Transcript Team 4 _ Final Presentation_ Synthetic Biology

DESIGNING A SYNTHETIC ORGANISM
Asfa A S (HT080934L)
Vasanth Natarajan (HT081073M)
Department of Chemical & Biomolecular Engineering
National University of Singapore
Vision of “SYNTHETIC BIOLOGY “
Recreate Life
Origin of Life
SYNTHETIC
BIOLOGY
Minimal
Genome
Designer Cells
Synthetic Biology
Synthetic biology is an ambitious and relatively new
field of biology that hopes to recreate life. The first and
foremost challenge in creating 'life in lab' lies in
identifying the minimum essential components that can
take on the essential properties of a living organism
What Defines LIFE
LIVING CELL
Autonomous
Replication
Darwinian
Evolution
Continued growth and
division dependent on
input
of
small
molecules and energy
Genetic and
phenotypic variation
for survival and
reproduction
Current Strategies
Bottom Up Approach
Top Down Approach
Strip down the genes
of an existing cell to
bare minimum enough
to sustain life
 Semi-synthetic
Design a protocell
Synthesizing cell from scratch
Synthesizing Life – Bottom Up Approach
RNA - store information
RNA – RNA polymerase –
replicate its own sequence
 2 RNA molecules – simplest cell
Assembly of single lipid
molecules/micelles
Gradual growth
Environmental factors to control
division
Szostak et al. , Nature 2001
Minimal Genome Concept
Aims to strip down a present day bacterium to its minimum
essential components pertaining to replication, transcription
and translation machinery.
Understand the basic components of the cell that makes it
living.
Provides a template genome that can be used to recreate life
A less complex cell that can be reliably modeled and
engineered to meet our requirements.
Essential Genes – A Comparative Study
450
400
Essential Genes
350
300
Craig Venter, 2005
250
Ehrlich SD,2003
200
Gil,2004
Koonin,1996
150
100
50
0
Studies on Minimal Genome
Mycoplasma genitalium – 482 protein coding genes – smallest genome
Craig Venter,2005 – 382 protein coding genes + 5 paralogous families –
Transposon mutagenesis
Ehrilch SD, 2003 – 271 essential genes in Bacillus subtilis – Gene knock out by
non replicating plasmid
Gil, 2004 – 206 essential genes – Comparison of Endosymbioints - Predicted
Koonin, 1996 – 256 essential genes – Comparison of M.genitalium and
H.influenzae - Predicted
Functional Groups
Main Roles
Intermediary metabolism
Transport and binding
proteins
Protein fate
Transcription
Cell envelope
Hypothetical proteins
Unknown function
Nucleosides and nucleotides
Energy metabolism
Protein synthesis
cell/organism defense
Synthesis of cofactors and
carriers
Cellular processes
Fats and phospholipid
metabolism
Regulatory function
DNA metabolism
Craig Venter,
2005
4
35
Ehrlich
SD, 2003
2
35
Koonin,
1996
0
7
Gil, 2004
24
12
35
47
37
16
29
95
1
7
16
7
35
47
36
8
21
11
1
2
3
1
23
43
20
1
1
7
1
1
10
3
35
45
29
8
10
10
1
3
6
6
5
3
0
2
2
5
2
25
2
12
1
3
2
11
2
32
Comparison of Functional Groups
Essential Genes – A Conclusive List
Different studies come up with a different number of essential genes.
Computation - Underestimates minimal genes - accounts only those
genes that have been conserved in evolution.
Transposon mutagenesis - Over estimates the genes – Classifies genes
that slow down growth as essential and essential genes that tolerate
mutation as non essential.
Antisense RNA - limited success rates
Most mutants produced are single mutants – synthetic lethality may
not be accounted
Construction of a single cell with systematic combination of
all the mutations in a single strain is beyond the scope of
present day technology.
Designing a Synthetic Organism
STRATEGY
Antisense RNA
Computationally
Predicted
Engineer the genome/add
new functions
Transposon Mutagenesis
Determine the
minimal genes
Gene knockout using non
replicating plasmid insertions
Synthesize and assemble
the genome
Genome Transplantation
SYNTHETIC
ORGANISM
Into a suitable propagating
cell that can take up the
genome
Success so far…
Infectious Virus Completely Synthesized – World’s First
Artificial Organism - 2002
3026 bp
1895 bp
2682 bp
cDNA - T7 RNA polymerase promoter constructed from 3 overlapping
DNA fragments.
Each fragment - overlapping 400-600 bp.
Each segment – 69 nt of + and – sequences
cDNA transcribed – Infectious RNA
Infection demonstrated in mice.
SYNTHETIC RNA => TRANSLATED => REPLICATED
=>ENCAPSIDATED INTO NEW COAT PROTEINS
Cello et al. Science, 2002
In the Future…
Mycoplasma laboratorium
Synthetic Genome
Only essential 382 genes
Complete synthesis,
cloning and sequential
assembly
Synthetic Algae
Biofuel
 Synthetic Genomics
Immortal synthetic organism
Military Purpose – Pentagon
Self killing switch
Proposed Applications
Biofuel – A dream in the making
Goals
 Seeks alternatives to fossil fuels
 Sustainability
 Cost reduction
Challenges
Microorganisms can be designed to make useful materials from
renewable materials (Sustainability) - to seek alternatives to fossil
fuels.
 In this case, designing a set of chemical pathways which allows
conversion of natural or waste materials for the production of Biofuels .
Biofuel – A dream in the making (contd.)
ZM - Z.mobilis
SC - S.cerevisiae
EC
- E.coli
 adh,pdc,pfl
 Genes that
are
important for ethanol
production
How to design a
synthetic organism by
adding new functions to
the existing genome ?
Biofuel – A Strategy for Designing synthetic organism
Synthetic
organism which
produces ethanol
with minimal
genes
Identified
Essential
gene list
Adh,pdc,
pfl
Add new genes to
the existing
prototype and
assemble genome
Genome
Transplantation
NO
YES
What next ?
Add few more
imp genes
success
Screen for viability
of cell , maximum
replication and
higher ethanol
production
Biomedical Applications
Devices
- For example, for tissue regeneration or tissue repair complex molecular
devices can be developed.
- Another example could be development of macromolecular assemblies to
sense the damage in blood vessels and repair them.
Novel Drug Release Technology
Smart Drugs -----> Synthetic molecular ensemble
 Encapsulates drug in an inactive form.
 Sensing disease indicators
The programmed module will make a decision
 Activates the drug . (Active only in cells affected by disease)
Inactive
form
Disease
Indicator
s
Active
form
Programmed
Module
Therapeutics
Genetic code
expansion
Environmental Applications
Bioremediation:
Treatment of environmental contaminants via biological systems.
 Rational modification of bacteria and other microorganisms to
eliminate toxic waste from soil.
For certain chemicals for which clean up is difficult, novel organisms
with specific wiring can be used.
Biosensing :
 Detect biotoxins
 Helps in detecting toxin levels in environment
The Hindering Factor
Obstacles
 Bio engineered systems remains
noisy
 Not easier to predict accurately
how a new system will behave
 Engineered organisms capable of
self replication and evolution
 Expensive , Unreliable and adhoc
biological systems
How to overcome ?
FORSEEN RISKS
 Some of the risks are indefinable at present – we cannot anticipate
certain risks at this early stage
 Accidental release of harmful organism
- Extinction of existing species
- Endemic
- Damaging/Disrupting the habitat ( Upset natural balance)
 Purposeful Design and release of harmful organism – Bioterrorism
 Bio-hacker culture
Control Measures
 To educate and train a responsible generation of bioengineers
and scientists
 Working with approved research facilities
 Controls and regulations can be imposed on part suppliers (eg .
screening of oligonucleotides )
 Strict laws and policies to be imposed.
 Incorporating novel genetic codes for high risk organisms to
avoid tampering.
Conclusion
Synthetic Biology – Greatest existing challenge
Synthetic and semi-synthetic approaches.
Discerning the minimal genome enhances better understanding
of cells
Engineered organism can be used for various applications in
fields of biomedicine and environment
Potential risks and hazards not clear
Key References
Cello J et al. Chemical Synthesis of Poliovirus cDNA: Generation of
Infectious Virus in the Absence of Natural Template. Science(2002), 297
Smith et al. Complete Chemical Synthesis, Assembly and Cloning of a
Mycoplasma genitalium Genome. Science(2008), 319
Koonin et al. A Minimal Gene Set for Cellular Life Derived by Comparison
of Complete Bacterial Genomes. PNAS(1996),93
Venter C.J et al. Essential Genes of a Minimal Bacterium.
PNAS(2006),103
Szostak et al. Synthesizing Life. Nature(2001),409
Ehrlich SD et al. Essential Bacillus subtilis genes.PNAS(2003),100
Gil et al. Determination of the Core of a Minimal Bacterial Gene Set.
Microbiology and Molecular Biology Reviews(2004),68
Thank You!