1. & Implications for Applied Microbiology
Janet Nguyen
Andrei Anghel
Nagham Chaban
Synthetic Life
& Implications for Applied Microbiology 1 1 1

2. 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
New and Emerging Science and Technology (NEST) High-Level Expert Group 2 2 2

4. 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 4 4 4

5. Overview of Presentation
Topics to be Covered:
Genome Synthesis
Intercellular Transplant
Potential Uses of Technology 5 5 5

6. Timeline of Advancements6 6

7. Experimental Organisms
Organisms were specifically chosen for:
Size of genome
Stability of genome in host
Speed of replication
Lack of cell wall 7 7 7

8. 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 8 8 8

9. Experimental Organisms
Mycoplasma genus 9 9 9

10. 1. Genome Synthesis
Janet Nguyen 10 10 10 10

11. 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
Watermark sequences: Replace regions experimentally demonstrated to not interfere with cell viability
Differences at 19 sites appeared harmless and were not corrected 11

12. Synthesis: Interesting watermarks
A code to interpret the rest of the watermarks and website address.
To live to err, to fall, to triumph, to recreate life out of life.
See things not as they are, but as they might be.
What I cannot build,
cannot understand.
3 months to find single bp deletion error
2008 genome - simply signed the names of contributing scientists as watermarks
With first genome, were criticized for trying not to say something more profound than signing the works.
Thus if the DNA sequence is GTTCGA, then GTT will be one letter and CGA will be another, and so on. 12

13. Synthesis: The Genome Mycoplasma mycoides JCVI-syn1.0
Watermark sequences: Replace regions experimentally demonstrated to not interfere with cell viability
Differences at 19 sites appeared harmless and were not corrected 13

14. Synthesis Overview 1. 1 kb fragments 2. 10 kb fragments
4. Complete genome 14

15. 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
each cassette was individually synthesized and sequence-verified by manufacturer
To aid in building process, DNA cassettes and assembly intermediates were designed to contain restriction sites at their termini, and recombined in the presence of vector elements to allow for growth and selection in yeast
Hierarchical strategy was designed to assemble the genome in three stages by transformation and homologous recombination in yeast from kb cassettes. 15

16. 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
Generally, at least one 10-kb assembled fragment could be obtained by screening 10 yeast clones. But rate of success varied from %
Plasmid DNA isolated form E.coli clones: digested to screen for cells containing a vector with an assembled 10-kb insert
19/111 had errors.Alternate clones selected, sequence-verified before next stage 16

17. 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
Generally, at least one 10-kb assembled fragment could be obtained by screening 10 yeast clones. But rate of success varied from %
Plasmid DNA isolated form E.coli clones: digested to screen for cells containing a vector with an assembled 10-kb insert
19/111 had errors.Alternate clones selected, sequence-verified before next stage 17

18. - all you need to add here is a origin of replication for yeast, and a yeast chromsomal centromere because it is eukaryotic. Turning it into a shuttle vector and it can propagate in either E.coli or yeast.

19. 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)
Generally, at least one 10-kb assembled fragment could be obtained by screening 10 yeast clones. But rate of success varied from %
Plasmid DNA isolated form E.coli clones: digested to screen for cells containing a vector with an assembled 10-kb insert
19/111 had errors.Alternate clones selected, sequence-verified before next stage 19

20. Synthesis Overview 1. 1 kb fragments 2. 10 kb fragments
4. Complete genome 20

21. 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)
Explain PCR: Since every 10-kb assembly intermediate was represented by a primar pair, the presence of all amplicons would suggest an assembled 100-kb intermediate. (generally, 25% or more of the clones screened contained all of the amplicons expected for a complete assembly).
Designed primer to represent a 10-kb fragment so that the presence of the PCR product would suggest an assembled 100-kb piece.
After PCR: One of these clones selected for further screening. Circular plasmid DNA was extracted and sized on agarose gel alongside supercoiled marker.
Successful second-stage assemblies with vector sequence are ~105 kb
When all amplicons were produced following multiplex PCR, second-stage assembly intermediate of the correct size was usually produced
In some cases, small deletions occurred. In others, multiple 10-kb fragments were assembled, which produced a larger second-stage assembly intermediate. These differences were easily detected on an agarose gel before complete genome assembly. 21

22. Synthesis Overview 1. 1 kb fragments 2. 10 kb fragments
4. Complete genome 22

23. 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
After first point: (Circular plasmids the size of the second-stage assemblies could be isolated from yeast spheroplasts after an alkaline-lysis procedure).
To further purify the 11 assembly intermediates, they were treated with exonuclease and passed through an anion-exchange column. .A small fraction of the total plasmid DNA, 1/100, was digested with NotI and analyzed by field-inversion gel electrophoresis. This method produced ~1ug of each assembly per 400 ml of yeast culture, ~10^11 cells.
Method above does not completely remove all linear yeast chromosomal DNA, which could substantially decrease yeast transformation and assembly efficiency 23

24. 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
Untrapped linear DNA can be separated out of the agarose plug by electrophoresis, thus enriching for the trapped circular molecules.
In this third/final stage of assembly, an additional vector sequence was not required b/c the yeast cloning elements were already present in assembly 24

25. Synthesis complete! Transplantation of genome Verification of genome
Next steps:
Transplantation of genome
Verification of genome

26. 2. Intercellular Transplantation
Andrei Anghel 26 26 26 26

27. Transplant
Overview
Dr. Carole Lartigue 27 27

28. 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 28 28 28

29. 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 29 29 29

30. 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 30 30 30

31. 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 31 31 31

32. 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 32 32 32

33. The Complete Synthetic Cell

34. The Complete Synthetic Cell

35. 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 35 35 35

36. Transplantation: Verification36 36 36

37. 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 with ×107 cells used
Yields began to plateau at high donor DNA concentrations 37 37 37

38. 3. Potential Uses of the Technology
Nagham Chaban 38 38 38 38

39. 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 39 39 39

40. 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. 40 40 40

41. 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! 41 41 41

42. 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) 42 42 42

43. 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 43 43 43

44. Applications: Food Products
ACTIVIA
People are infecting themselves with bacteria
Can improve digestion
People like this! 44 44 44

45. Applications: Energy Production
BIOFUELS
Important issue worldwide
Plants  biofuels
Plant biomass simple sugars
Fermented sugar  energy 45 45 45

46. 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 46 46 46

47. Overview ~1 million bp synthetic genome
Synthetic genome was transplanted into a cell of a different subspecies – booted up!
Vast implications/uses for applied microbiology
Synthetic biology can reshape our lives and transfer our society
Important concerns regarding religion (playing with god) should be discussed and addressed 47 47 47

48. Thank you for staying awake
Questions &
Ethics Discussion
Thank you for staying awake 48 48 48 48

49. 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 49 49 49

50. 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),
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),
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), 50 50 50 50