Synthetic genomics

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Synthetic genomics is a nascent field of synthetic biology that uses aspects of genetic modification on pre-existing life forms, or artificial gene synthesis to create new DNA or entire lifeforms.

Contents

Overview

Synthetic genomics is unlike genetic modification 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 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 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

Researchers were able to create a synthetic organism for the first time in 2010. [1] This breakthrough was undertaken by Synthetic Genomics, Inc., which continues to specialize in the research and commercialization of custom designed genomes. [2] It was accomplished by synthesizing a 600 kbp genome (resembling that of Mycoplasma genitalium , save the insertion of a few watermarks) via the Gibson Assembly method and Transformation Associated Recombination. [3]

Recombinant DNA technology

Soon after the discovery of restriction endonucleases and ligases, 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. [4] Although recombinant DNA technology is more commonly used in the construction of fusion proteins and plasmids, several techniques with larger capacities have emerged, allowing for the construction of entire genomes. [5]

Polymerase cycling assembly

Polymerase Cycling Assembly. Blue arrows represent oligonucleotides 40 to 60 bp with overlapping regions of about 20 bp. The cycle is repeated until the final genome is constructed. PCA illustrated by Nivin Nasri (edited).png
Polymerase Cycling Assembly. Blue arrows represent oligonucleotides 40 to 60 bp with overlapping regions of about 20 bp. The cycle is repeated until the final genome is constructed.

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

Gibson assembly method. The blue arrows represent DNA cassettes, which could be any size, 6 kb each for example. The orange segments represent areas of identical DNA sequences. This process can be carried out with multiple initial cassettes. GAM illustrated by Nivin Nasri.png
Gibson assembly method. The blue arrows represent DNA cassettes, which could be any size, 6 kb each for example. The orange segments represent areas of identical DNA sequences. This process can be carried out with multiple initial cassettes.

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

Gap Repair Cloning. The blue arrows represent DNA contigs. Segments of the same colour represent complementary or identical sequences. Specialized primers with extensions are used in a polymerase chain reaction to generate regions of homology at the terminal ends of the DNA contigs. GRC illustrated by Nivin Nasri.png
Gap Repair Cloning. The blue arrows represent DNA contigs. Segments of the same colour represent complementary or identical sequences. Specialized primers with extensions are used in a polymerase chain reaction to generate regions of homology at the terminal ends of the DNA contigs.

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]

Transformation-Associated Recombination. Cross over events occur between regions of homology across the cassettes and YAC vector, thereby connecting the smaller DNA sequences into one larger contig. TAR illustrated by Nivin Nasri (edited).png
Transformation-Associated Recombination. Cross over events occur between regions of homology across the cassettes and YAC vector, thereby connecting the smaller DNA sequences into one larger contig.

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 nucleobase) of DNA 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). [13] The two new artificial nucleotides or Unnatural Base Pair (UBP) were named d5SICS and dNaM . More technically, these artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA. [14] [15] In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a plasmid 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. [16] This is the first known example of a living organism passing along an expanded genetic code to subsequent generations. [14] [17] 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. [14] Then, the natural bacterial replication pathways use them to accurately replicate the plasmid 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 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

Related Research Articles

<span class="mw-page-title-main">Base pair</span> Unit consisting of two nucleobases bound to each other by hydrogen bonds

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 and RNA. Dictated by specific hydrogen bonding patterns, "Watson–Crick" base pairs allow the DNA helix to maintain a regular helical structure that is subtly dependent on its nucleotide sequence. The complementary nature of this based-paired structure provides a redundant copy of the genetic information encoded within each strand of DNA. The regular structure and data redundancy provided by the DNA double helix make DNA well suited to the storage of genetic information, while base-pairing between DNA and incoming nucleotides provides the mechanism through which DNA polymerase replicates DNA and RNA polymerase transcribes DNA into RNA. Many DNA-binding proteins can recognize specific base-pairing patterns that identify particular regulatory regions of genes.

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

<span class="mw-page-title-main">Yeast artificial chromosome</span> Genetically engineered chromosome derived from the DNA of yeast

Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of bacterial artificial chromosome

<span class="mw-page-title-main">DNA synthesis</span>

DNA synthesis is the natural or artificial creation of deoxyribonucleic acid (DNA) molecules. DNA is a macromolecule made up of nucleotide units, which are linked by covalent bonds and hydrogen bonds, in a repeating structure. DNA synthesis occurs when these nucleotide units are joined to form DNA; this can occur artificially or naturally. Nucleotide units are made up of a nitrogenous base, pentose sugar (deoxyribose) and phosphate group. Each unit is joined when a covalent bond forms between its phosphate group and the pentose sugar of the next nucleotide, forming a sugar-phosphate backbone. DNA is a complementary, double stranded structure as specific base pairing occurs naturally when hydrogen bonds form between the nucleotide bases.

A cDNA library is a combination of cloned cDNA fragments inserted into a collection of host cells, which constitute some portion of the transcriptome of the organism and are stored as a "library". cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism. Similarly, tissue-specific cDNA libraries can be produced. In eukaryotic cells the mature mRNA is already spliced, hence the cDNA produced lacks introns and can be readily expressed in a bacterial cell. While information in cDNA libraries is a powerful and useful tool since gene products are easily identified, the libraries lack information about enhancers, introns, and other regulatory elements found in a genomic DNA library.

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.

Xenobiology (XB) is a subfield of synthetic biology, the study of synthesizing and manipulating biological devices and systems. The name "xenobiology" derives from the Greek word xenos, which means "stranger, alien". Xenobiology is a form of biology that is not (yet) familiar to science and is not found in nature. In practice, it describes novel biological systems and biochemistries that differ from the canonical DNA–RNA-20 amino acid system. For example, instead of DNA or RNA, XB explores nucleic acid analogues, termed xeno nucleic acid (XNA) as information carriers. It also focuses on an expanded genetic code and the incorporation of non-proteinogenic amino acids, or “xeno amino acids” into proteins.

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

A genomic library is a collection of overlapping DNA fragments that together make up the total genomic DNA of a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. Next, the vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.

Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence.

Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing. This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.

<span class="mw-page-title-main">Functional cloning</span>

Functional cloning is a molecular cloning technique that relies on prior knowledge of the encoded protein’s sequence or function for gene identification. In this assay, a genomic or cDNA library is screened to identify the genetic sequence of a protein of interest. Expression cDNA libraries may be screened with antibodies specific for the protein of interest or may rely on selection via the protein function. Historically, the amino acid sequence of a protein was used to prepare degenerate oligonucleotides which were then probed against the library to identify the gene encoding the protein of interest. Once candidate clones carrying the gene of interest are identified, they are sequenced and their identity is confirmed. This method of cloning allows researchers to screen entire genomes without prior knowledge of the location of the gene or the genetic sequence.

Mycoplasma laboratorium or Synthia refers to a synthetic strain of bacterium. The project to build the new bacterium has evolved since its inception. Initially the goal was to identify a minimal set of genes that are required to sustain life from the genome of Mycoplasma genitalium, and rebuild these genes synthetically to create a "new" organism. Mycoplasma genitalium was originally chosen as the basis for this project because at the time it had the smallest number of genes of all organisms analyzed. Later, the focus switched to Mycoplasma mycoides and took a more trial-and-error approach.

<span class="mw-page-title-main">Nucleic acid analogue</span> Compound analogous to naturally occurring RNA and DNA

Nucleic acid analogues are compounds which are analogous to naturally occurring RNA and DNA, used in medicine and in molecular biology research. Nucleic acids are chains of nucleotides, which are composed of three parts: a phosphate backbone, a pentose sugar, either ribose or deoxyribose, and one of four nucleobases. An analogue may have any of these altered. Typically the analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canonical bases, and phosphate-sugar backbone analogues such as PNA, which affect the properties of the chain . Nucleic acid analogues are also called xeno nucleic acids and represent one of the main pillars of xenobiology, the design of new-to-nature forms of life based on alternative biochemistries.

Gibson assembly is a molecular cloning method that allows for the joining of multiple DNA fragments in a single, isothermal reaction. It is named after its creator, Daniel G. Gibson, who is the chief technology officer and co-founder of the synthetic biology company, Telesis Bio. The technology is more efficient than manual plasmid genetic recombination methods, but remains expensive as it is still under patent.

The minimal genome is a concept which can be defined as the set of genes sufficient for life to exist and propagate under nutrient-rich and stress-free conditions. Alternatively, it can also be defined as the gene set supporting life on an axenic cell culture in rich media, and it is thought what makes up the minimal genome will depend on the environmental conditions that the organism inhabits. By one early investigation, the minimal genome of a bacterium should include a virtually complete set of proteins for replication and translation, a transcription apparatus including four subunits of RNA polymerase including the sigma factor rudimentary proteins sufficient for recombination and repair, several chaperone proteins, the capacity for anaerobic metabolism through glycolysis and substrate-level phosphorylation, transamination of glutamyl-tRNA to glutaminyl-tRNA, lipid biosynthesis, eight cofactor enzymes, protein export machinery, and a limited metabolite transport network including membrane ATPases. Proteins involved in the minimum bacterial genome tend to be substantially more related to proteins found in archaea and eukaryotes compared to the average gene in the bacterial genome more generally indicating a substantial number of universally conserved proteins. The minimal genomes reconstructed on the basis of existing genes does not preclude simpler systems in more primitive cells, such as an RNA world genome which does not have the need for DNA replication machinery, which is otherwise part of the minimal genome of current cells.

Clyde A. Hutchison III is an American biochemist and microbiologist notable for his research on site-directed mutagenesis and synthetic biology. He is Professor Emeritus of Microbiology and Immunology at the University of North Carolina at Chapel Hill, distinguished professor at the J Craig Venter Institute, a member of the National Academy of Sciences, and a fellow of the American Academy of Arts and Sciences.

Synthetic genome is a synthetically built genome whose formation involves either genetic modification on pre-existing life forms or artificial gene synthesis to create new DNA or entire lifeforms. The field that studies synthetic genomes is called synthetic genomics.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

<span class="mw-page-title-main">Hachimoji DNA</span> Synthetic DNA

Hachimoji DNA is a synthetic nucleic acid analog that uses four synthetic nucleotides in addition to the four present in the natural nucleic acids, DNA and RNA. This leads to four allowed base pairs: two unnatural base pairs formed by the synthetic nucleobases in addition to the two normal pairs. Hachimoji bases have been demonstrated in both DNA and RNA analogs, using deoxyribose and ribose respectively as the backbone sugar.

References

  1. ↑ Hotz, Robert Lee. "Scientists Create Synthetic Organism". Wall Street Journal. ISSN   0099-9660 . Retrieved 2015-09-23.
  2. ↑ "Synthetic Genomics, Inc. - Our Business". www.syntheticgenomics.com. Retrieved 2015-09-26.
  3. ↑ Montague, Michael G; Lartigue, Carole; Vashee, Sanjay (2012-01-01). "Synthetic genomics: potential and limitations". Current Opinion in Biotechnology. 23 (5): 659–665. doi:10.1016/j.copbio.2012.01.014. PMID   22342755.
  4. ↑ Montague, Michael G; Lartigue, Carole; Vashee, Sanjay (2012). "Synthetic genomics: potential and limitations". Current Opinion in Biotechnology. 23 (5): 659–665. doi:10.1016/j.copbio.2012.01.014. PMID   22342755.
  5. ↑ Gibson, Daniel (2011). Synthetic Biology, Part B: Computer Aided Design and DNA Assembly; Chapter Fifteen - Enzymatic Assembly of Overlapping DNA Fragments. Academic Press. pp. 349–361. ISBN   978-0-12-385120-8.
  6. ↑ Stemmer, Willem P. C.; Crameri, Andreas; Ha, Kim D.; Brennan, Thomas M.; Heyneker, Herbert L. (1995-10-16). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene. 164 (1): 49–53. doi:10.1016/0378-1119(95)00511-4. PMID   7590320.
  7. ↑ Smith, Hamilton O.; Hutchison, Clyde A.; Pfannkoch, Cynthia; Venter, J. Craig (2003-12-23). "Generating a synthetic genome by whole genome assembly: φX174 bacteriophage from synthetic oligonucleotides". Proceedings of the National Academy of Sciences. 100 (26): 15440–15445. Bibcode:2003PNAS..10015440S. doi: 10.1073/pnas.2237126100 . ISSN   0027-8424. PMC   307586 . PMID   14657399.
  8. ↑ Gibson, Daniel G; Young, Lei; Chuang, Ray-Yuan; Venter, J Craig; Hutchison, Clyde A; Smith, Hamilton O (2009-04-12). "Enzymatic assembly of DNA molecules up to several hundred kilobases". Nature Methods. 6 (5): 343–345. doi:10.1038/nmeth.1318. PMID   19363495. S2CID   1351008.
  9. ↑ Kouprina, Natalay; Larionov, Vladimir (2003-12-01). "Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes". FEMS Microbiology Reviews. 27 (5): 629–649. doi: 10.1016/S0168-6445(03)00070-6 . ISSN   1574-6976. PMID   14638416.
  10. ↑ Marsischky, Gerald; LaBaer, Joshua (2004-10-15). "Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods". Genome Research. 14 (10b): 2020–2028. doi: 10.1101/gr.2528804 . ISSN   1088-9051. PMID   15489321.
  11. ↑ Gibson, Daniel G.; Benders, Gwynedd A.; Andrews-Pfannkoch, Cynthia; Denisova, Evgeniya A.; Baden-Tillson, Holly; Zaveri, Jayshree; Stockwell, Timothy B.; Brownley, Anushka; Thomas, David W. (2008-02-29). "Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome". Science. 319 (5867): 1215–1220. Bibcode:2008Sci...319.1215G. doi:10.1126/science.1151721. ISSN   0036-8075. PMID   18218864. S2CID   8190996.
  12. ↑ Gibson, Daniel G.; Glass, John I.; Lartigue, Carole; Noskov, Vladimir N.; Chuang, Ray-Yuan; Algire, Mikkel A.; Benders, Gwynedd A.; Montague, Michael G.; Ma, Li (2010-07-02). "Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome". Science. 329 (5987): 52–56. Bibcode:2010Sci...329...52G. doi:10.1126/science.1190719. ISSN   0036-8075. PMID   20488990.
  13. ↑ Malyshev, Denis A.; Dhami, Kirandeep; Quach, Henry T.; Lavergne, Thomas; Ordoukhanian, Phillip (24 July 2012). "Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet". Proceedings of the National Academy of Sciences of the United States of America. 109 (30): 12005–12010. Bibcode:2012PNAS..10912005M. doi: 10.1073/pnas.1205176109 . PMC   3409741 . PMID   22773812.
  14. 1 2 3 Malyshev, Denis A.; Dhami, Kirandeep; Lavergne, Thomas; Chen, Tingjian; Dai, Nan; Foster, Jeremy M.; Corrêa, Ivan R.; Romesberg, Floyd E. (May 7, 2014). "A semi-synthetic organism with an expanded genetic alphabet". Nature . 509 (7500): 385–8. Bibcode:2014Natur.509..385M. doi:10.1038/nature13314. PMC   4058825 . PMID   24805238.
  15. ↑ Callaway, Ewan (May 7, 2014). "Scientists Create First Living Organism With 'Artificial' DNA". Nature News. Huffington Post. Retrieved 8 May 2014.
  16. 1 2 Fikes, Bradley J. (May 8, 2014). "Life engineered with expanded genetic code". San Diego Union Tribune. Retrieved 8 May 2014.
  17. ↑ Sample, Ian (May 7, 2014). "First life forms to pass on artificial DNA engineered by US scientists". The Guardian. Retrieved 8 May 2014.
  18. ↑ Pollack, Andrew (May 7, 2014). "Scientists Add Letters to DNA's Alphabet, Raising Hope and Fear". New York Times. Retrieved 8 May 2014.
  19. ↑ ETH Zurich (1 April 2019). "First bacterial genome created entirely with a computer". EurekAlert! . Retrieved 2 April 2019.
  20. ↑ Venetz, Jonathan E.; et al. (1 April 2019). "Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality". Proceedings of the National Academy of Sciences of the United States of America . 116 (16): 8070–8079. doi: 10.1073/pnas.1818259116 . PMC   6475421 . PMID   30936302.
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