Synthetic Biology

1. Synthetic Biology

2. Synthetic Biology 1. The design and fabrication of biological components and systems that do not already exist in the natural world 2. The re-design and fabrication of existing biological systems.

3. Synthetic Biology vs. Systems Biology • Systems biology studies complex biological systems as integrated wholes, using tools of modeling, simulation, and comparison to experiment. The focus tends to be on natural systems, often with some (at least long term) medical significance. • Synthetic biology studies how to build artificial biological systems for engineering applications, using many of the same tools and experimental techniques. But the work is fundamentally an engineering application of biological science, rather than an attempt to do more science. The focus is often on ways of taking parts of natural biological systems, characterizing and simplifying them, and using them as a component of a highly unnatural, engineered, biological system. http://syntheticbiology.org/FAQ.html

4. Oil Eating Microorganisms Nature Biotechnology - 24, 952 - 953 (2006) doi:10.1038/nbt0806-952 Blueprint of an oil-eating bacterium Víctor de Lorenzo

5. MIT iGEM's Project: to sense and remove Hg ions from contaminated water. Two Cell System One cell will use the Mer promoter to sense the presence of Mercury ions, then activate the GFP fused downstream. Second cell uses surface display mechanism to exhibit a Mercury capturing peptide, extracting the Mercury from the water. Both cells also display polystyrene binding peptides, and will thus be attached to a polystyrene filter.

6. Genome Transplantation in Bacteria:Changing One Species to AnotherCarole Lartigue, John I. Glass,* Nina Alperovich, Rembert Pieper, Prashanth P. Parmar,Clyde A. Hutchison III, Hamilton O. Smith, J. Craig Venter

7. Mycoplasma mycoides Mycoplasmacapricolum ? Successful transplantation • Clean change of one bacterial species into another • No recombination between donor & recipient chromosomes

8. Why use these bacteria? • Small Bacteria – Goat & Bovine Pathogens • Small Genome • No cell wall 2. Degree of Relatedness - 76.4% of M. Mycoides genome could be mapped to M. Capricolum genome - Of 76.4% there was 91.5% nucleotide identity 3. Plasmids containing a M. mycoidesLC origin of replication complex (ORC) can be established in M. capricolum, whereas plasmids with an M. capricolumORC cannot be established in M. mycoides LC MycoplasmaMycoides (Donor) MycoplasmaCapricolum (Recipient)

9. Key Phases for Successful Transplantation • Isolation of intact donor genome • Preparation of recipient cells • Installation of isolated genome into recipient cells

10. Donor Genomic DNA Preparation 100 ul x 20 Centrifuged, resuspended incubated, mixed with LMP agarose tetM & lacZ Mycoides cells grown in 10ug/ml tetracycline Plugs solidified @ 4oC Lysed, Proteinase K, Wash (4x) PFGE DNA DNA Check for intact circular DNA

11. Intact DNA Remains in Plug • Intact Circular DNA stays in plug • - linear DNA, fragment, PRO, & RNA migrate B. Plasmid safe DNase digests band but not plug

12. Silver Staining Indicates Naked DNA • SDS-PAGE & silver stain of plugs • Plugs boiled in SDS before or after PFGE • DNase I treatment

13. LC-MS/MS Analysis of Plugs • Background of non M. Mycoides proteins run on PFGE • No M. mycoides proteins present in plugs not exposed to PFGE

14. Liberation of DNA From Agarose Plug Melted @ 65oC DNA DNA Incubated overnight w/ β-agarase I ~10ug DNA (8 x 109 genomes)

15. Key Phases for Successful Transplantation • Isolation of intact donor genome • Preparation of recipient cells • Installation of isolated genome into recipient cells

16. Preparation of Recipient Cells Washed, resuspended in CaCl Incubate 37oC, pH 6.2 Held on ice capricolum (recipient) PEG fusion Buffer 10ug transfer RNA Incubated 30min RT DNA DNA DNA mycoides (Donor) 400ul SP4- medium

17. DNA SP4 agar plates w/ tetracycline & Xgal

18. Genetic Analysis • Displayed expected specific amplification * IS1296, tetM, & lacZ may have recombined to destroy arginine deiminase gene

19. Southern Blot Analysis • Donor, Recipient & putative transplants digested with Hind III & run on 1% agarose gel A. • Transplants contain multiple copies of IS1296 insertion sequence • 59% (44 of 75) were same as donor DNA blot • Banding differences due to IS1296 transposition B. • No probe hybridized with WT M. capricolum • 92% clones were same as donor DNA blot

20. Transplant Genome Library Analysis Whole Genome Libraries from 2 Transplant Clones 1300 random Sequence reads from each transplant (1.09 million bp) all matched M. mycoides *20 identical regions b/w 395 & 972 bp

21. Currently.. • Isolated naked DNA from donor M. mycoides • Created chemically competent M. capricolumrecipient cells • Isolated putatively transformed colonies • Confirmed genotypic identity using PCR, southern blot, & library screening Successful Introduction of M. mycoides genome into M. capricolumfollowed by subsequent loss of capricolum genome during antibiotic selection

22. Colony Hybridization Top Probe w/ mycoides specific antibody (anti-VchL) Result: Bound M. Mycoides donor genome and transplants Did not bind capricolum colonies Bottom Probe w/ carpricolum specific antibodies (anti-VmcE & VmcF) Result: Bound WT capricolum colonies Did not bind mycoides donor genome or transplants

23. Proteomic Analysis – 2DE & MALDI-MS • Mycoides & transplants identical • Significant differences (50%) in capricolum • Mascot Algorithm -red = identical to both species -blue = unique to M. mycoides *there were nine protein spots with confidence scores that indicated they were derived from M. capricolum genes, each case proved to be an artifact of either sequencing errors or gene boundary annotation errors (table S2).

24. Optimization of Genome Transplantation 2000ng DNA = 1.6 x 1013 genomes

25. PEG Based Method – Capricolum Cells Fuse • PEG fusion buffer ([Tris 20 mM, NaCl 500 mM, MgCl2 20 mM, polyethylene glycol 8000 (PEG; USB Corporation no. 19959)10%] tetM CAT Successful fusion Prepared as recipient cells Incubation w/ fusion buffer tetM CAT Only colonies in 5% PEG grew 30X increase when CaCl2 added Plated on SP4 agar plates w/ tetracycline & chloramphenicol

26. Preparation of Recipient Cells Washed, resuspended in CaCl Incubate 37oC, pH 6.2 Held on ice Capricolum (recipient) *PEG fusion Buffer 10ug transfer RNA Incubated 30min RT DNA DNA DNA Mycoides (Donor) 400ul SP4- medium

27. Concluding Remarks • Transplant occurred but mechanism of transplant is still unclear - No demonstration of mosaicism • Other methods for transplantation - cation & detergent mediated transfection, electroporation, & compaction of genome methods all unsuccessful • Transplants performed without detergent & proteinase K treatment were unsuccessful - Improbability of finding naturally occurring free-floating, intact naked genomes limits this transplantation phenomenon to the laboratory

28. Registry of Standard Biological Parts Http://parts.mit.edu/registry/index.php/Part_Types

29. Synthetic Chromosome – Venter Institute • Synthetically created a chromosome that is 381 genes long and contains 580,000 base pairs • The DNA sequence is based on the bacterium Mycoplasma genitalium which the team pared down to the bare essentials needed to support life, removing a fifth of its genetic make-up. The wholly synthetically reconstructed chromosome, which the team have christened Mycoplasma laboratorium, has been watermarked with inks for easy recognition. • The new life form will depend for its ability to replicate itself and metabolise on the molecular machinery of the cell into which it has been injected, and in that sense it will not be a wholly synthetic life form. http://www.guardian.co.uk/science/2007/oct/06/genetics.climatechange