Transcript Synthetic Life - Colin Mayfield

Janet Nguyen
Andrei Anghel
Nagham Chaban
Synthetic Life
& Implications for Applied Microbiology
What is Synthetic Biology?
Definition:
Synthetic biology is the engineering of biology; the
synthesis of complex, biological based system, which
display function that does not exist in nature. In essence,
synthetic biology enables the design of biological system
in rational and systemic way
Drafted by the NEST High Level Expert Group
Overview of Presentation
J. Craig Venter Institute:
• Synthesized a novel 1.1 mbp genome
• Transplanted a synthetic genome into host cells and
completely replaced the host genome
• New cells were capable of self-replication and
expressed only novel genes
Overview of Presentation
Topics to be Covered:
1. Genome Synthesis
2. Intercellular Transplant
3. Potential Uses of
Technology
Timeline of Advancements
Experimental Organisms
• Organisms were specifically chosen
for:
 Size of genome
 Stability of genome in host
 Speed of replication
 Lack of cell wall
Experimental Organisms
• Donor: Mycoplasma mycoides
Subspecies: mycoides
Strain: Large Colony GM12
Replicates every 80 min
• Recipient: Mycoplasma capricolum
Subspecies: capricolum
Strain: California Kid (CK)
Replicates every 100 min
Experimental Organisms
• Mycoplasma genus
1. Genome Synthesis
Janet Nguyen
Synthesis: Designing the genome


M. mycoides JCVI-syn1.0
Biologically significant differences were
corrected

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Synthetic and wild type polymorphic at 19 sites
Watermark sequences


Sequences encode unique identifiers
Limits their translation into peptides
Synthesis: Interesting watermarks
I. A code to interpret the rest of the watermarks and
website address.
II. To live to err, to fall, to triumph, to recreate life out of
life.
III. See things not as they are, but as they might be.
IV. What I cannot build,
cannot understand.
Synthesis:
The Genome
Mycoplasma
mycoides
JCVI-syn1.0
Synthesis
Overview
1. 1 kb
fragments
2. 10 kb
fragments
3. 100 kb
fragments
4. Complete
genome
Synthesis: Strategy

Hierarchical strategy: 3 Stages


1 kb → 10 kb → 100 kb → genome (1000 kb)
Start with 1 kb fragments (n=1078) with 80 bp
overlaps to join to neighbours


chemically synthesized by Blue Heron
have restriction enzyme sites at termini
Synthesis: Stage 1 = 1 kb to 10 kb


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1-kb fragments and a
vector recombined in
vivo in yeast
Very active
recombination system!
Plasmid then transferred
to E.coli
Synthesis: Stage 1 = 1 kb to 10 kb



1-kb fragments and a
vector recombined in
vivo in yeast
Very active
recombination system!
Plasmid then transferred
to E.coli
Synthesis: Stage 1 = 1 kb to 10 kb

Recombinant plasmid isolated from E.coli clones


All first-stage assemblies sequenced


Plasmids digested to find cells with assembled 10 kb
insert
19/111 had errors
End of Stage 1: results in 10-kb fragments (n=109)
Synthesis
Overview
1. 1 kb
fragments
2. 10 kb
fragments
3. 100 kb
fragments
4. Complete
genome
Synthesis: Stage 2 = 10 kb to 100 kb

10 kb fragments and cloning vectors transformed
into yeast




100 kb assemblies not stably maintained in E.coli
Recombined plasmid extracted from yeast
Multiplex PCR
presence of a PCR product would suggest an assembled 100 kb

PCR products run on agarose gel

End of Stage 2: Results in 100 kb fragments (n=11)
Synthesis
Overview
1. 1 kb
fragments
2. 10 kb
fragments
3. 100 kb
fragments
4. Complete
genome
Synthesis: Complete genome
assembly


Isolated small quantities of each 100 kb fragment
Purification: exonuclease then anion-exchange
column
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Small fraction of total plasmid DNA (1/100) was digested
Then analyzed by gel electrophoresis
Result: 1ug of each assembly per 400ml of yeast culture
Not all yeast chromosomal DNA removed
Synthesis: Complete genome
assembly
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To further enrich for the 100 kb fragments:
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Trapped plasmids digested, releases inserts
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Sample of each fragment mixed with molten agarose
As agarose solidifies, fibers thread and “trap” circular plasmids
gel electrophoresis
transformed into yeast, no vector sequence required
Complete genome assembled in vivo in yeast, and
grown as yeast artificial chromosome
Synthesis complete!
Next steps:
• Transplantation of genome
• Verification of genome
2. Intercellular Transplantation
Andrei Anghel
Transplant
Overview
Dr. Carole Lartigue
Transplantation: Hurdles

Genetic modifications made to DNA template in
order to allow cloning entire chromosome as a
plasmid in yeast


Inactivation of recipient
cell restriction enzyme
Methylation of the
synthetic genome
Transplantation: Hurdles

Genetic modifications made to DNA template in
order to allow cloning entire chromosome as a
plasmid in yeast


Inactivation of recipient
cell restriction enzyme
Methylation of the
synthetic genome
Transplantation: Hurdles

Genetic modifications made to DNA template in
order to allow cloning entire chromosome as a
plasmid in yeast


Inactivation of recipient
cell restriction enzyme
Methylation of the
synthetic genome
Transplantation: Hurdles

Genetic modifications made to DNA template in
order to allow cloning entire chromosome as a
plasmid in yeast


Inactivation of recipient
cell restriction enzyme
Methylation of the
synthetic genome
Transplantation: Procedure
• Starved M. capricolum cells were mixed with
isolated, synthetic DNA
• Incubated for 3 hours at 37°C to allow recovery,
then plated until large blue colonies formed
• Blue colonies were then used to
inoculate selective broth tubes
The Complete Synthetic Cell
The Complete Synthetic Cell
Transplantation: Verification and
Efficiency
• Ensuring no false-positive results was crucial
• M. mycoides JCVI-syn1.0 was transformed with a vector
containing a selectable tetracycline-resistance marker
and a b-galactosidase gene for screening
• PCR experiments and Southern blot analysis of isolated
putative transplanted cells
• Multiple specific antibody reactions were carried out to
test for species specific proteins
Transplantation: Verification
Transplantation: Verification and
Efficiency
• Only 1 out of 48 yeast colonies contained a full genome
• Only 1 in 150,000 successful transplants in the most
efficient experiments
• Transplant yield was optimal
5×107 cells used
• Yields began to plateau at
donor DNA concentrations
with 107 high
3. Potential Uses of the Technology
Nagham Chaban
Uses of the Technology
• DNA is the software of life
• How could synthetic biology and DNA transfer
affect our lives?
• Creating synthetic bacteria and transferring
man-made DNA allowed the new bacteria to live
and replicate
• That was proof of principle
that life can be created from
a computer
Uses of the Technology
• Designing synthetic bacteria ensures that
synthetic DNA can be used for valuable things in
our lives
• The key is to understand how to change this
software in order to create synthetic life
• Can lead to powerful technology and many
applications and products: biofuel, medicines,
food, etc.
Applications: Medicine
MALARIA
• Kills many people
• Numerous malaria pathogens
are resistant to the first
generation drug
• Artemisinin is a second generation
drug that can treat malaria
• But there is always a problem!
Applications: Medicine
• Artemisinin is available in low quantity in nature
• Synthetic biology can be the solution by building
up a new biosynthetic pathway for this molecule
in microorganisms (i.e. yeast or E.coli)
Applications: Medicine
THERAPEUTIC BACTERIA
• Strange idea, we think of bacteria to be
associated with disease, not therapy
TUMOR-KILLING BACTERIA
• Creating a safe synthetic bacteria to be
injected into the bloodstream
• Travel to tumor, insert itself into cancer cell,
produce tumour-killing toxin
Applications: Food Products
ACTIVIA
• People are infecting themselves with bacteria
• Can improve digestion
• People like this!
Applications: Energy Production
BIOFUELS
• Important issue worldwide
• Plants  biofuels
• Plant biomass simple sugars
• Fermented sugar  energy
Applications: Risks
• Natural genome pool contamination
• Synthetic products released in the
environment should have a specific life span
• Creation of deadly pathogens: bio-terrorism
• Negative environmental impact
• Global monitoring and tracking of synthetic
products are necessary
Overview
1. ~1 million bp synthetic genome
2. Synthetic genome was transplanted into a cell
of a different subspecies – booted up!
3. Vast implications/uses for applied microbiology
4. Synthetic biology can reshape our lives and
transfer our society
5. Important concerns regarding religion (playing
with god) should be discussed and addressed
Questions &
Ethics Discussion
Thank you for staying awake
Discussion Points
•
What if a synthetic RNA can be designed to catalyze its
own reproduction within an artificial membrane?
•
No guarantee that a synthetic genome that works for one
organism (E. coli) will work in another (B. subtilis)
•
Cost/expenses
•
Religious/ethical issues
References
•
Gibson, D. G., Glass, J. I., Lartigue, C., Noskov, V. N., Chuang, R.,
Algire, M. A., et al. (2010). Creation of a bacterial cell controlled by a
chemically synthesized genome. Science, 329(5987), 52-56.
•
Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P.,
Hutchison III, C. A., et al. (2007). Genome transplantation in
bacteria: Changing one species to another. Science, 317(5838),
632-638.
•
Laitigue, C., Vashee, S., Algire, M. A., Chuang, R. -., Benders, G. A.,
Ma, L., et al. (2009). Creating bacterial strains from genomes that
have been cloned and engineered in yeast. Science, 325(5948),
1693-1696.