-Know that we can manipulate genomes by inserting or deleting certain genes.

-What about synthesizing an entirely novel genome using sequencing technology?

-M. Genitalium smallest number of genes.

-Wanted to show that an entire genome could be synthesized, assembled, cloned, and finally transplanted into a new species.

-Chose M. mycoides (donor) and M. capricolum (recipient) because they are fast growing bacterial species.

-Design based on previously obtained DNA sequences of wild type M. mycoides.

“The first species.... to have its parents be a computer"

-Ideally would be able to synthesize entire genome via oligonucleotide synthesis technology.

Overview of Steps: 1.Synthesize oligonucleotides with overlapping regions.2.Use homologous recombination to combine 1078 DNA cassettes (~1000bp) into 109 10,000bp assemblies3.Use homologous recombination to combine 109 10,000bp assemblies into 11 100kbp assemblies4.Use homologous recombination to combine 11 100kbp strands into 1 ~1.1Mbp genome

grown as a circular Yeast Plasmid

-Only require 40-50bp homology for recombination to occur.

-Recombination occurs between overlapping cassettes and vector elements to produce plasmid in yeast.

-Vector is transferred to E. Coli.

-Treat with NotI and screen for 10kb fragments.

-Sequence fragments to ensure there are no errors.

-Vectors containing the 10kbp inserts were pooled and transformed into yeast cells.

-Recombination occurs to produce the 100kbp strands.

-Cannot be maintained in E. Coli. Extract DNA and Use multiplex PCR to check for the presence of each 100kbp assembly.

-Use primer pair for each 10kbp assembly.

-Chose one candidate and size the circular plasmid. Expect ~105kbp.

M, S, and λ are ladders

-In the final step the initial challenge was the isolation of the 100kbp assemblies.

-Used FIGE to verify intermediate product.

-Had to further purify plasmids. Mixed with molten agarose.

-Transform into yeast.

-Screen for complete genome using restriction analysis with Asc I and BssH II and multiplex PCR.

L and λ are ladders, 235 is synthetic cellH=Host (M. Capricolum)

-Intact genomes transplanted into M. Capricolum.

-Replace the genome of a cell with one from another species by transplanting a whole genome as naked DNA.

-Polyethylene glycol–mediated transformation.

-Select with medium containing X-gal and tetracycline.

Two Methods:

Multiplex PCR with four primer pairs for watermark sequences.

Restriction enzyme analysis with Asc I and BssH II to look for expected sizes of fragments.

-In addition, one of the transplants is chosen and sequenced.

-No DNA from M. capricolum is found so complete replacement of genome occurred during transplantation.

-Produced synthetic cells capable of self replication.

-Cells follow the synthetic genome and are phenotypically similar to natural M. Mycoides.

-14 genes disrupted in total but growth pattern similar.

-Approach should be applicable to synthesis and transplantation of novel genomes.

-Restriction enzyme problems.

-Cytoplasm not synthetic.

-Synthesis cost.

-Ethical issues. Creating life?

Gibson, D; Glass, J; Lartigue, C; et al. “Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome.” Science. V. 329 p. 52-56. 2010.

Thomason, L; Court, D; Bubuneko, M; et al. “Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination.” Current Protocols in Molecular Biology. 78:1.16.1–1.16.24.

“DNA Oligo FAQ.” Invitrogen Life Technologies. Available at: http://www.invitrogen.com/site/us/en/home

/Products-and-Services/Product-Types/Primers-Oligos-Nucleotides. Accessibility

verified: December 2, 2012.