iGEM Journal Club
6/30/10
“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
CREATION OF A BACTERIAL
CELL CONTROLLED BY A
CHEMICALLY SYNTHESIZED
GENOME
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!
Synthetic
Wild-type
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?