palo alto high school uc berkeley
DESCRIPTION
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. LOCATION LOCATION LOCATION:. Directing Biology through Synthetic Assemblies and Organelles. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/1.jpg)
Palo Alto High SchoolUC Berkeley
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Univ. of Calif. San Francisco
Lincoln High School, S.F., CA
Lauren JannEric MeltzerJimmy HuangAlex Ng
Eric ChouRobert OvadiaMichael Chen
![Page 2: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/2.jpg)
LOCATIONLOCATIONLOCATION:
LOCATIONLOCATIONLOCATION:
Directing Biology through Synthetic Assemblies and
Organelles
![Page 3: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/3.jpg)
How does a cell carry out so many different processes?
![Page 4: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/4.jpg)
1. Proteincomplexes
One Simple Solution: Spatial Organization“Location, Location, Location”
2. compartments
How does a cell carry out so many different processes?
![Page 5: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/5.jpg)
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?
![Page 6: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/6.jpg)
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?
![Page 7: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/7.jpg)
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?
![Page 8: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/8.jpg)
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
![Page 9: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/9.jpg)
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!
![Page 10: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/10.jpg)
Now more on the two projects . . .
![Page 11: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/11.jpg)
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
![Page 12: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/12.jpg)
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
![Page 13: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/13.jpg)
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
![Page 14: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/14.jpg)
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
![Page 15: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/15.jpg)
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:
![Page 16: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/16.jpg)
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
![Page 17: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/17.jpg)
• 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
![Page 18: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/18.jpg)
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)
![Page 19: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/19.jpg)
BUILDING A NEW ORGANELLESTEP 1: create compartment with
novel molecular identity code
Introducing phosphoinositides
= Endoplasmic ReticulumPI
![Page 20: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/20.jpg)
BUILDING A NEW ORGANELLESTEP 1: create compartment with
novel molecular identity code
Introducing phosphoinositides
= Endoplasmic Reticulum
P
![Page 21: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/21.jpg)
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
![Page 22: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/22.jpg)
BUILDING A NEW ORGANELLESTEP 1: create compartment with
novel molecular identity code
???=
P
PI[5]P
![Page 23: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/23.jpg)
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
![Page 24: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/24.jpg)
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
![Page 25: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/25.jpg)
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)
![Page 26: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/26.jpg)
How can we detect the synthesome?
Receptor - tagged with GFP and lipid phosphatase
Specific lipid recognition domain (PH domain) - tagged with RFP
GFP
RFPP
![Page 27: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/27.jpg)
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
![Page 28: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/28.jpg)
GFP
Alpha-factor stimulation
GFPUndergoes efficient endocytosis
Before
After
Tagged receptor is properly localized and functional
Did we actually make a new compartment?
![Page 29: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/29.jpg)
Before
After
Alpha-factor stimulation
Tagged receptor is properly localized and functionalQ
uic
kTim
e™
and
aTIF
F (U
ncom
pre
sse
d) d
ecom
pre
ssor
are
need
ed
to se
e th
is pictu
re.
Qu
ickTim
e™
and
aTIF
F (U
ncom
pre
sse
d) d
ecom
pre
ssor
are
need
ed
to se
e th
is pictu
re.
We’re not sure
![Page 30: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/30.jpg)
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
![Page 31: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/31.jpg)
• 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!
![Page 32: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/32.jpg)
Special Thanks to:Julie ReisGeorge Cachianes
![Page 33: Palo Alto High School UC Berkeley](https://reader036.vdocuments.net/reader036/viewer/2022062408/56813f92550346895daa80ea/html5/thumbnails/33.jpg)
Step 2: Using the SynthesomeSome Thoughts
Level of Complexity
1. Simple “Scaffold” (3D -> 2D)
2. Fusion to extracellularTail of receptor
3. Import System