1. Synthetic Biology

2. The Big Picture Want synthetic genomes to use as ‘biofactories,’ producing materials useful to humans Want the minimal genome to use as the building block for synthetic life Need ability to synthesize genomes to find the minimal genome

3. Early efforts 1979- Synthesis of 207 bp gene of tyrosine suppressor tRNA (Khorana et al) 1990- Synthesis of a 32kb polyketide gene cluster (Kodumal et al) 2002- Full length, infectious polio virus (Cello et al) 2003- φX174 bacteriophage synthesis (Venter et al)

4. Minimal genome? “The ‘minimal genome’ approach seeks to estimate the smallest number of genetic elements sufficient to build a modern-type free-living cellular organism.” (Mushegian) Essential set of survival genes

5. Prerequisites Knowledge of existing genomes Define shorter list of key players by dry-lab comparative analysis. Protein sequence similarities. Key: homology is the basic concept of any evolutionary analysis

7. Why Mycoplasma Part of the mollicutes- generally known as mycoplasma. Wall-less Evolved by massive genome reduction Obligate parasites Smallest known genome of any free living organism capable of growing in axenic culture Lack genomic redundancy

8. Testing for non-essential genes Used transposon mutagenesis to systematically disrupt genes Looked for mutants that survived after 4 weeks (in order to detect slow growing mutants) Detected about 100 non- essential genes Statistically approaching saturation

9. What genes are nonessential? 48% of genes found were hypothetical proteins or encoded proteins of unknown function Some of those that were identified: DNA metabolism, transporters, recombination, DNA repair Some genes identified in the study may be essential for long term survival. Metabolic genes… DNA repair… Found more genetic redundancy than previously thought

11. Why care about artificial chromosomes? 100 genes are not essential, but… In combination? Need to be able to efficiently assemble reduced genomes with combinations of these genes missing

12. http://www.youtube.com/watch?v=iQ1VNEgcW E8

14. M. Genitalium JCVI-1.0 Contains functional copies of all wild type genes except MG408 MG408 disrupted by antibiotic marker to block pathogenicity and allow selection “Watermarks” in intergenetic regions Designation?

16. 5-7 kb cassettes Partition up the 583 kb M. genitalium genome into 101 pieces. Overlaps 80-360 bp Boundaries between genes- why? Insertion of aminoglycoside resistance into MG408

17. The Artificial Chromosome: How Did They Do It? In the past… showed 5-7 kb fragments could be assembled de novo. Take these small fragments and join them in- vitro to make larger assemblies… …And larger… Until you can synthesize a ~583 kb genome

18. Five Stage Assembly

19. In-Vitro Recombination and Vector Insertion

20. In-Vitro Recombination 3’ exonuclease “chew-back” using T4 polymerase without dNTPs Annealing Repair gaps w/ Taq pol. and Taq ligase

21. Vector Prep and Insertion To prepare the BAC using primers with “tails” homologous ends of the A assembly. Also engineer in Not 1 sites. More about that later… Clone into E. coli and amplify plasmid copies

22. What About The Not 1 Sites?

23. In-Vivo Recombination In Yeast ½ genomes assembled by in-vitro recombination did not work well. Why? TAR cloning: homologous recombination in- vivo ¼ genomes combined to form whole genome in circular pTARBAC3 vector This is cool because you are combining 6 pieces of DNA at once One of the ¼ genomes is cut. Why?

25. Whole Genome: QC and Recovery Screened yeast transformants by PCR + Southern Blot Positive clones tested for stability by Southern Blotting of subclones Selected one of the clones for shotgun sequencing – Isolate and enrich artificial chromosome – Purify – Shotgun sequence: 7x coverage

26. ERROR! Errors in sequence supplied to contractors Errors in cassettes synthesized by contractors From repair of assembly junctions From propagation of assemblies in E. coli and yeast Sequenced assemblies at various stages: mostly correct, a few errors. These were corrected

27. Bioethics “…progress in science and technology often outpaces the relevant ethical, legal and moral discourse, and regulation…” (Gabrielle et. al)

28. Great promise... Renewable fuel sources Pharmaceuticals Chemical detoxification Environmental control Beneficial microbes

29. ... or great risk? Synthetic pathogens Genetic transfer (similar to GMO arguments) Economic risk Patent/ownership

30. Regulation Self-governance US National Science Advisory Board for Biosecurity (NSABB) Trade regulation (one for you, two for me)

31. Isolation Physical isolation Biological isolation – New genetic code – Engineered nutrient dependency – Programmed cell death – Microbial ‘bouncers’ – Remove genetically mobile elements

32. NSABB’s ‘Experiments of Concern’ Include experiments that might create knowledge, products or technologies that could enhance the harmful consequnces of a biological agent of toxin; disrupt immunity or the effectiveness of an immunization without clinical and/or agricultural justification; introduce resistance of a biological agent against useful prophylactic or therapeutic interventions, or facilitate their ability to evade detection methodologies; increase the stability, transmissibility or the ability to disseminate a biological agent or toxin; alter the host range or tropism of a biological agent or toxin, enhance the susceptibility of a host population; generate a new pathogenic agent or toxin; or reconstitute an eradicated or extinct biological agent (NSABB, 2007).

33. References Gibson, D.G. Et al. (2008) Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome. Glass, J. I. et al. (2005) Essential Genes of a Minimal Genome. PNAS 103, 425-430

34. Questions?