synthetic life & implications for applied microbiology janet nguyen andrei anghel nagham chaban
TRANSCRIPT
Synthetic Life& Implications for Applied Microbiology
Janet NguyenAndrei AnghelNagham Chaban
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
• 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
Experimental Organisms
1. Genome Synthesis
Janet Nguyen
Synthesis: Designing the genome
M. mycoides JCVI-syn1.0
Biologically significant differences were corrected 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
SynthesisOverview
1. 1 kb fragments
2. 10 kb fragments
3. 100 kb fragments
4. Complete genome
Hierarchical strategy: 3 Stages1 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: Strategy
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 Plasmids digested to find cells with assembled 10 kb
insert
All first-stage assemblies sequenced 19/111 had errors
End of Stage 1: results in 10-kb fragments (n=109)
Synthesis: Stage 1 = 1 kb to 10 kb
SynthesisOverview
1. 1 kb fragments
2. 10 kb fragments
3. 100 kb fragments
4. Complete genome
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: Stage 2 = 10 kb to 100 kb
SynthesisOverview
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 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
To further enrich for the 100 kb fragments: Sample of each fragment mixed with molten agarose As agarose solidifies, fibers thread and “trap” circular plasmids
Trapped plasmids digested, releases inserts 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
TransplantOverview
Dr. Carole Lartigue
Transplantation: Hurdles
Transplantation: Hurdles
Transplantation: Hurdles
Transplantation: Hurdles
• 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
Transplantation: Procedure
The Complete Synthetic Cell
The Complete Synthetic Cell
• 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 and Efficiency
Transplantation: Verification
• 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 with 107 - 5×107 cells used
• Yields began to plateau at high donor DNA concentrations
Transplantation: Verification and Efficiency
3. Potential Uses of the Technology
Nagham Chaban
• 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
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
ACTIVIA•People are infecting themselves with bacteria•Can improve digestion•People like this!
Applications: Food Products
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 microbiology4. Synthetic biology can reshape our lives and
transfer our society5. 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.