Synthetic genomics
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Synthetic genomics is a nascent field of
Overview
Synthetic genomics is unlike
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 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 models and decreasing computational costs the field of synthetic genomics is beginning to enter a productive stage of vitality.
History
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Researchers were able to create a synthetic organism for the first time in 2010.
Recombinant DNA technology
Soon after the discovery of
Polymerase cycling assembly
![](http://upload.wikimedia.org/wikipedia/commons/thumb/c/c2/PCA_illustrated_by_Nivin_Nasri_%28edited%29.png/220px-PCA_illustrated_by_Nivin_Nasri_%28edited%29.png)
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 and a standard ligase; and (c) denaturation at 95 °C, forming progressively longer contiguous strands and ultimately resulting in the final genome.[6] PCA was used to generate the first synthetic genome in history, that of the Phi X 174 virus.[7]
Gibson assembly method
![](http://upload.wikimedia.org/wikipedia/commons/thumb/e/e7/GAM_illustrated_by_Nivin_Nasri.png/220px-GAM_illustrated_by_Nivin_Nasri.png)
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 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.[8]
Transformation-associated recombination
![](http://upload.wikimedia.org/wikipedia/commons/thumb/8/86/GRC_illustrated_by_Nivin_Nasri.png/220px-GRC_illustrated_by_Nivin_Nasri.png)
The goal of transformation-associated recombination (TAR) technology in synthetic genomics is to combine DNA contigs by means of homologous recombination 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.[9]
![](http://upload.wikimedia.org/wikipedia/commons/thumb/c/c4/TAR_illustrated_by_Nivin_Nasri_%28edited%29.png/220px-TAR_illustrated_by_Nivin_Nasri_%28edited%29.png)
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 in which specialized primers with extensions beyond the sequence of the DNA target are utilized.[10] 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 Mycoplasma genitalium genome in 2008, the first synthetic organism ever created.[11] Similar steps were taken in synthesizing the larger Mycoplasma mycoides genome a few years later.[12]
Unnatural base pair (UBP)
An unnatural base pair (UBP) is a designed subunit (or
The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids 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 proteins.[16] 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.[18]
Computer-made form
In April 2019, scientists at ETH Zurich 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.[19][20]
See also
- Artificial gene synthesis
- Artificially Expanded Genetic Information System
- Bioroid
- Genetic engineering
- Hachimoji DNA
- Synthetic biological circuit
- Synthetic genomes
References
- ISSN 0099-9660. Retrieved 2015-09-23.
- ^ "Synthetic Genomics, Inc. - Our Business". www.syntheticgenomics.com. Retrieved 2015-09-26.
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- ^ PMID 24805238.
- ^ Callaway, Ewan (May 7, 2014). "Scientists Create First Living Organism With 'Artificial' DNA". Nature News. Huffington Post. Retrieved 8 May 2014.
- ^ a b Fikes, Bradley J. (May 8, 2014). "Life engineered with expanded genetic code". San Diego Union Tribune. Retrieved 8 May 2014.
- ^ Sample, Ian (May 7, 2014). "First life forms to pass on artificial DNA engineered by US scientists". The Guardian. Retrieved 8 May 2014.
- ^ Pollack, Andrew (May 7, 2014). "Scientists Add Letters to DNA's Alphabet, Raising Hope and Fear". New York Times. Retrieved 8 May 2014.
- EurekAlert!. Retrieved 2 April 2019.
- PMID 30936302.