Download Minimal genome? - Western Washington University

Synthetic Biology
The Big Picture
• Want synthetic genomes to use as
‘biofactories,’ producing materials useful to
humans
• Want the minimal genome to use as the
building block for synthetic life
• Need ability to synthesize genomes to find the
minimal genome
Early efforts
• 1979- Synthesis of 207 bp gene of tyrosine
suppressor tRNA (Khorana et al)
• 1990- Synthesis of a 32kb polyketide gene
cluster (Kodumal et al)
• 2002- Full length, infectious polio virus (Cello
et al)
• 2003- φX174 bacteriophage synthesis (Venter
et al)
Minimal genome?
“The ‘minimal genome’ approach seeks to estimate the
smallest number of genetic elements sufficient to build a
modern-type free-living cellular organism.” (Mushegian)
Essential set of survival genes
Prerequisites
• Knowledge of existing genomes
• Define shorter list of key players by dry-lab
comparative analysis.
• Protein sequence similarities.
• Key: homology is the basic concept of any
evolutionary analysis
Why Mycoplasma
• Part of the mollicutes- generally known as
mycoplasma.
• Wall-less
• Evolved by massive genome reduction
• Obligate parasites
• Smallest known genome of any free living
organism capable of growing in axenic culture
• Lack genomic redundancy
Testing for non-essential genes
• Used transposon mutagenesis
to systematically disrupt genes
• Looked for mutants that
survived after 4 weeks (in order
to detect slow growing
mutants)
• Detected about 100 nonessential genes
• Statistically approaching
saturation
What genes are nonessential?
• 48% of genes found were hypothetical proteins or
encoded proteins of unknown function
• Some of those that were identified: DNA
metabolism, transporters, recombination, DNA
repair
• Some genes identified in the study may be
essential for long term survival. Metabolic
genes… DNA repair…
• Found more genetic redundancy than previously
thought
Why care about artificial
chromosomes?
• 100 genes are not essential, but…
• In combination?
• Need to be able to efficiently assemble
reduced genomes with combinations of these
genes missing
http://www.youtube.com/watch?v=iQ1VNEgcW
E8
M. Genitalium JCVI-1.0
• Contains functional copies of all wild type
genes except MG408
• MG408 disrupted by antibiotic marker to
block pathogenicity and allow selection
• “Watermarks” in intergenetic regions
• Designation?
5-7 kb cassettes
• Partition up the 583 kb M. genitalium genome into
101 pieces.
• Overlaps 80-360 bp
• Boundaries between genes- why?
• Insertion of aminoglycoside resistance into MG408
The Artificial Chromosome: How Did
They Do It?
• In the past… showed 5-7 kb fragments could
be assembled de novo.
• Take these small fragments and join them invitro to make larger assemblies…
• …And larger…
• Until you can synthesize a ~583 kb genome
Five Stage Assembly
In-Vitro Recombination and Vector
Insertion
In-Vitro Recombination
• 3’ exonuclease “chew-back”
using T4 polymerase without
dNTPs
• Annealing
• Repair gaps w/ Taq pol. and
Taq ligase
Vector Prep and Insertion
• To prepare the BAC
using primers with
“tails” homologous
ends of the A assembly.
• Also engineer in Not 1
sites. More about that
later…
• Clone into E. coli and
amplify plasmid copies
What About The Not 1 Sites?
In-Vivo Recombination In Yeast
• ½ genomes assembled by in-vitro
recombination did not work well. Why?
• TAR cloning: homologous recombination invivo
• ¼ genomes combined to form whole genome
in circular pTARBAC3 vector
• This is cool because you are combining 6
pieces of DNA at once
• One of the ¼ genomes is cut. Why?
Whole Genome: QC and Recovery
• Screened yeast transformants by PCR +
Southern Blot
• Positive clones tested for stability by Southern
Blotting of subclones
• Selected one of the clones for shotgun
sequencing
– Isolate and enrich artificial chromosome
– Purify
– Shotgun sequence: 7x coverage
ERROR!
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•
Errors in sequence supplied to contractors
Errors in cassettes synthesized by contractors
From repair of assembly junctions
From propagation of assemblies in E. coli and
yeast
• Sequenced assemblies at various stages:
mostly correct, a few errors. These were
corrected
Bioethics
“…progress in science and technology often
outpaces the relevant ethical, legal and moral
discourse, and regulation…” (Gabrielle et. al)
Great promise . . .
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Renewable fuel sources
Pharmaceuticals
Chemical detoxification
Environmental control
Beneficial microbes
. . . or great risk?
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Synthetic pathogens
Genetic transfer (similar to GMO arguments)
Economic risk
Patent/ownership
Regulation
• Self-governance
• US National Science
Advisory Board for
Biosecurity (NSABB)
• Trade regulation (one
for you, two for me)
Isolation
• Physical isolation
• Biological isolation
– New genetic code
– Engineered nutrient dependency
– Programmed cell death
– Microbial ‘bouncers’
– Remove genetically mobile elements
NSABB’s ‘Experiments of Concern’
• Include experiments that might create knowledge, products or
technologies that could enhance the harmful consequnces of a
biological agent of toxin; disrupt immunity or the effectiveness of
an immunization without clinical and/or agricultural justification;
introduce resistance of a biological agent against useful prophylactic
or therapeutic interventions, or facilitate their ability to evade
detection methodologies; increase the stability, transmissibility or
the ability to disseminate a biological agent or toxin; alter the host
range or tropism of a biological agent or toxin, enhance the
susceptibility of a host population; generate a new pathogenic
agent or toxin; or reconstitute an eradicated or extinct biological
agent (NSABB, 2007).
References
• Gibson, D.G. Et al. (2008) Complete Chemical
Synthesis, Assembly, and Cloning of a
Mycoplasma genitalium Genome.
• Glass, J. I. et al. (2005) Essential Genes of a
Minimal Genome. PNAS 103, 425-430
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Questions?