Production of Synthetic Cells “ Microplasma Laboratorium ”

1. Production of Synthetic Cells “MicroplasmaLaboratorium” Curran Rhodes

2. Background -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.

3. Gibson et. al -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"

4. Making the Genome • -Ideally would be able to synthesize entire genome via oligonucleotide synthesis technology. • Overview of Steps: • Synthesize oligonucleotides with overlapping regions. • Use homologous recombination to combine 1078 DNA cassettes (~1000bp) into 109 10,000bp assemblies • Use homologous recombination to combine 109 10,000bp assemblies into 11 100kbp assemblies • Use homologous recombination to combine 11 100kbp strands into 1 ~1.1Mbp genome

5. grown as a circular Yeast Plasmid

6. Homologous Recombination 1 -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.

7. Homologous Recombination 2 -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.

8. Intermediate Analysis M, S, and λ are ladders

9. Homologous Recombination 3 -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.

10. Restriction Enzyme Analysis In Yeast L and λ are ladders, 235 is synthetic cell H=Host (M. Capricolum)

11. Transplantation -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.

12. Characterization 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.

14. Results -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.

15. Complications -Restriction enzyme problems. -Cytoplasm not synthetic. -Synthesis cost. -Ethical issues. Creating life?

16. References 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.