iGEM Journal Club6/30/10

“Even in simple bacterial cells, do the chromosomes contain the entire genetic repertoire?

If so, can a complete genetic system be reproduced by chemical synthesis starting with only the digitized DNA sequence contained in a computer?”

Mycoplasma

• Genus of bacteria containing the smallest known free-living bacterium (M. genitalium)

• Causative agents of caprine/bovine diseases

• Small genomes (0.58-1.38kb)– M. genitalium: | 582,970bp– M. capricolum: | 1,155,500bp– M. mycoides: | 1,077,947bp

Step 1: Bacterial genome transplant

• 2007 – Venter Institute announces the successful transplantation of a Mycoplasma genome from one species to another

Step 2: The Minimal Genome Project

• January 2008 – Venter Institute reports having synthesized the complete 582,970 base pair chromosome of M. genitalium

• Overlapping "cassettes" of 5 to 7kb were assembled from chemically synthesized oligos

• Cassettes were joined by in vitro recombination to produce intermediate assemblies, then inserted into E.coli

• Intermediate assemblies inserted into yeast and combined via transformation-associated

recombination cloning

Genome Design

• Two finished M. mycoides genome sequences exist, differing at 95 sites

• Sequence differences between the synthetic cassettes and CP001668 that occurred at 19 sites appeared harmless– These provide 19 markers for the synthetic genome

• Four watermark sequences were designed to replace one or more cassettes in harmless regions– Tet resistance and X-Gal blue selection

Genome assembly

Stage 1 – Cassettes

• 1,078 one-kb cassettes

• Cassettes and a vector were recombined in yeast and transferred to E. coli.

• Plasmid DNA was then isolated from individual E. coli clones and digested to screen for cells containing a vector with an assembled 10-kb insert.

Stage 2 – 10kb Intermediates

• 109 ten-kb sections

• Too large for E.coli, so DNA was extracted from yeast to assemble 100kb sections

• Analyzed by multiplex PCR

Stage 3 – 100kb intermediates

• 11 100kb sections

• Isolated in circular plasmid form

• Removed linear yeast chromosomes by pooling samples in solidifying agarose

• Multiplex PCR and restriction to verify full assembly

Obstacles

• Bacterial genomes grown in yeast are unmethylated and thus not protected from the single restriction system of the recipient cell– Methylate the donor DNA with purified methylases or

crude M. mycoides or M. capricolum extracts– Disrupt the recipient cell’s restriction system

• Success was thwarted for many weeks by a single base pair deletion in the essential gene dnaA. – One wrong base out of over one million in an

essential gene rendered the genome inactive!

Successful Transplants!

Wild-typeSynthetic

Notable results

• Sequence matched the intended design with the exception of: – The 19 known polymorphisms– 8 new single nucleotide polymorphisms – E. coli transposon insertion – 85-bp duplication

• No sequences from M. capricolum (recipient)• Proteomic analysis shows almost the exact

protein pattern as WT M. mycoides

Implications

• Synthesis of a custom organism is possible

• Our sequencing technology is accurate enough to produce an entire genome

• DNA synthesis costs “will follow what has happened with DNA sequencing and continue to exponentially decrease”

• Synthetic biology is now in the public eye

Critiques

• Patents may stifle synthetic biology in this field

• Currently, far less efficient than gene insertion to achieve similar goals

Questions• Is it appropriate to “play god” by creating life?

• Does this further blur the line between living things and machines?

• Is the risk of bioterrorism/biowarfare worth continuing this technology? What additional safeguards should be put in place?

• What advantages does the synthetic genome approach have over gene insertion?