Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

1. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome iGEM Journal Club 6/30/10

2. “Even in simple bacterial cells, do the chromosomes contain the entire genetic repertoire? If so, can a complete genetic system be reproduced by chemical synthesis starting with only the digitized DNA sequence contained in a computer?”

3. Mycoplasma • Genus of bacteria containing the smallest known free-living bacterium (M. genitalium) • Causative agents of caprine/bovine diseases • Small genomes (0.58-1.38kb) • M. genitalium: | 582,970bp • M. capricolum: | 1,155,500bp • M. mycoides: | 1,077,947bp

4. Precursor Studies

5. Step 1: Bacterial genome transplant • 2007 – Venter Institute announces the successful transplantation of a Mycoplasma genome from one species to another

6. Step 2: The Minimal Genome Project • January 2008 – Venter Institute reports having synthesized the complete 582,970 base pair chromosome of M. genitalium • Overlapping "cassettes" of5 to 7kb were assembled from chemically synthesizedoligos • Cassettes were joined by in vitro recombination to produceintermediate assemblies, then inserted into E.coli • Intermediate assemblies inserted into yeast and combined via transformation-associatedrecombination cloning

7. Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

8. Genome Design • Two finished M. mycoides genome sequences exist, differing at 95 sites • Sequence differences between the synthetic cassettes and CP001668 that occurred at 19 sites appeared harmless • These provide 19 markers for the synthetic genome • Four watermark sequences were designed to replace one or more cassettes in harmless regions • Tet resistance and X-Gal blue selection

9. Genome assembly

10. Stage 1 – Cassettes • 1,078 one-kb cassettes • Cassettes and a vector were recombined in yeast and transferred to E. coli. • Plasmid DNA was then isolated from individual E. coli clones and digested to screen for cells containing a vector with an assembled 10-kb insert.

11. Stage 2 – 10kb Intermediates • 109 ten-kb sections • Too large for E.coli, so DNA was extracted from yeast to assemble 100kb sections • Analyzed by multiplex PCR

12. Stage 3 – 100kb intermediates • 11 100kb sections • Isolated in circular plasmid form • Removed linear yeast chromosomes by pooling samples in solidifying agarose • Multiplex PCR and restriction to verify full assembly

13. Obstacles • Bacterial genomes grown in yeast are unmethylated and thus not protected from the single restriction system of the recipient cell • Methylate the donor DNA with purified methylases or crude M. mycoides or M. capricolum extracts • Disrupt the recipient cell’s restriction system • Success was thwarted for many weeks by a single base pair deletion in the essential gene dnaA. • One wrong base out of over one million in an essential gene rendered the genome inactive!

14. Successful Transplants! Synthetic Wild-type

15. Notable results • Sequence matched the intended design with the exception of: • The 19 known polymorphisms • 8 new single nucleotide polymorphisms • E. coli transposon insertion • 85-bp duplication • No sequences from M. capricolum (recipient) • Proteomic analysis shows almost the exact protein pattern as WT M. mycoides

16. Implications • Synthesis of a custom organism is possible • Our sequencing technology is accurate enough to produce an entire genome • DNA synthesis costs “will follow what has happened with DNA sequencing and continue to exponentially decrease” • Synthetic biology is now in the public eye

17. Critiques • Patents may stifle synthetic biology in this field • Currently, far less efficient than gene insertion to achieve similar goals

18. Questions • Is it appropriate to “play god” by creating life? • Does this further blur the line between living things and machines? • Is the risk of bioterrorism/biowarfare worth continuing this technology? What additional safeguards should be put in place? • What advantages does the synthetic genome approach have over gene insertion?