production of synthetic cells “ microplasma laboratorium ”
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-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.
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