Download Harvard iGEM 2006

Harvard iGEM 2006
Harvard iGEM 2006
Our Vision: modular drug delivery
1.
2.
cell A
cell B
cell C
packaging
DNA Nanostructures
Harvard iGEM 2006
targeting
Why DNA?
•
•
•
•
•
Strong Watson-Crick base pairing
Covalent modifications
Relatively inexpensive
Self-assembles
Highly programmable
DNA Nanostructures
Harvard iGEM 2006
How: engineered crossing over
How: scaffolded origami
DNA Nanostructures
Harvard iGEM 2006
Our Design: a novel structure
DNA Nanostructures
Harvard iGEM 2006
courtesy Shawn Douglas
Our Design: a novel structure
DNA Nanostructures
Harvard iGEM 2006
courtesy Shawn Douglas
Evidence: EM images
15.5 nm
DNA Nanostructures
Harvard iGEM 2006
Evidence: EM images
27.6 nm
30.5 nm
Evidence: protection assay
Protected biotin on the inside
of our container from large
streptavidin beads
Streptavidin bead
Payload protected
DNA Nanostructures
Harvard iGEM 2006
Streptavidin bead
Control: biotin bound
Harvard iGEM 2006
Adaptamers
Thrombin aptamer
Streptavidin aptamer
Thrombin
Linking region
Streptavidin
Aptamer: nucleic acid sequence that can bind a
substrate with high specificity and affinity.
Why develop adaptamers?
• To offer a new way to target substrates to
cells.
• To create a tool for studying cell-cell
interactions.
• To provide a foundation for engineered
catalysts.
Cell Surface Targeting
Harvard iGEM 2006
Adaptamer testing
streptavidin
adaptamer
Adaptamer + thrombin
thrombin
anti-thrombin antibody
(-) control: naked beads
Cell Surface Targeting
Harvard iGEM 2006
Bead surface
Adaptamer quenching
Displacement strand
+
Relative Fluorescence Units
3500
3000
2500
Fluorescence emitted from
anti-thrombin antibodies.
2000
1500
1000
500
+
Cell Surface Targeting
Harvard iGEM 2006
0
Adaptamer Displacement Background
added
strand added
Streptavidin on the cell surface
Streptavidin
• Binds strongly to biotin
molecule
• Used to bind biotinylated
nucleic acids or peptides
McDevitt, 1999
Big idea:
Streptavidin protein expressed on the cell surface
can be used to target any biotinylated DNA/protein
to the cell surface.
Cell Surface Targeting
Harvard iGEM 2006
Lpp-OmpA surface display
Surface protein
J. Francisco
C. Earhart
G. Georgiou
1992
extracellular
outer
membrane
Lpp
(lipoprotein)
signal peptide
periplasm
OmpA
(outer membrane protein A)
transmembrane domains
Cell Surface Targeting
Harvard iGEM 2006
BioBricks assembly
of protein domains
I. Phillips
P. Silver
2006
Standard BioBrick
Protein domain BioBrick
5’…TCTAGAG
XbaI
TACTAGT…3’
SpeI
5’…TCTAGA_
XbaI
_ACTAGT…3’
SpeI
6-bp mixed site
Reading frame maintained
TCTAGT
Mixed
Lac
Promoter RBS
Cell Surface Targeting
Harvard iGEM 2006
Lpp
OmpA
Streptavidin
Expression of fusion
protein in frame
• Inducible expression
• Histidine tag at end, so
construct in frame
• Streptavidin domain
recognized by Ab
Anti-Histidine
Cell Surface Targeting
Harvard iGEM 2006
Anti-Streptavidin
Harvard iGEM 2006
From cyanobacteria...
• Photosynthetic
• Circadian rhythm
• Evolved over billions of years
...to E. coli
• Model organism for synthetic
biology
• BioBrick registry
Cyanobacterial Oscillator
Harvard iGEM 2006
Applications of a Bio-oscillator
12PM
12PM
6PM
6AM
•Clock
•Nightlight
•Timed drug delivery
•Pharmaceutical
processes
•Bio-circuitry
•Investigate natural
systems
6PM
output
12AM
http://www.cellzome.com/img/
pathways.jpg
Cyanobacterial Oscillator
Harvard iGEM 2006
12AM
Time
Cyanobacterial
Bio-oscillator
The Repressilator
Fluorescence
Elowitz et al. 2000
`
10h
Cyanobacterial Oscillator
Harvard iGEM 2006
Time
24h
The Kai Clock in Cyanobacteria
P
P
P
P
P
P
Phosphorylation of KaiC
•KaiC autophosphorylates and
dephosphorylates
•KaiA promotes phosphorylation
•KaiB inhibits KaiA
•Transcription-translation independent
•Period: 14-60 h
b
Time
Cyanobacterial Oscillator
Harvard iGEM 2006
Achievements
Goal: reconstitute the cyanobacteria Kai oscillator in E. coli
1. Created KaiA, KaiB, and KaiC BioBricks.
2. Combined the above with registry parts to form functional
BioBricks.
3. Expressed Kai proteins in E. coli and verified interaction.
Cyanobacterial Oscillator
Harvard iGEM 2006
Results: Constructs Created
We’ve made the following constructs:
Kai genes
Lac promoter + Kai genes
KaiA + KaiC and KaiB + KaiC
(with promoters)
Cyanobacterial Oscillator
Harvard iGEM 2006
Results: Proteins Interact in E. coli
Western blot image
• Constructs transformed in E. coli
• Cultures sampled at OD 0.5
• Western blot probed with anti-KaiC antibodies
Cyanobacterial Oscillator
Harvard iGEM 2006
Results: Proteins Interact in E. coli
Western blot image
P
P
P
P
Cyanobacterial Oscillator
Harvard iGEM 2006
P
P
P
P
P
Results: Proteins Interact in E. coli
Western blot image
P
P
P
P
Cyanobacterial Oscillator
Harvard iGEM 2006
P
P
P
P
P
Results: Proteins Interact in E. coli
Western blot image
P
P
P
P
Cyanobacterial Oscillator
Harvard iGEM 2006
P
P
P
P
P
Results: Proteins Interact in E. coli
Western blot image
Conclusion:
• KaiA and KaiC are expressed and interacting
• KaiB not verified, but results consistent with
predictions
Cyanobacterial Oscillator
Harvard iGEM 2006
Further challenges
Verify oscillation in E. coli.
Solution: pulsed expression
1
Percentage of phosphorylated
KaiC
Synchronization problem
• Between cells
• Within cell
Computer model simulating constant production
of KaiC in a single cell
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
P
0.1
0
1
25
49
73
P97
P
121
145
Time (h)
P
Time
Cyanobacterial Oscillator
Harvard iGEM 2006
P
P169
193
217
Harvard iGEM 2006
Acknowledgements
Teaching Fellows
Advisors
Nicholas Stroustrup
Shawn Douglas
Chris Doucette
George Church
Pamela Silver
William Shih
Radhika Nagpal
Jagesh Shah
Alain Viel