Plans Today:
iGEM 2012 v.6.0
The first exam …
Special Interest Short Reports (5%)
Transition in class format
Topics
Synthetic cells,
Safe cells,
Metagenomics
Glycome in synthetic biology.
October 10, 2012
Class #11: iGEM and The Synthetic Cell
Plans Today:
iGEM 2012 v.6.0
The first exam …
Special Interest Short Reports (5%)
Transition in class format
Topics
Synthetic cells,
Safe cells,
Metagenomics
Glycome in synthetic biology.
Oct 17 – Dali Lama visit – no class
Oct 24 – Kathy Williams, PhD, J.D.
Patent Law
Oct 31 – Halloween and the remarkable
coincidence of the Mid-term creative
project! Part 1
Midterm Project Rules and Regulations.
(This carefully crafted plan supersedes the plan in the syllabus.)
Rule #1 Be Creative!
Rule #2 Be Brief.
Rule #3 (the fine print): Students will present a creative project making use
of the materials covered in class, and beyond. The emphasis is creativity,
and the intent is stimulation. Hopefully this project will evolve continuously
from discussions in class and between class members and may form the
basis for your final project. The project format has two parts: On Oct 31 each student will get 60 seconds to present an overview of their project.
Feedback from the audience is encouraged to help each student maximize
the impact of their project. Being as this is Halloween day,
costumes/props/enhancements are encouraged to facilitate this
communication (for extra credit of course)! On November 5th, a one page
written description of the project is due, hopefully incorporating any
comments/improvements resulting from the oral presentation. Both parts of
the presentation are evaluated and constitute 10% of the final grade for the
course.
The Future of Synthetic Biology
Key considerations:
1. Levels of expression needs to be regulated precisely
2. Host pathways need to be known to minimize deleterious interactions
3. Part characterization is essential
4. Appropriate Chassis is key –
5. So is safety…..
Synthetic cells?
http://www.spiegel.de/international
Why create a synthetic cell?
Why create a minimal cell?
Why M. genitalium?
Mycoplasma
• Genus of bacteria containing the smallest known free-living
bacterium (M. genitalium)
• Parasitic bacteria of primates, genital and respiratory tracts
• Small genomes (0.58-1.38kb)
– M. genitalium:
582,970bp
– M. capricolum: 1,155,500bp
– M. mycoides:
1,077,947bp
– Nanoarchaeum equitans is a species of tiny microbe,
discovered in 2002 in a hydrothermal vent; its genome
is only 490,885 nucleotides long; but has an obligate
host
•Whole genome sequencing could be accomplished by
shot-gun (random) sequencing approaches
Gene Map of M. genitalium
Gene
ontology
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium.
Glass J I et al. PNAS 2006;103:425-430
©2006 by National Academy of Sciences
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium.
Glass J I et al. PNAS 2006;103:425-430
©2006 by National Academy of Sciences
Metabolic pathways and substrate transport mechanisms encoded by M. genitalium.
Glass J I et al. PNAS 2006;103:425-430
©2006 by National Academy of Sciences
Legend to molecular anatomy
(previous slide)
Metabolic pathways and substrate transport mechanisms
encoded by M. genitalium. Metabolic products are colored red,
and mycoplasma proteins are black. White letters on black boxes
mark nonessential functions or proteins based on our current
gene disruption study. Question marks denote enzymes or
transporters not identified that would be necessary to complete
pathways, and those missing enzyme and transporter names are
colored green. Transporters are colored according to their
substrates: yellow, cations; green, anions and amino acids;
orange, carbohydrates; purple, multidrug and metabolic end
product efflux. The arrows indicate the predicted direction of
substrate transport. The ABC type transporters are drawn as
follows: rectangle, substrate-binding protein; diamonds,
membrane-spanning permeases; circles, ATP-binding subunits.
Glass J I et al. PNAS 2006;103:425-430
Biology of The Mycoplasmas
•The Mycoplasmas evolved through a process of degenerate evolution from Gram-positive bacteria. They
have lost many genes such as those involved with cell wall synthesis and various biosynthetic pathways,
as they adapted to a more parasitic lifestyle. This allows them to have some of the smallest genomes of
any bacterial organism.
•Their UGA codon is used as another codon for tryptophan instead of a stop codon found in other
organisms.[1]
•M. genitalium lack any genes for amino acid biosynthesis and contain few genes for nucleic acid,
vitamins, and fatty acid biosynthesis. They must acquire these components from their host or through an
artificial medium. This makes them difficult to study in the lab under in vitro conditions, due to their strict
growth requirements.
•They also lack genes for oxidative metabolism (Kreb cycle), gluconeogenesis, catalase and
peroxidase, or other toxic oxygen protective enzymes. They also appear to possess few regulatory
proteins, such as two-component systems and no identifiable transcription factors.
•They do however possess the genes necessary for DNA replication, transcription, and translation, but
even these contain a minimal set of rRNA and tRNA genes.[10] They also contain genes for glycolysis,
phospholipids metabolism, and for converting vitamins to cofactors. They have less DNA repair genes
compared to E. coli and H. influenzae. It is assumed however that the genes detected such as uracil DNA
glycosylase, exinuclease ABC genes, and recA must be essential for proper DNA repair in all organisms.[9]
•While M. genitalium have been able to reduce its genome during the course of its evolution as a parasite, it
must maintain genes necessary for this mode of life. A significant portion of its genome is devoted to
the transport of nutrients from its host such as glucose and fructose, as well as genes for attachment
organelles, adhesins, and antigenic variation to evade the host immune system. In fact it’s estimated that
5% of its total DNA is devoted to repeat fragments used for antigenic recombination of adhesin proteins.[1]
Due to its minimal set of genes needed for protein synthesis and DNA replication, protein synthesis and cell
replication occur much slower compared to E. coli. M. genitalium grow much more slowly with a generation
time of about 24 hours.[1]
Comparison of
two bacterial genomes:
Haemophilus influenzae
and
Mycoplasma genitalium
H. influenzae was the first free-living
organism to have its entire genome
sequenced: 1,830,140 base pairs of
DNA in a single circular chromosome that
contains 1740 protein-coding genes, 58
tRNA genes, and 18 other RNA genes.
The sequencing method used was whole
genome shotgun (1995). Discovered in
1892, mistakenly thought to be causative
agent of influenza until 1933, when the
influenza virus was discovered.
E.coli? Genome, 4,639,221 bp; genes
4,288 genes
Linear representation of the synthesized circular genome
Genetic Watermark: So that the assembled genome would be recognizable
as synthetic, four of the ordered DNA sequences contained strings of bases
that, in code, spell out an e-mail address, the names of many of the people
involved in the project, and a few famous quotations.
DNA shuttling
The Flow of stepwise construction
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
Wildtype
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
How many of the genes are essential for viability?
How to create a minimal Genome
Venter’s group estimated that M. genitalium might be able to survive on only 265 to 350
genes (Science, 10 December 1999, p. 2165). But testing one gene at a time could not
tell the researchers exactly what would constitute such a minimal genome. It’s possible,
for example, that any one of five genes can do some vital task. Tested one at a time, all
five may seem unnecessary, but if all of them are removed, the microbe dies. “We didn’t
have a way of doing cumulative knockouts to get down to just having a chromosome with
just 300 genes in it,” Venter explains. “So we don’t know which are the right 300 genes, or
even if 300 is the right number. That study drove my thinking that we needed a synthetic
genome.”
The biggest genome synthesized to date is that of the 7500-nucleotide poliovirus
(Science, 9 August 2002, p. 1016). “It’s not trivial to do these syntheses,”
Transposon insertions in M. genitaium.
Transposon insertions have been identified in 140
different genes in M. genitalium, so that the minimal
(essential) genome may be 470 – 140 ~ 350 genes
What is essential?
What is redundancy?
Conditions for testing?
Compensatory gene activity?