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.