” use “synthesome”

STEP 2: Recruit proteins to carryout any synbio process of choice (e.g. drug or biofuel factory)

A quick recap

OUR FOCUS (Ask about in questions)

BUILDING A NEW ORGANELLESTEP 1: create compartment with

novel molecular identity code

Introducing phosphoinositides

= Endoplasmic ReticulumPI

BUILDING A NEW ORGANELLESTEP 1: create compartment with

novel molecular identity code

Introducing phosphoinositides

= Endoplasmic Reticulum

P

BUILDING A NEW ORGANELLESTEP 1: create compartment with

novel molecular identity code

P

PP

Late Endosome

Plasma Membrane

P

Early Endosome

P

PI[3,5]P

PI[4,5]P

PI[3]P

PI[3]P

PI[3,5]P

PI[4]P

PI[4,5]P

BUILDING A NEW ORGANELLESTEP 1: create compartment with

novel molecular identity code

???=

P

PI[5]P

BUILDING A NEW ORGANELLESTEP 1: create compartment with

novel molecular identity code

Synthesome=

Can we create a synthetic membrane compartment containing this novel phospholipid?

P

PI[5]P

Taking advantage of nature:The Ste2 Receptor Endocytosis

Pathway

Endocytosis

Early Endosome

Late Endosome

Vacuole (Lysosome)

Stimulation with Mating Factor

P

P5

P 33

P

P5P5

3’ phosphoinositide phosphatase (MTM)

desiredspecies

PI[3,5]P2 PI[5]P

Found in “higher” eukaryotes

The Ste2 Receptor

3

Strategy: Recruit lipid phosphatase to receptor (via

zipper) to convert late endosomes to new organelle

Vacuole (Lysosome)

Endocytosis

Early Endosome

Stimulation with Mating Factor

P 3Late

Endosome

P

P53

P5

NEW LIPID=> NEW COMPARTMENT

Ste2 Receptor + MTM Phosphatase(via zipper recruitment)

How can we detect the synthesome?

Receptor - tagged with GFP and lipid phosphatase

Specific lipid recognition domain (PH domain) - tagged with RFP

GFP

RFPP

Project 2 -- Milestones for creating compartment with new

lipid identity

1. Fuse Ste2 receptor to GFP and zipper

2. Create and tether lipid phosphatase to Ste2 via zipper

3. Confirm receptor assembly is functional

4. Observe blocking of endosome / vacuole fusion

5. Use RFP-tagged PI[5]P binding domain to detect new

lipid

6. Use biochemical assays to detect new lipid

GFP

Alpha-factor stimulation

GFPUndergoes efficient endocytosis

Before

After

Tagged receptor is properly localized and functional

Did we actually make a new compartment?

Before

After

Alpha-factor stimulation

Tagged receptor is properly localized and functionalQ

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We’re not sure

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

• 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

UCSF iGEM 2007 - Overall Summary

- all-purpose chassis for housing whatever synthetic system you could imagine!

Special Thanks to:Julie ReisGeorge Cachianes

Step 2: Using the SynthesomeSome Thoughts

Level of Complexity

1. Simple “Scaffold” (3D -> 2D)

2. Fusion to extracellularTail of receptor

3. Import System