Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

1. Creation of a Bacterial Cell Controlledby a Chemically Synthesized Genome Presidential Commission for the Study of Bioethical Issues Dan Gibson, +21, Ham Smith and Craig Venter Science (2010) 329: 52 New Directions: The Ethics of Synthetic Biology and Emerging Technologies December 2010 20.020(S11) 02.09.11

2. Summary • Synthesis and transplantation of M. mycoides genomeinto closely related species, M. capricolum • Rationally designed some DNA elements • Tools used to generate transplanted DNA include chemical DNA synthesis, cloning in E. coli, recombination in S. cerevisiae • Tools used to transplant into recipient cell include in vitro processing of DNA, agar plug purification of large DNA assemblies and inactivation of host restriction system

3. Background: writing DNA vs reading DNA Productivity measured in bp of oligos/person/day Cost measured in $/bp of genes

4. Background: reading DNA vs writing DNA

5. Goal: build a working cell that uses a synthetic genome • Design, synthesis and assembly of largest functional genome to date aka “Darwin is dead” art from Drew Endy

6. Goal: build a working cell that uses a synthetic genome • Design, synthesis and assembly of largest functional genome to date aka “Darwin is dead”

7. Goal: build a working cell that uses a synthetic genome • Design, synthesis and assembly of largest functional genome to date “JCVI-syn1.0” Previous successes * Gibson et al. Science 2008 Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome ( ~600, 000 bp in yeast) * Lartigue et al. Science 2007 Genome Transplantation in Bacteria: Changing One Species to Another

8. Approach • Writing DNA 2 polished M. mycoides genomes CP001621 CP001668 (aka YCpMmyc1.1) 4 “watermark” sequences Also wrote in TetR and LacZ Itaya Nature Biotechnology : (2010) 28: 687–689

9. Approach • Writing DNA • Assembling DNA Cloning + recombination for 10, 100 kb and 1Mb fragments Chemical synthesis of ~1kb sequences Itaya Nature Biotechnology : (2010) 28: 687–689

10. Approach • Writing DNA • Assembling DNA • Transplanting DNA to M. capricolum In vitro methylation and deprotonation Agarose plug isolation of DNA inactivated restriction enzyme gene (MCAP0050) Itaya Nature Biotechnology : (2010) 28: 687–689

11. Technical Achievement (1): Assembly Figure 1 Science (2010) 329: 52

12. Technical Achievement (1): Assembly Success rate varied (10-100%) 19/111 assemblies were corrected for sequence errors Method: mixed 10X 1080 bp cassettes  10 kb 80-bp overlaps to adjacent cassettes Recombined in S. cerevisiae ~10 candidates transferred to E. coli Figure 2 Science (2010) 329: 52

13. Technical Achievement (1): Assembly Success rate usually 25% Incorrect instances include partial deletions and duplications Method 10X 10 kb assemblies 100 kb, with 10 specific primer pairs Muliplex PCR Recombined in S. cerevisiae Unstable in E. coli Figure 2 Science (2010) 329: 52

14. Technical Achievement (1): Assembly 1/48 “sMmYCp235” produced all 11 amplicons seen in WT genome (Fig. 3A) Method: 100 kb assemblies 1.08 Mb Spheroplast S. cerevisiae (~1ug DNA/400 ml cells) Separate circular from linear, release 100 kb insert from circular, pool and co-transform, check assembly junctions Figure 2 Science (2010) 329: 52

15. Technical Achievement (2): Transplantation Modify recipient restriction system Scored for TetR and blue on X-gal Lartigue (2007) http://www.nytimes.com/imagepages/2007/06/29/science/20070629CELL_GRAPHIC.html

16. Technical Achievement (2): Transplantation 1.0 WT PCR for watermarks Digests of genome plugs Figure 4 and 5 Science (2010) 329: 52

17. Conclusions • According to JCVI: “The synthetic cell is called Mycoplasma mycoides JCVI-syn1.0 and is the proof of principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome.”

18. Conclusions • According to Venter on CNN: “We built it from four bottles of chemicals.” “So it's the first living self-replicating cell that we have on the planet whose DNA was made chemically and designed in the computer.” “So it has no genetic ancestors. Its parent is a computer.” http://www.cnn.com/2010/HEALTH/05/21/venter.qa/index.html

19. Conclusions • According to Jim Collins (BU): “This is an important advance in our ability to re-engineer organisms, not make new life from scratch…Although some of us in synthetic biology have delusions of grandeur, our goals are much more modest." http://www.nature.com/news/2010/100520/full/news.2010.255.html

20. Perspectives • “Build a cell” (1) mycoides and capricolum nearly identical (2) “synthetic genome” is knock-off • Cost ($ and time)

23. 5 ethical principles • Relevant to considering the social implications of emerging technologies: • public beneficence • responsible stewardship • intellectual freedom and responsibility • democratic deliberation • justice and fairness • Intended to illuminate and guide public policy choices

24. 18 recommendations Public beneficence: maximize public benefits and minimize public harm (1) Public Funding Review and Disclosure (2) Support for Promising Research (3) Innovation Through Sharing

25. 18 recommendations Responsible stewardship: concern for those who are not in a position to represent themselves, and for the environment (4) Coordinated Approach to Synthetic Biology (5) Risk Assessment Review and Field Release Gap Analysis (6) Monitoring, Containment, and Control (7) Risk Assessment Prior to Field Release (8) International Coordination and Dialogue (9) Ethics Education (10) Ongoing Evaluation of Objections

26. 18 recommendations Intellectual freedom and responsibility: use creative potential in morally accountable ways (11) Fostering Responsibility and Accountability (12) Periodic Assessment of Security and Safety Risks (13) Oversight Controls

27. 18 recommendations Democratic deliberation: collaborative decision making that embraces respectful debate of opposing views and active participation by citizens (14) Scientific, Religious, and Civic Engagement (15) Information Accuracy (16) Public Education

28. 18 recommendations Justice and fairness: distribution of benefits and burdens across society (17) Risks in Research (18) Risks and Benefits in Commercial Production and Distribution