Download BUILT-IN BIOSAFETY DESIGN Ollie Wright - 29/04/13

BUILT-IN BIOSAFETY DESIGN
Ollie Wright - 29/04/13
INTRODUCTION
• Two
concerns from Asilomar:
that outcompete
indigenous organisms
• GMMOs
• Genetic
contamination via
horizontal gene
transfer (HGT)
1975
Asilomar
Courtesy of Google
IGEM
• iGEM
2012
• Physical
containment
• Amber
suppression
• Endonuclease
bacteriocin
toxins
Weakness
1
&
2
X
3
5
4
BENCHMARKS
1x10-8
per cell generation
Full thyA deletion
Moe-Behrens, G. H. G., Davis, R., & Haynes, K. A. (2013). Preparing synthetic biology
for the world. Frontiers Microbiol, 4, 5. doi:10.3389/fmicb.2013.00005
• Gain-of-function
much harder to evolve than loss-of-function
HOW TO IMPROVE?
•
Use microbes with low environmental retention times, rather than
relying on kill switches
•
Remove conjugation/transduction machinery
•
Remove homologous sequences to minimise recombination
•
Transformation is harder to mitigate:
•
Minimise genes with an obvious selection advantage
•
Include genes with an obvious selection disadvantage
FOCUS ON PLASMIDS?
•3
advantages of plasmids over genomic integrants:
• Easy
control of gene dosage
flanking homologous sequence as physically
isolated from genome
• No
• Imperfect
retention; reversion of host to near wild-type
CONDITIONAL ORIGIN OF REPLICATION
COR
•
Initiation of replication protein
provided in trans to origin
sequence
•
DIAL strains from Anderson lab
•
E.g. ColE2, R6K
ca
rg
o
•
rep
RBS
COR
auxo
Kittleson, J. T., Cheung, S., & Anderson, J. C. (2011). Rapid optimization of gene
dosage in E. coli using DIAL strains. J Biol Eng, 5, 10. doi:10.1186/1754-1611-5-10
Chromosome
AUXOTROPHY
auxo
Host strain auxotrophic for
essential metabolite (rich media
compatible)
•
Metabolic gene provided in trans
by plasmid
•
ca
rg
o
•
E.g. thyA (thymidine), dapA
(diaminopimelic acid) or thiL (thiamine
pyrophosphate)
rep
antitoxin
auxo
COR
F
R
T
selection
F
R
T
Chromosome
TOXIN-ANTITOXIN
auxo
Toxin-antitoxin pair
•
Usually bicistronic, but can shift
antitoxin to chromosome
•
E.g. Zeta/Epsilon (cell wall synthesis),
Kid/Kis (endoribonuclease)
antitoxin
toxin
rep
ca
rg
o
•
COR
F
R
T
selection
F
R
T
Chromosome
SYSTEM REDUNDANCY
auxo
COR
•
Auxotrophy
•
Toxin-antitoxin pair as bonus
ca
rg
o
•
toxin
COR
rep
antitoxin
Chromosome
ACCESS EFFICACY OF DESIGN
•
If selection coefficient is weak, traditionally need large number of microbial
generations before sampling - in the order of 10 to 1,000 years, if ever...
•
As proof-of-principle, need to make a worst-case-scenario system with a strong
selection coefficient - sample after hours/days
•
If a biosafety design passes this worst-case-scenario, a strong indicator of suitability
THE FUTURE
• Semantic
containment:
• Codon
refactoring to
shuffle natural genetic code
GG
G
CCC
Courtesy of Google
• Xeno-nucleic
acids that
uses orthogonal machinery
Schmidt, M., & de Lorenzo, V. (2012). Synthetic constructs in/for the environment:
managing the interplay between natural and engineered biology. FEBS Lett, 586(15),
2199–2206.
TAKE HOME MESSAGES
• General
recommendations for the here-and-now:
• System
redundancy - don’t rely on linear chains
• Deletions
preferable - regaining function is evolutionary
more difficult than inactivation (i.e. kill switch)
• Minimise
fitness cost of incorporating biosafety to
promote practical use
ACKNOWLEDGEMENTS
CSynBI, Imperial College
http://tinyurl.com/synbiosafety
http://www3.imperial.ac.uk/syntheticbiology
Sponsors