Synthetic genomics is a nascent field of

synthetic biology Synthetic biology (SynBio) is a multidisciplinary field of science that focuses on living systems and organisms. It applies engineering principles to develop new biological parts, devices, and systems or to redesign existing systems found in nat ...

that uses aspects of

genetic modification Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including th ...

on pre-existing life forms, or artificial gene synthesis to create new DNA or entire lifeforms.

Overview

Synthetic genomics is unlike

in the sense that it does not use naturally occurring genes in its life forms. It may make use of custom designed base pair series, though in a more expanded and presently unrealized sense synthetic genomics could utilize genetic codes that are not composed of the two base pairs of

DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...

that are currently used by life. The development of synthetic genomics is related to certain recent technical abilities and technologies in the field of genetics. The ability to construct long

base pair A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...

chains cheaply and accurately on a large scale has allowed researchers to perform experiments on genomes that do not exist in nature. Coupled with the developments in

protein folding Protein folding is the physical process by which a protein, after Protein biosynthesis, synthesis by a ribosome as a linear chain of Amino acid, amino acids, changes from an unstable random coil into a more ordered protein tertiary structure, t ...

models and decreasing computational costs the field of synthetic genomics is beginning to enter a productive stage of vitality.

History

Researchers were able to create a synthetic organism for the first time in 2010. This breakthrough was undertaken by Synthetic Genomics, Inc., which continues to specialize in the research and commercialization of custom designed genomes. It was accomplished by synthesizing a 600 kbp genome (resembling that of ''

Mycoplasma genitalium ''Mycoplasma genitalium'' (also known as ''MG','' Mgen, or since 2018, ''Mycoplasmoides genitalium'') is a sexually transmitted, small and pathogenic bacterium that lives on the mucous epithelial cells of the urinary and genital tracts in ...

'', save the insertion of a few watermarks) via the Gibson Assembly method and Transformation Associated Recombination.

Recombinant DNA technology

Soon after the discovery of restriction endonucleases and

ligase In biochemistry, a ligase is an enzyme that can catalyze the joining ( ligation) of two molecules by forming a new chemical bond. This is typically via hydrolysis of a small pendant chemical group on one of the molecules, typically resulting i ...

s, the field of genetics began using these molecular tools to assemble artificial sequences from smaller fragments of synthetic or naturally-occurring DNA. The advantage in using the recombinatory approach as opposed to continual DNA synthesis stems from the inverse relationship that exists between synthetic DNA length and percent purity of that synthetic length. In other words, as you synthesize longer sequences, the number of error-containing clones increases due to the inherent error rates of current technologies. Although recombinant DNA technology is more commonly used in the construction of fusion proteins and

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...

s, several techniques with larger capacities have emerged, allowing for the construction of entire genomes.

Polymerase cycling assembly

Polymerase cycling assembly (PCA) uses a series of oligonucleotides (or oligos), approximately 40 to 60 nucleotides long, that altogether constitute both strands of the DNA being synthesized. These oligos are designed such that a single oligo from one strand contains a length of approximately 20 nucleotides at each end that is complementary to sequences of two different oligos on the opposite strand, thereby creating regions of overlap. The entire set is processed through cycles of: (a) hybridization at 60 °C; (b) elongation via

Taq polymerase ''Taq'' polymerase is a thermostable DNA polymerase I named after the thermophilic eubacterial microorganism ''Thermus aquaticus,'' from which it was originally isolated by master's student Alice Chien et al. in 1976. Its name is often abbr ...

and a standard ligase; and (c) denaturation at 95 °C, forming progressively longer contiguous strands and ultimately resulting in the final genome. PCA was used to generate the first synthetic genome in history, that of the Phi X 174 virus.

Gibson assembly method

The Gibson assembly method, designed by Daniel Gibson during his time at the J. Craig Venter Institute, requires a set of double-stranded DNA cassettes that constitute the entire genome being synthesized. Note that cassettes differ from contigs by definition, in that these sequences contain regions of homology to other cassettes for the purposes of recombination. In contrast to Polymerase Cycling Assembly, Gibson Assembly is a single-step, isothermal reaction with larger sequence-length capacity; ergo, it is used in place of Polymerase Cycling Assembly for genomes larger than 6 kb. A T5 exonuclease performs a chew-back reaction at the terminal segments, working in the 5' to 3' direction, thereby producing complementary overhangs. The overhangs hybridize to each other, a

Phusion DNA polymerase Nu jazz (also spelt nü jazz or known as jazztronica, or future jazz) is a genre of jazz and electronic music. The music blends jazz elements with other musical styles, such as funk, electronic music, and free improvisation. Nu jazz typically ve ...

fills in any missing nucleotides and the nicks are sealed with a ligase. However, the genomes capable of being synthesized using this method alone is limited because as DNA cassettes increase in length, they require propagation in vitro in order to continue hybridizing; accordingly, Gibson assembly is often used in conjunction with transformation-associated recombination (see below) to synthesize genomes several hundred kilobases in size.

Transformation-associated recombination

The goal of transformation-associated recombination (TAR) technology in synthetic genomics is to combine DNA contigs by means of

homologous recombination Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in Cell (biology), cellular organi ...

performed by the yeast artificial chromosome (YAC). Of importance is the CEN element within the YAC vector, which corresponds to the yeast centromere. This sequence gives the vector the ability to behave in a chromosomal manner, thereby allowing it to perform homologous recombination. First, gap repair cloning is performed to generate regions of homology flanking the DNA contigs. Gap Repair Cloning is a particular form of the

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed st ...

in which specialized primers with extensions beyond the sequence of the DNA target are utilized. Then, the DNA cassettes are exposed to the YAC vector, which drives the process of homologous recombination, thereby connecting the DNA cassettes. Polymerase Cycling Assembly and TAR technology were used together to construct the 600 kb ''

'' genome in 2008, the first synthetic organism ever created. Similar steps were taken in synthesizing the larger ''

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'' genome a few years later.

Unnatural base pair (UBP)

An unnatural base pair (UBP) is a designed subunit (or

nucleobase Nucleotide bases (also nucleobases, nitrogenous bases) are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic building blocks of nuc ...

) of

which is created in a laboratory and does not occur in nature. In 2012, a group of American scientists led by Floyd E. Romesberg, a chemical biologist at the Scripps Research Institute in San Diego, California, published that his team designed an unnatural base pair (UBP). The two new artificial nucleotides or ''Unnatural Base Pair'' (UBP) were named d5SICS and dNaM. More technically, these artificial

nucleotides Nucleotides are Organic compound, organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both o ...

bearing hydrophobic

s, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA. In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a

containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed, and inserted it into cells of the common bacterium ''E. coli'' that successfully replicated the unnatural base pairs through multiple generations. This is the first known example of a living organism passing along an expanded genetic code to subsequent generations. This was in part achieved by the addition of a supportive algal gene that expresses a nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into ''E. coli'' bacteria. Then, the natural bacterial replication pathways use them to accurately replicate the

containing d5SICS–dNaM. The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

s which can be encoded by DNA, from the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

s. The artificial strings of DNA do not encode for anything yet, but scientists speculate they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses.

Computer-made form

In April 2019, scientists at

ETH Zurich ETH Zurich (; ) is a public university in Zurich, Switzerland. Founded in 1854 with the stated mission to educate engineers and scientists, the university focuses primarily on science, technology, engineering, and mathematics. ETH Zurich ran ...

reported the creation of the world's first bacterial genome, named '' Caulobacter ethensis-2.0'', made entirely by a computer, although a related viable form of ''C. ethensis-2.0'' does not yet exist.In addition to the synthetic design, the team used advanced computer algorithms to map out the entire genome's structure optimising it for stability and function. While the bacterium itself has not been successfully created yet, this achievement represents a major step toward the goal of producing fully synthetic organisms. The development of C. ethensis-2.0 exemplifies the potential of synthetic genomics to revolutionise biotechnology, offering possibilities for customised microorganisms in fields such as medicine, energy and environmental science.

References

External links

Synthetic Genomes: Technologies and Impact
- A 2004 study completed for the DOE on the subject.
Effects of Developments in Synthetic Genomics: Hearing before the Committee on Energy and Commerce, House of Representatives, One Hundred Eleventh Congress, Second Session, May 27, 2010
{{emerging technologies, topics=yes, biomed=yes Algae biomass producers Genetic engineering Genome editing Synthetic biology