Download Lezione 7-8 6 Novembre 2009

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
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: [email protected]
siete tenuti a partecipare, l’argomento è parte del corso, venite per
tempo per trovare posto in aula che sarà affollata
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
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
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. [email protected]
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 microarraysynthesized 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
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.
fig genome assembly
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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 JCVI1.1, 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).
Figure construction of synt genome
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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.
images of M.mycoides JCVI-syn1.0 and WT
M. mycoides
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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.