Past%20

1
Past Near-Future Synthetic Biology Projects
iGEM Harvard Thu 2-Jun-2005 1000-1030 AM
Thanks to Washington U,
Harvard-MIT Broad Inst., DARPA-BioSpice, DOE-GTL,
EU-MolTools, NGHRI-CEGS, NHLBI-PGA,
NIGMS-SysBio, PhRMA, Lipper Foundation
Agencourt, Ambergen, Atactic, BeyondGenomics,
Caliper, Genomatica, Genovoxx, Helicos, MJR,
NEN, Nimblegen, SynBioCorp, ThermoFinnigan,
Xeotron/Invitrogen For more info see
arep.med.harvard.edu
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Avoiding the tarpits
How experiments can go wrong 1. Abstraction
decouple design/fabrication 2. Protein
overproduction 3. Protein destabilizers How they
go right 1. Repeating previous work 2. Keep it
complex 3. Training pets to do what they do
naturally Reliable methods under-utilized in
iGEM 1. PCR 2. Recombineering 3. Selection
3
Integration details
Basic lab hygiene 1. Positive AND negative
controls 2. Communicate quantitatively (including
variance) 3. Quality testing from start (
continuity) Incompatibilities 1.
Intra/extra/non-cellular (e.g. redox,
detergents) 2. Moving a portion of an
interacting set (e.g. promoters) 3. Codon
usage 4. Protein stability, aggregation 5.
Cross-talk (always plan "background")
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Engineering Biological Systems

Action Specificity KO "Design" Small
molecules (drugs) Fast Varies
Varies Hard Antibodies
Fast Varies Varies
Hard RNAi
Slow Varies Medium
OK Riboregulators Fast
Varies Medium /- Insertion "traps"
Slow Yes Varies
Random Recombination Slow
Perfect Complete Easy Proteasome
targeting Fast Excellent
Medium Easy Physical environment Varies
Microfabrication Varies
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Proteasome targeting(via drug homologous
recombination)
Janse, DM, Crosas,B Finley,D Church, GM
(2004) Localization to the Proteasome is
Sufficient for Degradation.
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Programmable ligand-controlled riboregulators of
eukaryotic gene expression.
Bayer Smolke 2005 Nat Biotech. 23337-43.
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Integrase Counter Team (IGEM
Summer '04)
http//theory.med.harvard.edu/SynBio/

• Boston University
• Will Blake
• Jim Flanigon
• Farren Isaacs
• Ellen OShaughnessy
• Neil Patel
• Margot Schomp
• Jim Collins

• Harvard University
• John Aach
• Patrik D'haeseleer
• Gary Gao
• Jinkuk Kim
• Xiaoxia Lin
• Nathan Walsh
• George Church

Gardner et al.2000 Nature 403339 Construction
of a genetic toggle switch in E.coli
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In vivo Counter Designs
1 2 3
Riboswitch counter ?
0 1 1 0
?Integrase bit counter
Cell-cycle counter ?
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Integrase advantages

• High fidelity site specific and directional
  recombination (as opposed to homologous
  recombination)
• Reversible excision just as reliable as
  integration
• Specific each integrase recognize its own att
  sites, but no others
• Numerous over 300 known Tyr integrases and 30
  known Ser integrases
• Efficient very few other factors needed to
  integrate or excise
• Extensively used Phage systems well
  characterized and used extensively in genetic
  engineering (e.g., the GATEWAY cloning system by
  Invitrogen)

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Integrase/Excisionase structure
l Integrase (int)
l Excisionase (xis)

• Mol Cell. 2003 Jul12(1)187-98. A conformational
  switch controls the DNA cleavage activity of
  lambda integrase. Aihara H, Kwon HJ, Nunes-Duby
  SE, Landy A, Ellenberger T.
• Sam MD, Cascio D, Johnson RC, Clubb RT. Crystal
  structure of the excisionase-DNA complex from
  bacteriophage lambda. J Mol Biol. 2004 Apr
  23338(2)229-40.

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Design Phage Int/Xis system
Phage attachment sites attP
P
P
O
O
B
B
attB Bacterial attachment sites
Int

Xis
Int
Integrated Left attachment sites attL
Integrated Right attachment sites attR
P
B
O
P
O
B
Stably integrated prophage
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Int/Xis system with inverted att sites
Phage attachment sites attP
Bacterial attachment sites attB
0
O
O
P
P
B
B
Int

Xis
Int
Integrated Right attachment site attR
Integrated Left attachment site attL
1
P
B
P
B
O
O
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Design 1 Bit counter initial concept
0
0
0
0
1
1
Int1
Xis1
Int2
Xis3
Int2
Xis2

• Counting mechanism
• Initial state 0 0 0
• Pulse 1 1 0 0
• Pulse 2 0 1 0
• etc. . . .
• Race condition problems between each Int and Xis

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Design 2 Full Cycle of Two ½-bits
State Pulse Products
0
0
1A Int2
0
1
2A Int1 Xis1 Rpt2
1
1
1B Int2 Xis2 Rpt1
1
0
2B Int1
0
0
int2
xis2
reporter1
int2
1
attR1 term attL1
xis1
reporter2
int1
2
attP2 term attB2
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BioBricks
Basic Part Types Basic Part Types Basic Part Types Composite Part Types Composite Part Types Composite Part Types
Type Type Description Type Type Description
R Regulatory Operator region E Reporter Compound reporter devices
B RBS Ribosome binding site Q Inverter Inverter and logic
C CDS Protein coding sequence   Composite Other composite parts
B Terminator Transcriptional terminator I Project Student projects
  RNA RNA binding sites and coding G Generator TIPS-to-Protein converter
F Signalling Cell-cell signalling   Measurement Performance measurement constructs
E Reporter Basic reporter CDS T Temporary Temporary and trial parts
M Tag Tag or Modifier S Intermediate Generated during assembly
V Plasmid Plasmids   Other Parts not yet classified
V Cells Cell strains
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Design Composite half bits in BioBricks

• Two 2kb composite parts

? Xis AAV
ECFP AAV
? Int LVA
p22 attP
Reverse Terminator
p22 attB (rev comp)
? Half Bit
BBa_E0024
BBa_I11020
BBa_I11021
BBa_I11033
BBa_B0025
BBa_I11032
BBa_I11060
P22 Xis AAV
EYFP AAV
p22 Int LVA
? attP
Terminator
? attB (rev comp)
p22 Half Bit
BBa_E0034
BBa_I11030
BBa_I11031
BBa_I11023
BBa_B0013
BBa_I11022
BBa_I11061
Lewis and Hatfull, Nuc. Acid Res., 2001, Vol. 29,
2205-2216 Andersen, Applied and Environmental
Microbiology, 1998, 2240-2246
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Integrase counter ODE Stochastic modeling
The simulation is sensitive to the relative
degradation rates of Int and Xis. Previously
Int was less stable, but in this simulation the
stabilities are equal.
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Synthesis TestingCan Int Xis control GFP
expression?
PLlacO
PLtetO
attP
Int
GFP_AAV
attB
pBAD
Xis
pSC101
Kan
Lutz and Bujard, Nuc. Acids Res., 1997, Vol. 25,
No. 6 1203-1210
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Invitrogen Gateway Vectors

Parr RD, Ball JM.(2003) Plasmid 49179. Nakayama
M, Ohara O. (2003) BBRC 312825
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A synthetic oscillatory network of
transcriptional regulators
SsrA 11-aa 'lite' tags reduce repressor
half-life from gt 60 min to 4 min.
Insets normalized autocorrelation of the first
repressor
Continuous model Stochastic similar
parameters
Elowitz Leibler, (Pub), Nature 2000403335-8
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Synthetic oscillator network
Controls with IPTG Variable amplitude
period in sib cells
Single cell GFP levels
Elowitz Leibler, Nature 2000403335-8
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Reconstitution of Circadian Oscillation of
Cyanobacterial KaiC Phosphorylation in vitro
Nakajima, et al. Science. 2005 Apr
15308(5720)414-5
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