QuickTime™ and a
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Univ. of Calif. San Francisco
QuickTime™ and a
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Lauren Jann Eric Chou
Eric Meltzer
Robert Ovadia
Jimmy Huang Michael Chen
Alex Ng
Lincoln High School, S.F., CA
Palo Alto High School
UC Berkeley
LOCATION
LOCATION
LOCATION:
Directing Biology through
Synthetic Assemblies and
Organelles
How does a cell carry out so
many different processes?
How does a cell carry out so
many different processes?
2. compartments
1. Protein
complexes
One Simple Solution: Spatial Organization
“Location, Location, Location”
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”
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”
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
scaffold
Trying to use scaffold as “molecular
breadboard” to build new cellular circuits
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!
Now more on the two projects . . .
PROJECT 1: Using a protein scaffold
to rewire a MAP kinase signaling
pathway
Pheromone
Pheromone
Receptor
Receptor
Scaffold
This pathway
requires scaffold
protein (Ste5) that
binds & organizes
all three kinases
MAP3K
MAP2K
MAP2K
MAPK
mating response
Scaffold is like
“molecular
breadboard”
MAP3K
OUTPUT
MAPK
Scaffold
MODEL SYSTEM: Yeast mating pathway - example
of conserved MAP kinase cascade found in all
eukaryotes
mating response
NO OUTPUT
GOAL: Alter pathway output by
recruiting new negative effector proteins
to scaffold
HOW: Add new interaction site to scaffold - leucine
zipper
Pheromone
synthetic
recruitment
site (leucine
zipper)
negative
effector
(+zipper)
Scaffold
Receptor
MAP3K
MAP2K
MAPK
Repression
mating response
WHAT EFFECTORS?
Use bacterial enzymes that suppress
MAPK pathways in the human immune
system
Pheromone
Scaffold
Receptor
Toolkit: “Borrow” bacterial enzymes that
are known to act on human MAPK
signaling:
MAP3K
MAP2K
MAPK
– OspF
• MAPK Phosphothreonine Lyase
P
MAPK
MAPK
Irreversibly removes
phosphorylated side chain
mating response
How important is recruitment of
effectors to scaffold?
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
Predictions:
RESULT: As predicted, recruitment of
negative effectors to scaffold strongly
represses pathway output
Pathway Output
(GFP Fluorescence)
OspF
irreversible
6000
No Effector
5000
Unrecruited
4000
3000
2000
Recruited
1000
0
0
20
40
60
80
100
120
140
Time
Bottom-line:
Recruited -- strong repression
Unrecruited -- weak or no repression
MORE COMPLEX REWIRING: Can
we build negative feedback loop ?
RESULTS:
Pathway Output
(GFP Fluorescence)
DESIGN:
Express negative effector from promoter
activated by pathway
1900
Wild-type
OspF
1700
Unrecruited
1500
1300
Recruited
1100
900
GFP expression
stops
700
500
0
20
40
60
80
100
120
140
Time
NEGATIVE FEEDBACK LOOP
YIELDS ADAPTATION:
Initial response like wild-type, BUT
then at ~40 min automatically
stops expressing more GFP
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.
PROJECT 2:
BUILDING A NEW ORGANELLE
FOR SYNTHETIC BIOLOGY
A quick recap
wild-type cell
create “synthesome”
use “synthesome”
STEP 1: Create spatially
distinct membrane
compartment that has
unique molecular identity
STEP 2: Recruit proteins to
carryout any synbio
process of choice (e.g.
drug or biofuel factory)
OUR FOCUS
(Ask about in questions)
BUILDING A NEW ORGANELLE
STEP 1: create compartment with
novel molecular identity code
Introducing phosphoinositides
PI
=
Endoplasmic Reticulum
BUILDING A NEW ORGANELLE
STEP 1: create compartment with
novel molecular identity code
P
Introducing phosphoinositides
=
Endoplasmic Reticulum
BUILDING A NEW ORGANELLE
STEP 1: create compartment with
novel molecular identity code
P
P
PI[3,5]P
Late Endosome
P
P
PI[4,5]P
Plasma Membrane
PI[3]P
P
PI[3,5]P
PI[3]P
Early Endosome
PI[4]P
PI[4,5]P
BUILDING A NEW ORGANELLE
STEP 1: create compartment with
novel molecular identity code
P
PI[5]P
=
???
BUILDING A NEW ORGANELLE
STEP 1: create compartment with
novel molecular identity code
P
PI[5]P
=
Synthesome
Can we create a synthetic
membrane compartment containing
this novel phospholipid?
Taking advantage of nature:
The Ste2 Receptor Endocytosis Pathway
Stimulation with
Mating Factor
The Ste2
Receptor
Endocytosis
P
3
3
5
P
P
Late
Endosome
Early
Endosome
PI[3,5]P2
5
P
3P
3’ phosphoinositide
phosphatase (MTM)
Found in “higher”
eukaryotes
5
P
PI[5]P
desired
species
Vacuole
(Lysosome)
Strategy: Recruit lipid phosphatase to
receptor (via zipper) to convert late
endosomes to new organelle
Stimulation with
Mating Factor
Endocytosis
Ste2 Receptor
+ MTM Phosphatase
(via zipper recruitment)
P
3
5P
P
3
Late
Endosome
Early
Endosome
5P
NEW LIPID
=> NEW COMPARTMENT
Vacuole
(Lysosome)
How can we detect the synthesome?
Specific lipid recognition domain
(PH domain) - tagged with
RFP
P
RFP
GFP
Receptor - tagged with GFP and
lipid phosphatase
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
Tagged receptor is properly localized
and functional

Undergoes efficient endocytosis
GFP
Before
Alpha-factor
stimulation
After
Did we actually make a
new compartment?
Before
Alpha-factor
stimulation
After

We’re not sure
QuickTime™ and a
QuickTime™ and a
TIFF (Uncompressed) decompressor
TIFF (Uncompressed) decompressor
are needed to see this picture.are needed to see this picture.
Tagged receptor is properly localized
and functional
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
Positive controls fail; switching to GFP
new lipid
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
- all-purpose chassis for housing whatever synthetic
identities
system you could imagine!
Special Thanks to:
Julie Reis
George Cachianes
Step 2: Using the Synthesome
Some Thoughts
1.
Simple “Scaffold”
(3D -> 2D)
Level of Complexity
2. Fusion to extracellular
Tail of receptor
3. Import System