harvard igem 2006
DESCRIPTION
Harvard iGEM 2006. Our Vision: modular drug delivery. 1. 2. cell A. cell B. cell C. targeting. packaging. Why DNA ?. Strong Watson-Crick base pairing Covalent modifications Relatively inexpensive Self-assembles Highly programmable. How: engineered crossing over. - PowerPoint PPT PresentationTRANSCRIPT
Harvard iGEM 2006
Harvard iGEM 2006
DNA Nanostructures
Our Vision: modular drug delivery
1. 2.
packaging targeting
cell A
cell B
cell C
Harvard iGEM 2006
DNA Nanostructures
• Strong Watson-Crick base pairing• Covalent modifications• Relatively inexpensive• Self-assembles• Highly programmable
Why DNA?
How: engineered crossing over
Harvard iGEM 2006
DNA Nanostructures
How: scaffolded origamiN. Seeman
P. RothemundW. Shih
et. al
Harvard iGEM 2006
DNA Nanostructures
Our Design: a novel structure
courtesy Shawn Douglas
Harvard iGEM 2006
DNA Nanostructures
Our Design: a novel structure
courtesy Shawn Douglas
Harvard iGEM 2006
DNA Nanostructures
Evidence: EM images
15.5 nm
Evidence: EM images
27.6 nm
30.5 nm
Harvard iGEM 2006
DNA Nanostructures
Evidence: protection assayProtected biotin on the inside of our container from large streptavidin beads
Streptavidin beadStreptavidin bead
Payload protected Control: biotin bound
Harvard iGEM 2006
Thrombin
Streptavidin
Thrombin aptamerStreptavidin aptamer
Linking region
Adaptamers
Aptamer: nucleic acid sequence that can bind a substrate with high specificity and affinity.
Harvard iGEM 2006
Cell Surface Targeting
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.
Adaptamer testing
Bead surface
adaptamer
streptavidin
thrombin
anti-thrombin antibody
Adaptamer + thrombin
(-) control: naked beads
Harvard iGEM 2006
Cell Surface Targeting
Adaptamer quenching
+Displacement strand
+0
500
1000
1500
2000
2500
3000
3500
Adaptamer added
Displacement strand added
Background
Re
lati
ve F
luo
res
cen
ce
Un
its Fluorescence emitted from
anti-thrombin antibodies.
Harvard iGEM 2006
Cell Surface Targeting
Streptavidin on the cell surface
McDevitt, 1999
Streptavidin• Binds strongly to biotin molecule• Used to bind biotinylated nucleic acids or peptides
Big idea:Streptavidin protein expressed on the cell surface can be used to target any biotinylated DNA/protein to the cell surface.
Harvard iGEM 2006
Cell Surface Targeting
periplasm
extracellular
outermembrane
Lpp-OmpA surface displayJ. Francisco
C. EarhartG. Georgiou
1992
Lpp(lipoprotein) signal peptide
OmpA (outer membrane protein A)transmembrane domains
Surface protein
Harvard iGEM 2006
Cell Surface Targeting
BioBricks assemblyof protein domains
5’…TCTAGAG XbaI
TACTAGT…3’ SpeI
Standard BioBrick
5’…TCTAGA_ XbaI
_ACTAGT…3’ SpeI
Protein domain BioBrick
TCTAGT Mixed
6-bp mixed siteReading frame maintained
I. PhillipsP. Silver
2006
Promoter RBS Lpp OmpA Streptavidin
Promoter RBS Lpp OmpA Streptavidin
Lac
Harvard iGEM 2006
Cell Surface Targeting
Expression of fusion protein in frame
Anti-Histidine Anti-Streptavidin
• Inducible expression• Histidine tag at end, so construct in frame• Streptavidin domain recognized by Ab
Harvard iGEM 2006
Harvard iGEM 2006
Cyanobacterial Oscillator
From cyanobacteria...
...to E. coli
• Photosynthetic
• Circadian rhythm
• Evolved over billions of years
• Model organism for synthetic biology
• BioBrick registry
Harvard iGEM 2006
Cyanobacterial Oscillator
Time
Applications of a Bio-oscillator
•Clock•Nightlight•Timed drug delivery•Pharmaceutical processes•Bio-circuitry•Investigate natural systems
12PM 12PM
6PM 6AM 6PM
12AM12AM
output
http://www.cellzome.com/img/pathways.jpg
Harvard iGEM 2006
Cyanobacterial Oscillator
The Repressilator Cyanobacteria Bio-oscillator
LacLac
λ-cIλ-cI
TetTetElowitz et al. 2000
`
Time
Fluo
resc
ence
10h 24h
Harvard iGEM 2006
Cyanobacterial Oscillator
P P
P
P
P
P
The Kai Clock in Cyanobacteria•KaiC autophosphorylates and dephosphorylates•KaiA promotes phosphorylation•KaiB inhibits KaiA•Transcription-translation independent•Period: 14-60 h
Time
Phos
phor
ylati
on o
f Kai
C
b
Harvard iGEM 2006
Cyanobacterial Oscillator
AchievementsGoal: 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.
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Constructs Created
We’ve made the following constructs:
Kai genes
Lac promoter + Kai genes
KaiA + KaiC and KaiB + KaiC (with promoters)
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Proteins Interact in E. coli
• Constructs transformed in E. coli• Cultures sampled at OD 0.5• Western blot probed with anti-KaiC antibodies
Western blot image
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Proteins Interact in E. coliWestern blot image
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Proteins Interact in E. coliWestern blot image
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Proteins Interact in E. coliWestern blot image
Harvard iGEM 2006
Cyanobacterial Oscillator
Results: Proteins Interact in E. coliWestern blot image
Conclusion:• KaiA and KaiC are expressed and interacting• KaiB not verified, but results consistent with predictions
Harvard iGEM 2006
Cyanobacterial Oscillator
P P
P
P
P
P
Computer model simulating constant production of KaiC in a single cell
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1 25 49 73 97 121 145 169 193 217
Time (h)
Per
cen
tag
e o
f p
ho
sph
ory
late
d
Kai
C
Further challengesVerify oscillation in E. coli.
Synchronization problem• Between cells• Within cell
Time
Solution: pulsed expression
Harvard iGEM 2006
AcknowledgementsAdvisors
George ChurchPamela SilverWilliam Shih
Radhika NagpalJagesh Shah
Alain Viel
Teaching FellowsNicholas Stroustrup
Shawn DouglasChris Doucette