Synthetic Biology

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=iQ1VNEgcWE8

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?