Lezione 7-8 16 Novembre 2010

1. Lezione 7-8 16 Novembre 2010 corso di genomica a.a. 2010/11 aula 6 ore 14.00-16.00 corso di laurea specialistica magistrale Biotecnologia Industriale

2. la lezione di Giovedì 18-11-2010 Facoltà di Medicina e Chirurgia INAUGURAZIONE DELL'ANNO ACCADEMICO 2010 - 2011 Giovedì, 18 Novembre 2010 Il Preside di Facoltà, Prof. Giuseppe Novelli ha il piacere di invitare la S.V. alla giornata di Inaugurazione dell' Anno Accademico 2010 – 2011. ore 12,00 Aula Golgi e Aula Fleming Lectio Magistralis CLYDE A. HUTCHISON J. Craig Venter Institute, San Diego - CA “Building a cell controlled by a synthetic genome” Presiede il Magnifico Rettore, Prof. Renato Lauro Via Montpellier, 1 00133 - Roma 06.2020064 E-mail: eventi.medicina@med.uniroma2.it siete tenuti a partecipare, l’argomento è parte del corso, venite per tempo per trovare posto in aula che sarà affollata

3. titoli dei lavori di CA Hutchison • - Creation of a bacterial cell controlled by a chemically synthesized genome. 2010 • - Cloning whole bacterial genomes in yeast. 2010 • - Creating bacterial strains from genomes that have been cloned and engineered in yeast. 2009 • Enzymatic assembly of DNA molecules up to several hundred kilobases. 2009 • One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. 2008 • Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. 2008 • Genome transplantation in bacteria: changing one species to another. 2007 • Essential genes of a minimal bacterium. 2006 • - Crowding and function reunite. G. J. Pielak and A. C. Miklos (2010) • PNAS 107, 17457-17458 6

4. micoplasma sintetico? Essential genes of a minimal bacterium. The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA Science. 2010 Jul 2;329(5987):52-6. Epub 2010 May 20. Abstract We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication. http://en.wikipedia.org/wiki/Mycoplasma_laboratorium su questo sito c’è una sintesi di tutto il piano sperimentale di Hutchison con la descrizione della strategia, tecniche e risultati in forma semplice e di base

5. si apre un dibattito Science. 2010 Jul 2;329(5987):38-9. Genetic technologies. Synthetic "life," ethics, national security, and public discourse. Cho MK, Relman DA. Stanford University, Stanford, CA 94305, USA. micho@stanford.edu Comment on: * Science. 2010 Jul 2;329(5987):52-6. Synthetic biology and the ethics of knowledge. T. Douglas and J. Savulescu (2010) J. Med. Ethics 36, 687-693 High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides. A. Y. Borovkov, A. V. Loskutov, M. D. Robida, K. M. Day, J. A. Cano, T. Le Olson, H. Patel, K. Brown, P. D. Hunter, and K. F. Sykes (2010) Nucleic Acids Res. 38, e180

6. in natura tutto funziona? One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome Daniel G. Gibson,a1 Gwynedd A. Benders,b Kevin C. Axelrod,a Jayshree Zaveri,a Mikkel A. Algire,a Monzia Moodie,a Michael G. Montague,a J. Craig Venter,a Hamilton O. Smith,b and Clyde A. Hutchison, IIIb1 We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments. Keywords: in vivo DNA assembly, genome synthesis, combinatorial assembly, yeast transformation, Mycoplasma genitalium, synthetic biology Proc Natl Acad Sci U S A. 2008 December 23; 105(51): 20404–20409.

7. fig genome assembly

8. legend to figure 25 frgm assemble Construction of a synthetic M. genitalium genome in yeast. Yeast cells were transformed with 25 different overlapping A-series DNA segments (blue arrows; ≈17 kb to ≈35 kb each) composing the M. genitalium genome. To assemble these into a complete genome, a single yeast cell (tan) must take up at least one representative of the 25 different DNA fragments and incorporate them in the nucleus (yellow), where homologous recombination occurs. This assembled genome, called JCVI-1.1, is 590,011 bp, including the vector sequence (red triangle) shown internal to A86–89. The yeast propagation elements contained within the vector are an origin of replication (ARSH4), a centromere (CEN6), and a histidine-selectable marker (HIS3). In addition to full assembly of the genome as depicted here, some yeast cells may take up fewer than 25 different pieces and produce subassemblies of the genome by a mechanism such as NHEJ (see text). Others may take up more than 25 fragments and produce more than one assembled molecule per cell (not illustrated).

9. Figure construction of synt genome

10. Strategia di assemblaggio The assembly of a synthetic M. mycoides genome in yeast. A synthetic M. mycoides genome was assembled from 1078 overlapping DNA cassettes in three steps. In the first step, 1080-bp cassettes (orange arrows), produced from overlapping synthetic oligonucleotides, were recombined in sets of 10 to produce 109 ~10-kb assemblies (blue arrows). These were then recombined in sets of 10 to produce 11 ~100-kb assemblies (green arrows). In the final stage of assembly, these 11 fragments were recombined into the complete genome (red circle). With the exception of two constructs that were enzymatically pieced together in vitro (27) (white arrows), assemblies were carried out by in vivo homologous recombination in yeast. Major variations from the natural genome are shown as yellow circles. These include four watermarked regions (WM1 to WM4), a 4-kb region that was intentionally deleted (94D), and elements for growth in yeast and genome transplantation. In addition, there are 20 locations with nucleotide polymorphisms (asterisks). Coordinates of the genome are relative to the first nucleotide of the natural M. mycoides sequence. The designed sequence is 1,077,947 bp. The locations of the Asc I and BssH II restriction sites are shown. Cassettes 1 and 800-810 were unnecessary and removed from the assembly strategy (11). Cassette 2 overlaps cassette 1104, and cassette 799 overlaps cassette 811.

11. images of M.mycoides JCVI-syn1.0 and WT M. mycoides

12. legend to M. mycoides Images of M. mycoides JCVI-syn1.0 and WT M. mycoides. To compare the phenotype of the JCVI-syn1.0 and non-YCp WT strains, we examined colony morphology by plating cells on SP4 agar plates containing X-gal. Three days after plating, the JCVI-syn1.0 colonies are blue because the cells contain the lacZ gene and express β-galactosidase, which converts the X-gal to a blue compound (A). The WT cells do not contain lacZ and remain white (B). Both cell types have the fried egg colony morphology characteristic of most mycoplasmas. EMs were made of the JCVI-syn1.0 isolate using two methods. (C) For scanning EM, samples were postfixed in osmium tetroxide, dehydrated and critical point dried with CO2, and visualized with a Hitachi SU6600 SEM at 2.0 keV. (D) Negatively stained transmission EMs of dividing cells with 1% uranyl acetate on pure carbon substrate visualized using JEOL 1200EX CTEM at 80 keV. To examine cell morphology, we compared uranyl acetate–stained EMs of M. mycoides JCVI-syn1.0 cells (E) with EMs of WT cells made in 2006 that were stained with ammonium molybdate (F). Both cell types show the same ovoid morphology and general appearance. EMs were provided by T. Deerinck and M. Ellisman of the National Center for Microscopy and Imaging Research at the University of California at San Diego.