ELSI: Risks and Rewards of Synthetic Biology

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ELSI Risks and Rewards of Synthetic Biology
iGEM Harvard Mon 6-Jun-2005 930-1100 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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Harvard iGEM
Tag! / BioWire (Signal Transfer) Paula, Patrick,
Jenny, Kang-Xing Thermal / Diagnostic Detection
Chris, Ives, Thomas, Danny, Connie Cell Density
Detection Yin, Hing, Orr, Sasha
June 2 1000-1045 (George, Biolabs Main Lecture
Hall) Overview of synthetic biology -- Slides
1  June 6 930-1100 (George) Risks and
Rewards -- Slides2 
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Why IGEM competition should not be like
Core-War'84, Robot Wars'94, etc.
Computer Viruses and Hacking Take 1.6 Trillion
Toll on Worldwide Economy http//seclists.org/list
s/isn/2000/Jul/0070.html
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3 Exponential technologies(synergistic)
Computation Communication (bits/sec)
E.coli
operons
Synthesis (daltons)
tRNA
urea
B12
Analysis (bp/)
telegraph
tRNA
Shendure J, Mitra R, Varma C, Church GM, 2004
Nature Reviews of Genetics. Carlson 2003
Kurzweil 2002 Moore 1965
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It seemed like a good idea at the time.
Crops River life Grain trade Livestock Fertilizer
Nuclear Power Tankers Pets
Malaria Cholera Yersinia Flu HIV Anoxic
fish TMI, Chernobyl Mussels sea
snakes Australian herbicide
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Responsible Conduct In Research
"If scientists find that their discoveries have
implications for some important aspect of public
affairs, they have a responsibility to call
attention to the public issues involved .. A good
example is the response of biologists to the
development of recombinant DNA technologies --
first calling for a temporary moratorium on the
research and then helping to set up a regulatory
mechanism to ensure its safety."
http//www.aaes.org/membership/index.asp
http//www.nap.edu/readingroom/books/obas/
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Synthetic Biology Policy

• Closed vs open -- International transparency
• Lower costs availability impact on biohackers
• Rapid response tools
• Culture of cooperation
• Professional society with code of ethics
• Licensing monitoring vendors, signatures/IDs

NHGRI ELSI 1990-present Synthetic Biology 1.0
June 2004 Sloan Foundation Study Science
Technology Expert Partnership Jun'05 Center for
Strategic and International Studies (CSIS)
Carnegie Foundation The J. Craig Venter
Institute National Science Advisory Board for
Biosecurity (NSABB) Jul'05
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1977
1995 cult Aum Shinrikyo, aerosolize anthrax
botulinum in Tokyo on 8 occasions.
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Bio-risks, Biosecurity
Accidental Bio-Risks IL4-pox Purposeful
Bio-Risks "the most powerful..called Anthrax 836
isolated ironically after another
accident..disinfected the sewer but ..one of the
rodents captured in the Kirov sewers had
developed a new strain, more virulent than the
original. The army immediately ordered him to
cultivate the new strains." --Ken Alibek in
"Biohazard" Uncertain Bio-Risks Gene therapy,
GMOs, Ecology
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Accidental Bio-risk in research
"Recently immunized genetically resistant mice
with the virus expressing IL-4 also resulted in
significant mortality due to fulminant mousepox."
Jackson et al. (2001) J Virol. 751205-10.
A smallpox victim in Gloucester, 1923
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Uncertain Bio-risks
Gene therapy Immune reactions (Penn)
Insertions in tumor suppressor genes
onocogenes Genetically modified organisms (GMO)
uncontrolled spread of Ice-nucleation ice
resistance (AGS) Herbicide resistance
(Monsanto) Allergens Pharmaceuticals
vaccines Drought resistance Eco-engineering
Ocean fertilization
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ELSI Safe Synthetic Biology

• Church, G.M. (2004) A synthetic biohazard
  non-proliferation proposal.
• http//arep.med.harvard.edu/SBP
• Monitor oligo synthesis via expansion of
• Controlled substances, Select Agents,
  Recombinant DNA
• Computational tools are available small number
  of reagent, instrument synthetic DNA suppliers
  at present.
• System modeling checks for
• synthetic biology projects
• Multi-auxotrophy novel genetic codes
• prevent functional transfer of DNA to other
  cells.

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Encourage responsibility in the entire
bio-supply-chain via licensing
1. Chemicals Phosphoramidites 2. Instruments 3.
Synthetic oligonucleotides 4. Synthetic
genes/genomes 5. Design ordering software
(check for select agents) 6. Educational goals
(defense not offense)7. Intellectual property
know-how 8. Engineering standards societies 9.
Network of trainees (no trainee left behind)
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Defense not offense(albeit a perfect defense can
allow first strikes)
How would you make cells resistant to all viruses
(phages)? Auto-destruct at a specific conditions
(temperature) Toxin-antitoxin pairs RelB/E,
MazE/, yeeU/V, CcdB inhibits GyrA
Auxotrophs (metabolic dependencies) Inexpensive
monitoring diagnostics, bio-weather-map Optima
l biosynthesis e.g. artemesinin (malaria) Explore
epitopes vaccines
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Safety via blocking exchange
Can we make a cell which is resistant to all
viruses and incapable of functional DNA
exchange in or out? Options Restriction
enzymes (mainly incoming) Chirality (very
challenging) Genetic code remapping
Micrococcus luteus is naturally missing 6
codons UUA(L), CUA(L), AUA(I), GUA(V), CAA(Q),
AGA(R). Kowal, AK, Oliver, JS NAR 1997, 25
4685
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108 TypeIIS Endonucleases

• FokI GGATG (N9/13) 5 4
• BsmFI GGGAC (N10/14) 5 4 NEB SAGE
• MmeI TCCRAC (N20/18) 5.5 -2 NEB LongSage
• EcoP15I CAGCAG (N25/27) 6 2 homemade
  SuperSage
• BceAI ACGGC (N12/14) 5 2
• Bce83I CTTGAG (N16/14) 6 -2
• AcuI CTGAAG (N16/14) 6 -2
• AarI CACCTGC(N4/8) 7 4
• SceI TAGGG_ATAACAGGGTAAT 18 -4
• BsaXI (N9/12)AC(N5)CTCC(N10/7) 63-3 NEB
• Velculescu etal. Science. 1995 270484.
• SahaS,etal. Nat Biotec. 2002 20508
• Matsumura etal.PNAS 2003 10015718

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Mirror world enzyme, parasite,
predator resistance access 2n diastereomers (n
chiral atoms)

• L-amino acids D-ribose (rNTPs, dNTPs)
• Transition EF-Tu, peptidyl transferase,
  DNA-ligase
• D-amino acids L-ribose (rNTPs, dNTPs)

Dedkova, et al. (2003) Enhanced D-amino acid
incorporation into protein by modified
ribosomes. J Am Chem Soc 125, 6616-7
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Free up 1 to 15 codons in vivo
1

3
2
4


M. luteus missing 6 codons UUA(L), CUA(L),
AUA(I), GUA(V), CAA(Q), AGA(R).



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Up to 760K Oligos/Chip18 Mbp for 1K raw (6-18K
genes)
5000 lower oligo costs

• lt1K Oxamer Electrolytic acid/base
• 8K Atactic/Xeotron/Invitrogen
• Photo-Generated Acid
• Sheng , Zhou, Gulari, Gao (U.Houston)
• 24K Agilent Ink-jet standard reagents
• 48K Febit
• 100K Metrigen
• 380K Nimblegen Photolabile 5'protection
• Nuwaysir, Smith, Albert

Tian, Gong, Church
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Improving DNA synthesis accuracy
Method
Bp/error Chip assembly only 160
Hybridization-selection
1,400 MutS-gel-shift 10,000 PCR
35 cycles 10,000 MutHLS cleavage
100,000 Tian Church 2004 Nature Carr
Jacobson 2004 NAR Smith Modrich 1997
PNAS http//www.invitrogen.com/content.cfm?pageid
453
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CAD-PAM Computer aided Design -Polymerase
Assembly Multiplexing
50 75 125 225 425 825
1002(n-1)
For tandem, inverted and dispersed repeats
Focus on 3' ends, hierarchical assembly,
size-selection and scaffolding. Mullis 1986
CSHSQB, Dillon 1990 BioTech, Stemmer 1995 Gene
Tian et al. 2004 Nature, Kodumal et 2004 PNAS
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Genome assembly
PCR
in vitro
in vivo
50 HS PAM 425 MutS PAM 10K anneal
100K l-red 5Mbp
USER-S1 USER-S1
USER-5'only One pool 480 pools
480 genomic 48
1 of 117K universal primers
primer pairs
HSHybridization-Selection USERUracil DNA
glycosylase EndoVIII to remove
flanking primer pairs
Isaacs, Carr, Emig, Gong, Tian, Jacobson, Church
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Genome assembly alternatives
1. tet
2. kan
3. tet
Serial electroporation 48 stages 1 strain
(1.6 days/stage) vs. Conjugation 7 stages
48 gt 24 gt 12 gt 6 gt 3 gt 2 gt 1 strains
Reppas Church
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All 30S-Ribosomal-protein DNAs(codon
re-optimized)
1.7 kb
0.3 kb
14.6 kb
Atactic lt4K chip
s19
0.3kb
Nimblegen 95K chip
Tian, Gong, Sheng , Zhou, Gulari, Gao, Church
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Extreme mRNA makeover for protein expression in
vitro
RS-2,4,5,6,9,10,12,13,15,16,17,and 21 detectable
initially. RS-1, 3, 7, 8, 11, 14, 18, 19, 20
initially weak or undetectable. Solution
Iteratively resynthesize all mRNAs with less
mRNA structure.
Western blot based on His-tags
Tian Church
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Genome engineering Cross-feeding Auxotrophs
SecondPassage
First Passage
?trp/?tyrA pair of genomes shows the best
co-growth (syntrophs)
Reppas Lin
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Co-evolution of syntrophicTrp-/Tyr- genome pair
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Plone-bead FISSeq SBE SBL Consider
amplification , homopolymer, context errors?

• of bases sequenced (total Mbp) 23 (no)
  10.8 (yes)
• bases sequenced (unique) 73 b 4.7 Mb
  (72)
• Average fold coverage 324,000
  2.3
• Pixels used per bead (analysis) 3.6
  3.6
• Read Length (bp) 14 24
• Indels 0.6 ?
• Substitutions (raw error-rate) 4e-5
  1e-2
• Throughput (kb/min) 360 10
• Speed/cost ratio relative to 1100
  32
• current ABI capillary sequencing _at_ 0.75
  kb/min/device

Shendure Porreca
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Plone analysis of synthetic evolved E.coli Tyr-
/ Trp- pair
374,449 GgtA MhpT 5'UTR
3-hydroxyphenylpropionic acid transport
986,327 TgtG OmpF Promoter-10 Outer
membrane porin 1,990,682 GgtA PgsA ArggtCys
Essential phospholipid synthesis
protein 3,599,626 GgtC FtsX LeugtVal
Essential cell division protein 3,957,957 CgtT
PpiC 5'UTR Peptidyl-prolyl
cis-trans isomerase (stress) 4,002,444 GgtC
YigI Arg-gtGly Unknown conserved
protein
Reppas, Shendure, Porreca
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Harvard iGEM
Tag! / BioWire (Signal Transfer) Paula, Patrick,
Jenny, Kang-Xing Thermal / Diagnostic Detection
Chris, Ives, Thomas, Danny, Connie Cell Density
Detection Yin, Hing, Orr, Sasha
June 2 1000-1045 (George, Biolabs Main Lecture
Hall) Overview of synthetic biology -- Slides
1  June 6 930-1100 (George) Risks and
Rewards -- Slides2 
31
Defense not offense(albeit a perfect defense can
allow first strikes)
How would you make cells resistant to all viruses
(phages)? Auto-destruct at a specific conditions
(temperature) Toxin-antitoxin pairs RelB/E,
MazE/, yeeU/V, CcdB inhibits GyrA
Auxotrophs (metabolic dependencies) Inexpensive
monitoring diagnostics, bio-weather-map Optima
l biosynthesis e.g. artemesinin (malaria) Explore
epitopes vaccines
32
.