A brief history of synthetic biology
Discovery of CRISPRFirst bacteria with
unnatural amino acid.
Transgenosis in
plants and animals.
Cameron et al: Nat Rev Microbiol 2014
DNA synthesis is the largest segment within enabling products segment, whereas oligonucleotide synthesis is expected to be fastest growing market at
57.8% CAGR during 2014 and 2020. Chassis organism would be the fastest growing core product during the forecast period with synthetic DNA occupying
largest market share. Other core products included in the study are synthetic genes, synthetic scells, and XNA. Biofuels, within enabled product segment,
is expected to exhibit tremendous growth; registering a CAGR of 110.1% during forecast period. However, synthetic biology-based pharmaceuticals and
diagnostics products will generate largest amount of revenue within enabled product segment followed by agriculture and chemicals sub-segments.
Synthetic Biology Market is Expected to Reach $38.7 Billion, Globally, by 2020 - Allied Market Research
Bacterial genome
assembly
Hierarchical organization of living matter
• the blueprint: DNA
...AACGGCTAATCTGG...
• the effectors: proteins
• nanomachines: protein complexes
• cellular factories: cellular facotries
• autonomous units of life: cells
• organisms and ecosystems
The top-down approach derives a synthetic cell from a biological cell by manipulating, for
example, its genes and protein content. This type of synthetic cell is typically living and still
closely related to its biological ancestor. Top down synthetic biology involves using metabolic
and genetic engineering techniques to impart new functions to living cells.
The bottom-up approach, on the contrary, starts with nonliving matter. Its most basic synthetic cell
is merely a cell-sized compartment. Cell-like functionality is derived by reconstituting functional
modules, made from natural or artificial molecular building blocks. The complexity of the synthetic
cell is increased step-by-step, by including more and more components. The ultimate goal is
contraction of an artificial cell (reconstruction of life).
Top-down and botom-up approaches in synthetic biology
Enabling technologies: DNA assembly
Polymerase chain reaction (PCR)
Amplification of up to 20 kbDNA fragment from pre-existing template (genomic DNA,
cDNA library, cloned fragment)
Site directed mutagenesis
Transformation
Synthetic genes: Polymerase chain assembly
Price of synthetic fragments up to 3 kb is €0.10 per base pair.
Restriction cloning
in bacteriain vitro
PCR-based cloning
• DNA of interest can be flanked by any restriction site.
Gateway cloning
λ bacteriophage site specific integration system
DNA assembly with multisite Gateway cloning
The multisite Gateway system relies on five sets of specific and non-crossreacting att sequnces.
Golden Gate Assembly
• Seamless assembly
Sequence and ligation independent cloning (SLIC)
Gibson Assembly
• Seamless assembly
Gibson assembly was used to synthetise 16.3 kb mouse mitochondrial genome directly from 60-mer oligos.
Bacterial artificial chromosomes (BACs)
• BACs are plasmids costructed with the replication
origin of E. Coli F-factor, and so can be maintained
in single copy per cell
• They can keep DNA fragments up to 300 kb
• Recombinant BACs are transformed into E. coli by
electroporation
• Once in the cell, BAC replicates as an F-factor
Yeast artificial chromosomes (YACs)
• YACs contain yeast centromeric DNA, telomeres and
selctable markers
• YACs cab be shittled between yeast and E. coli
• They can keep DNA fragments up to 2000 kb
In vivo cloning: Recombineering
Recombineering takes advantage of cellular homologus recombination machinery to assemble desired
DNA molecules in cells (bacteria, yeast) based on sequence homology.
In bacteria can efficincy of homologous recombination further be increased by coexpression of phage
recombinat system (lambda red recombineering)
Towards synthetic genomes: genome transplantation
Mycoplasma is a genus of bacteria that lack a cell wall around their cell membranes. This characteristic makes them naturally
resistant to antibiotics that target cell wall synthesis. Mycoplasmas have very small genomes.
Lartigue et al: Sceince 2007: replaced the genome of Mycoplasma capricolum with one from another species - Mycoplasma
mycoides virtually transforming one spceies in another.
• Intact circular genome free of proteins was isoleted through puls field gel electrophoresis.
• Isolted genome of M. mycoides was transplanted into M. capricolum cells by polyethylene glycol–mediated transformation.
Assembly of a synthetic genome of Mycoplasma genitalium
• Mycoplasma genitalium: small pathogenic bacterium that lives on the skin cells of the urinary and genital tracts in humans. It has
600 kb genome encoding 500 genes- the smallest genome of a free living creature.
• Overlapping “cassettes” of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by
in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb (“1/8 genome”) 144 kb (“1/4
genome”), which were all cloned as bacterial artificial chromosomes in Escherichia coli.
• Clones of all four 1/4 genomes was assembled by transformation associated recombination cloning in the yeast
Saccharomyces cerevisiae, then isolated and sequenced.
Whole genome assembly in yeast
Gibson et al: Science 2008
Creation of bacteria with fully synthetic genome
It took geneticist Craig Venter 15 years and US$40 million to synthesize the genome of a bacterial parasite. Venter’s team wrote a few
coded 'watermarks' into the genome sequence, which spelled out the names of the team members, as well as several famous quotes.
But besides these tweaks and a few other changes, the synthetic M. mycoides genome was identical to its blueprint.
• M. genitalium grows so slow that one experiment can take weeks to complete. The team decided to change microbes in midstream,
sequencing the 1-million-base genome of the faster-growing M. mycoides and beginning to build a synthetic copy of its chromosome.
• After engeneering artificial genome in yeast, one must be able to transplant the bacterial chromosome from yeast back into a recipient bacterial cell.
Lartigue et al: Science 2009
• Synthetic E. coli genome that uses only 61 of the 64 available
codons in its protein-coding sequences, replacing two serine
codons and one stop codon with synonyms.
Synthetic genome of E. coli with reduced codon usage
• DNA was computationally designed, chemically synthesized and
assembled in 100-kilobase fragments in vectors in S. cerevisiae
• These vectors were then taken up by E. coli and integrated into
the genome in the direct place of the equivalent natural region.
Iterating this process five times resulted in 500-kilobase sections
of DNA being replaced by synthetic versions.
• Eight strains of E. coli were produced in this way, each harbouring
synthetic DNA sections that covered a different region of the
genome. These sections were then combined using conjugation
to make the complete synthetic genome.
Fredens et al: Nature 2019
An udergrad student project: Synthetic yeast chromosome
Each student makes their own stretch of the yeast genome, which involves stitching together very short lengths of DNA created by a DNAsynthesis
machine into ever-larger chunks. These chunks are then incorporated into the yeast chromosome, a few at a time, through a process
called homologous recombination. Eventually, this results in an entirely synthetic chromosome.
The 272,871–base pair designer eukaryotic chromosome, synIII, which is based on the 316,617–base pair native S. cerevisiae chromosome III.
Annaluru et al., Science 2014
Genome editing technologies
• Synthetic genome technology is still expensive, laborious and limited to a few organims.
• Genome editing is affordable, relatively easy and applicable in a wide range of organisms.
MAGE: Multiple automated genomic engineering
• Allelic conversion mediated by directing oligonucleotides to the lagging strand of the replication fork during DNA replication that are delivered
to a cell by electroporatin.
• MAGE enables the rapid and continuous generation of sequence diversity at many targeted chromosomal locations across a large
population of cells through the repeated introduction of synthetic DNA.
• The conversion is facilitated by the bacteriophage λ-Red ssDNA-binding protein β in E. coli and conversion effcinecies can reach up to 30%.
• Up to 50 loci can be simultaneously modified in a single cell.
• MAGE has also beed developed for budding yeast with symultaneous incorporation of up to 12 oligos
Wang et al., Nature 2009 Barbieri et al., Cell 2017
Gene targeting
 Targeted gene knock-outs
 Conditional gene knoc-outs
 Gene tagging
 Amino acid substitutions
 ...
Gene targeting is extremely ineffcient in majority of eukaryotes
Efficient gene targeting is limited to a few model systems (yeast, ES in mouse, chicken DT40 cell line,
moss Physcomitrella patens, ...)
NHEJ is prevalent mode of repair in
organisms with complex genome
Inactivation of NHEJ can boost GT in some organisms
Break induction in targeted locus boosts gene targeting
Interest in programmable nucleases that can cleave sequence of choice.
Zinc-finger nucleases
• Expensive, laborious and time
consuming in making construts
• Target each site separately
• Low efficiency
• Fewer off-target mutations
• Target any genomic location
Zinc-fingr: a small protein strucutral motif coordinated by one or more zinc ions. Involved in DNA/RNA
binding. A singel zinc finger can recognize 2 or 3 bases, but does not bind very tightly to nucleic acids.
DNA binding specificity is achived by combining multiple zinc-fingers.
Zinc-finger nucelases are formed by combining multiple zinc-fingers with the nuclease domain of FokI restriction
endonuclease. FokI acts as a dimer.
Transcription activator-like effector nucleases (TAELNs)
• Expensive, laborious and time consuming in
making construts (easier than zinc-fingers)
• Target each site separately
• Low efficiency
• Fewer off-target mutations
• Target any genomic location
TAL-effectors: proteins are secreted by Xanthomonas bacteria when they infect plants, where they bind to promoters
and activate genes that aid infection. The DNA binding domain contains a repeated highly conserved 33–34 amino
acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the Repeat Variable
Diresidue (RVD), are highly variable and show a strong correlation with specific nucleotide recognition.
• Sensitive to DNA methylation
CRISPR-Cas: Prokaryotic adaptive immune system
Programmable CRISPR-Cas9 nuclease
• Target site must have PAM sequence
• Off target cleavage
• Cheap and easy to make costructs
• Highly versitile
It works!!!!
Applications of CRISPR-Cas9 technology
Multiplex genome engineering using CRISPR/Cas systen
Simultaneous expression of multiple sgRNAs allows effcient targeting of entire gene families.
Genome engineering with the CRISPR-Cas system
(A) E. coli harbors a fission BAC containing a double selection cassette. During fission, (i)
Cas9 induces six cuts (black triangles), splitting the genome into fragment 1 (light gray,
containing oriC indicated by black line) and fragment 2 (dark gray) and the fission BAC
into four pieces (linker sequence 1, linker sequence 2, and two copies of rpsL). (ii)
Homology regions (HRs) between fragments and their cognate linkers. (iii) Lambda red
recombination joins fragments and linkers to yield chromosomes 1 and 2 (Chr. 1 and
Chr. 2). Junctions 1 and 2 (j1 and j2) are new junctions.
(A) E. coli with two chromosomes (Chr. 1 ~3.45 Mb and Chr. 2 ~0.54 Mb) was generated by fission. The
sequence of Chr. 2 is watermarked as described in the text. The color-coding is as in Fig. 1A; a pheS*-KanR
double selection cassette (purple and yellow, respectively) is shown. A fusion sequence, consisting of a pheS*HygR
(purple and blue, respectively) double selection cassette flanked by HR1 and HR2, is introduced in the
indicated positions and orientation in Chr. 1 by lambda-red recombination. Cas9 spacer-directed cleavage (black
arrows), lambda-red recombination, and selection for fusion products through the loss of pheS* on 4-chlorophenylalanine
yield the indicated products. (i) Regenerating the original genomic arrangement, (ii) translocation
of the 0.54-Mb segment 700 kb away from its original position, and (iii) inversion of the 0.54-Mb segment.
Chromosome fission Chromosome fusion
Wang et al., Science 2019
March 2019: A food service company in the Midwestern region of the United States is now using an oil
made from genetically edited soybeans in its sauces, dressings, and fryer. By using gene-editing technology
to deactivate two genes found in soybeans, Minnesota-based agriculture company Calyxt says it’s created a
soybean oil with no trans fats and more heart-healthy fats than traditional soybean oils.
Are genome edited organisms GMOs?
transgene free CRISPR/Cas editing
Recommended reading:
Lartigue et al. (2009) Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast. Science 325:1693
Gibson et al. (2008) Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome. Science 319:1215
Marraffini et al. (2015) CRISPR-Cas immunity in prokaryotes. Nature 526:55