Mycoplasma laboratorium

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Mycoplasma laboratorium
Scientific classification OOjs UI icon edit-ltr.svg
Domain: Bacteria
Phylum: Mycoplasmatota
Class: Mollicutes
Order: Mycoplasmatales
Family: Mycoplasmataceae
Genus: Mycoplasma
Species:
M. mycoides
Subspecies:
M. m. JCVI-syn1.0
Trinomial name
Mycoplasma mycoides JCVI-syn1.0
Gibson et al., 2010 [a 1]
Synonyms [a 2]

Mycoplasma laboratoriumReich, 2000

Mycoplasma laboratorium or Synthia [b 1] 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. [b 2]

Contents

To identify the minimal genes required for life, each of the 482 genes of M. genitalium was individually deleted and the viability of the resulting mutants was tested. This resulted in the identification of a minimal set of 382 genes that theoretically should represent a minimal genome. [a 3] In 2008 the full set of M. genitalium genes was constructed in the laboratory with watermarks added to identify the genes as synthetic. [b 3] [a 4] However M. genitalium grows extremely slowly and M. mycoides was chosen as the new focus to accelerate experiments aimed at determining the set of genes actually needed for growth. [b 4]

In 2010, the complete genome of M. mycoides was successfully synthesized from a computer record and transplanted into an existing cell of Mycoplasma capricolum that had had its DNA removed. [b 5] It is estimated that the synthetic genome used for this project cost US$40 million and 200 man-years to produce. [b 4] The new bacterium was able to grow and was named JCVI-syn1.0, or Synthia. After additional experimentation to identify a smaller set of genes that could produce a functional organism, JCVI-syn3.0 was produced, containing 473 genes. [b 2] 149 of these genes are of unknown function. [b 2] Since the genome of JCVI-syn3.0 is novel, it is considered the first truly synthetic organism.

Minimal genome project

The production of Synthia is an effort in synthetic biology at the J. Craig Venter Institute by a team of approximately 20 scientists headed by Nobel laureate Hamilton Smith and including DNA researcher Craig Venter and microbiologist Clyde A. Hutchison III. The overall goal is to reduce a living organism to its essentials and thus understand what is required to build a new organism from scratch. [a 3] The initial focus was the bacterium M. genitalium, an obligate intracellular parasite whose genome consists of 482 genes comprising 582,970 base pairs, arranged on one circular chromosome (at the time the project began, this was the smallest genome of any known natural organism that can be grown in free culture). They used transposon mutagenesis to identify genes that were not essential for the growth of the organism, resulting in a minimal set of 382 genes. [a 3] This effort was known as the Minimal Genome Project. [a 5]

Choice of organism

Mycoplasma

Mycoplasma is a genus of bacteria of the class Mollicutes in the division Mycoplasmatota (formerly Tenericutes), characterised by the lack of a cell wall (making it Gram negative) due to its parasitic or commensal lifestyle. In molecular biology, the genus has received much attention, both for being a notoriously difficult-to-eradicate contaminant in mammalian cell cultures (it is immune to beta-lactams and other antibiotics), [a 6] and for its potential uses as a model organism due to its small genome size. [a 7] The choice of genus for the Synthia project dates to 2000, when Karl Reich coined the phrase Mycoplasma laboratorium. [a 2]

Other organisms with small genomes

As of 2005, Pelagibacter ubique (an α-proteobacterium of the order Rickettsiales) has the smallest known genome (1,308,759 base pairs) of any free living organism and is one of the smallest self-replicating cells known. It is possibly the most numerous bacterium in the world (perhaps 1028 individual cells) and, along with other members of the SAR11 clade, are estimated to make up between a quarter and a half of all bacterial or archaeal cells in the ocean. [a 8] It was identified in 2002 by rRNA sequences and was fully sequenced in 2005. [a 9] It is extremely hard to cultivate a species which does not reach a high growth density in lab culture. [a 10] [a 11] Several newly discovered species have fewer genes than M. genitalium, but are not free-living: many essential genes that are missing in Hodgkinia cicadicola , Sulcia muelleri , Baumannia cicadellinicola (symbionts of cicadas) and Carsonella ruddi (symbiote of hackberry petiole gall psyllid, Pachypsylla venusta [a 12] ) may be encoded in the host nucleus. [a 13] The organism with the smallest known set of genes as of 2013 is Nasuia deltocephalinicola , an obligate symbiont. It has only 137 genes and a genome size of 112 kb. [a 14] [b 6]

species namenumber of genessize (Mbp)
Candidatus Hodgkinia cicadicola Dsem 1690.14
Candidatus Carsonella ruddii PV 1820.16
Candidatus Sulcia muelleri GWSS 2270.25
Candidatus Sulcia muelleri SMDSEM 2420.28
Buchnera aphidicola str. Cinara cedri 3570.4261
Mycoplasma genitalium G37 4750.58
Candidatus Phytoplasma mali 4790.6
Buchnera aphidicola str. Baizongia pistaciae 5040.6224
Nanoarchaeum equitans Kin4-M 5400.49

Techniques

Several laboratory techniques had to be developed or adapted for the project, since it required synthesis and manipulation of very large pieces of DNA.

Bacterial genome transplantation

In 2007, Venter's team reported that they had managed to transfer the chromosome of the species Mycoplasma mycoides to Mycoplasma capricolum by:

The term transformation is used to refer to insertion of a vector into a bacterial cell (by electroporation or heatshock). Here, transplantation is used akin to nuclear transplantation.

Bacterial chromosome synthesis

In 2008 Venter's group described the production of a synthetic genome, a copy of M. genitalium G37 sequence L43967, by means of a hierarchical strategy: [a 16]

The genome of this 2008 result, M. genitalium JCVI-1.0, is published on GenBank as CP001621.1. It is not to be confused with the later synthetic organisms, labelled JCVI-syn, based on M. mycoides. [a 16]

Synthetic genome

In 2010 Venter and colleagues created Mycoplasma mycoides strain JCVI-syn1.0 with a synthetic genome. [a 1] Initially the synthetic construct did not work, so to pinpoint the error—which caused a delay of 3 months in the whole project [b 4] —a series of semi-synthetic constructs were created. The cause of the failure was a single frameshift mutation in DnaA, a replication initiation factor. [a 1]

The purpose of constructing a cell with a synthetic genome was to test the methodology, as a step to creating modified genomes in the future. Using a natural genome as a template minimized the potential sources of failure. Several differences are present in Mycoplasma mycoides JCVI-syn1.0 relative to the reference genome, notably an E.coli transposon IS1 (an infection from the 10kb stage) and an 85bp duplication, as well as elements required for propagation in yeast and residues from restriction sites. [a 1]

There has been controversy over whether JCVI-syn1.0 is a true synthetic organism. While the genome was synthesized chemically in many pieces, it was constructed to match the parent genome closely and transplanted into the cytoplasm of a natural cell. DNA alone cannot create a viable cell: proteins and RNAs are needed to read the DNA, and lipid membranes are required to compartmentalize the DNA and cytoplasm. In JCVI-syn1.0 the two species used as donor and recipient are of the same genus, reducing potential problems of mismatches between the proteins in the host cytoplasm and the new genome. [a 17] Paul Keim (a molecular geneticist at Northern Arizona University in Flagstaff) noted that "there are great challenges ahead before genetic engineers can mix, match, and fully design an organism's genome from scratch". [b 4]

Watermarks

A hidden watermark on a semiconductor chip from 1976, acting as a signature of its creators. In an analogous way, JC Venter and his team added watermarks using stop codons to sign their creation. Hidden watermark.jpg
A hidden watermark on a semiconductor chip from 1976, acting as a signature of its creators. In an analogous way, JC Venter and his team added watermarks using stop codons to sign their creation.

A much publicized feature of JCVI-syn1.0 is the presence of watermark sequences. The 4 watermarks (shown in Figure S1 in the supplementary material of the paper [a 1] ) are coded messages written into the DNA, of length 1246, 1081, 1109 and 1222 base pairs respectively. These messages did not use the standard genetic code, in which sequences of 3 DNA bases encode amino acids, but a new code invented for this purpose, which readers were challenged to solve. [b 7] The content of the watermarks is as follows:

  1. Watermark 1: an HTML document which reads in a Web browser as text congratulating the decoder, and instructions on how to email the authors to prove the decoding.
  2. Watermark 2: a list of authors and a quote from James Joyce: "To live, to err, to fall, to triumph, to recreate life out of life".
  3. Watermark 3: more authors and a quote from Robert Oppenheimer (uncredited): "See things not as they are, but as they might be".
  4. Watermark 4: more authors and a quote from Richard Feynman: "What I cannot build, I cannot understand".

JCVI-syn3.0

Gene functions in the minimal genome of the synthetic organism, Syn 3. Syn3 genome.svg
Gene functions in the minimal genome of the synthetic organism, Syn 3.

In 2016, the Venter Institute used genes from JCVI-syn1.0 to synthesize a smaller genome they call JCVI-syn3.0, that contains 531,560 base pairs and 473 genes. [b 8] In 1996, after comparing M. genitalium with another small bacterium Haemophilus influenzae, Arcady Mushegian and Eugene Koonin had proposed that there might be a common set of 256 genes which could be a minimal set of genes needed for viability. [b 9] [a 19] In this new organism, the number of genes can only be pared down to 473, 149 of which have functions that are completely unknown. [b 9] As of 2022 the unknown set has been narrowed to about 100. [b 10] In 2019 a complete computational model of all pathways in Syn3.0 cell was published, representing the first complete in silico model for a living minimal organism. [a 20]

Concerns and controversy

Reception

On Oct 6, 2007, Craig Venter announced in an interview with UK's The Guardian newspaper that the same team had synthesized a modified version of the single chromosome of Mycoplasma genitalium chemically. The synthesized genome had not yet been transplanted into a working cell. The next day the Canadian bioethics group, ETC Group issued a statement through their representative, Pat Mooney, saying Venter's "creation" was "a chassis on which you could build almost anything. It could be a contribution to humanity such as new drugs or a huge threat to humanity such as bio-weapons". Venter commented "We are dealing in big ideas. We are trying to create a new value system for life. When dealing at this scale, you can't expect everybody to be happy." [b 11]

On May 21, 2010, Science reported that the Venter group had successfully synthesized the genome of the bacterium Mycoplasma mycoides from a computer record and transplanted the synthesized genome into the existing cell of a Mycoplasma capricolum bacterium that had had its DNA removed. The "synthetic" bacterium was viable, i.e. capable of replicating. [b 1] Venter described it as "the first species.... to have its parents be a computer". [b 12]

The creation of a new synthetic bacterium, JCVI-3.0 was announced in Science on March 25, 2016. It has only 473 genes. Venter called it “the first designer organism in history” and argued that the fact that 149 of the genes required have unknown functions means that "the entire field of biology has been missing a third of what is essential to life". [a 21]

Press coverage

The project received a large amount of coverage from the press due to Venter's showmanship, to the degree that Jay Keasling, a pioneering synthetic biologist and founder of Amyris commented that "The only regulation we need is of my colleague's mouth". [b 13]

Utility

Venter has argued that synthetic bacteria are a step towards creating organisms to manufacture hydrogen and biofuels, and also to absorb carbon dioxide and other greenhouse gases. George M. Church, another pioneer in synthetic biology, has expressed the contrasting view that creating a fully synthetic genome is not necessary since E. coli grows more efficiently than M. genitalium even with all its extra DNA; he commented that synthetic genes have been incorporated into E.coli to perform some of the above tasks. [b 14]

Intellectual property

The J. Craig Venter Institute filed patents for the Mycoplasma laboratorium genome (the "minimal bacterial genome") in the U.S. and internationally in 2006. [b 15] [b 16] [a 22] The ETC group, a Canadian bioethics group, protested on the grounds that the patent was too broad in scope. [b 17]

Similar projects

From 2002 to 2010, a team at the Hungarian Academy of Science created a strain of Escherichia coli called MDS42, which is now sold by Scarab Genomics of Madison, WI under the name of "Clean Genome. E.coli", [b 18] where 15% of the genome of the parental strain (E. coli K-12 MG1655) were removed to aid in molecular biology efficiency, removing IS elements, pseudogenes and phages, resulting in better maintenance of plasmid-encoded toxic genes, which are often inactivated by transposons. [a 23] [a 24] [a 25] Biochemistry and replication machinery were not altered.

Related Research Articles

<span class="mw-page-title-main">Craig Venter</span> American Scientist

John Craig Venter is an American scientist. He is known for leading one of the first draft sequences of the human genome and led the first team to transfect a cell with a synthetic chromosome. Venter founded Celera Genomics, the Institute for Genomic Research (TIGR) and the J. Craig Venter Institute (JCVI). He was the co-founder of Human Longevity Inc. and Synthetic Genomics. He was listed on Time magazine's 2007 and 2008 Time 100 list of the most influential people in the world. In 2010, the British magazine New Statesman listed Craig Venter at 14th in the list of "The World's 50 Most Influential Figures 2010". In 2012, Venter was honored with Dan David Prize for his contribution to genome research. He was elected to the American Philosophical Society in 2013. He is a member of the USA Science and Engineering Festival's advisory board.

<i>Mycoplasma genitalium</i> Species of bacterium

Mycoplasma genitalium is a sexually transmitted, small and pathogenic bacterium that lives on the mucous epithelial cells of the urinary and genital tracts in humans. Medical reports published in 2007 and 2015 state that Mgen is becoming increasingly common. Resistance to multiple antibiotics, including the macrolide azithromycin, which until recently was the most reliable treatment, is becoming prevalent. The bacterium was first isolated from the urogenital tract of humans in 1981, and was eventually identified as a new species of Mycoplasma in 1983. It can cause negative health effects in men and women. It also increases the risk for HIV spread with higher occurrences in those previously treated with the azithromycin antibiotics.

<i>Escherichia coli</i> Enteric, rod-shaped, gram-negative bacterium

Escherichia coli ( ESH-ə-RIK-ee-ə KOH-lye) is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes such as EPEC and ETEC are pathogenic, can cause serious food poisoning in their hosts and are occasionally responsible for food contamination incidents that prompt product recalls. Most strains are part of the normal microbiota of the gut and are harmless or even beneficial to humans (although these strains tend to be less studied than the pathogenic ones). For example, some strains of E. coli benefit their hosts by producing vitamin K2 or by preventing the colonization of the intestine by pathogenic bacteria. These mutually beneficial relationships between E. coli and humans are a type of mutualistic biological relationship—where both the humans and the E. coli are benefitting each other. E. coli is expelled into the environment within fecal matter. The bacterium grows massively in fresh fecal matter under aerobic conditions for three days, but its numbers decline slowly afterwards.

<i>Mycoplasma</i> Genus of bacteria

Mycoplasma is a genus of bacteria that, like the other members of the class Mollicutes, lack a cell wall, and its peptidoglycan, around their cell membrane. The absence of peptidoglycan makes them naturally resistant to antibiotics such as the beta-lactam antibiotics that target cell wall synthesis. They can be parasitic or saprotrophic. Several species are pathogenic in humans, including M. pneumoniae, which is an important cause of "walking" pneumonia and other respiratory disorders, and M. genitalium, which is believed to be involved in pelvic inflammatory diseases. Mycoplasma species are among the smallest organisms yet discovered, can survive without oxygen, and come in various shapes. For example, M. genitalium is flask-shaped, while M. pneumoniae is more elongated, many Mycoplasma species are coccoid. Hundreds of Mycoplasma species infect animals.

<span class="mw-page-title-main">Synthetic biology</span> Interdisciplinary branch of biology and engineering

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

Mollicutes is a class of bacteria distinguished by the absence of a cell wall. The word "Mollicutes" is derived from the Latin mollis, and cutis. Individuals are very small, typically only 0.2–0.3 μm in size and have a very small genome size. They vary in form, although most have sterols that make the cell membrane somewhat more rigid. Many are able to move about through gliding, but members of the genus Spiroplasma are helical and move by twisting. The best-known genus in the Mollicutes is Mycoplasma. Colonies show the typical "fried-egg" appearance.

<span class="mw-page-title-main">J. Craig Venter Institute</span> Non-profit genomics research institute

The J. Craig Venter Institute (JCVI) is a non-profit genomics research institute founded by J. Craig Venter, Ph.D. in October 2006. The institute was the result of consolidating four organizations: the Center for the Advancement of Genomics, The Institute for Genomic Research (TIGR), the Institute for Biological Energy Alternatives, and the J. Craig Venter Science Foundation Joint Technology Center. It has facilities in Rockville, Maryland, and San Diego, California.

An artificial cell, synthetic cell or minimal cell is an engineered particle that mimics one or many functions of a biological cell. Often, artificial cells are biological or polymeric membranes which enclose biologically active materials. As such, liposomes, polymersomes, nanoparticles, microcapsules and a number of other particles can qualify as artificial cells.

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.

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">Prokaryote</span> Unicellular organism lacking a membrane-bound nucleus

A prokaryote is a single-cell organism whose cell lacks a nucleus and other membrane-bound organelles. The word prokaryote comes from the Ancient Greek πρό (pró), meaning 'before', and κάρυον (káruon), meaning 'nut' or 'kernel'. In the two-empire system arising from the work of Édouard Chatton, prokaryotes were classified within the empire Prokaryota. However in the three-domain system, based upon molecular analysis, prokaryotes are divided into two domains: Bacteria and Archaea. Organisms with nuclei are placed in a third domain: Eukaryota.

<i>Mycoplasma mycoides</i> Species of bacterium

Mycoplasma mycoides is a bacterial species of the genus Mycoplasma in the class Mollicutes. This microorganism is a parasite that lives in ruminants. Mycoplasma mycoides comprises two subspecies, mycoides and capri, which infect cattle and small ruminants such as goats respectively.

Synthetic mycoides refers to an artificial life form created by Craig Venter at the J Craig Venter Institute in May 2010. A synthetic genome was transferred into an empty cell to form the bacterium, which was capable of self replication and functioned solely from the transferred chromosomes.

Bacterial genomes are generally smaller and less variant in size among species when compared with genomes of eukaryotes. Bacterial genomes can range in size anywhere from about 130 kbp to over 14 Mbp. A study that included, but was not limited to, 478 bacterial genomes, concluded that as genome size increases, the number of genes increases at a disproportionately slower rate in eukaryotes than in non-eukaryotes. Thus, the proportion of non-coding DNA goes up with genome size more quickly in non-bacteria than in bacteria. This is consistent with the fact that most eukaryotic nuclear DNA is non-gene coding, while the majority of prokaryotic, viral, and organellar genes are coding. Right now, we have genome sequences from 50 different bacterial phyla and 11 different archaeal phyla. Second-generation sequencing has yielded many draft genomes ; third-generation sequencing might eventually yield a complete genome in a few hours. The genome sequences reveal much diversity in bacteria. Analysis of over 2000 Escherichia coli genomes reveals an E. coli core genome of about 3100 gene families and a total of about 89,000 different gene families. Genome sequences show that parasitic bacteria have 500–1200 genes, free-living bacteria have 1500–7500 genes, and archaea have 1500–2700 genes. A striking discovery by Cole et al. described massive amounts of gene decay when comparing Leprosy bacillus to ancestral bacteria. Studies have since shown that several bacteria have smaller genome sizes than their ancestors did. Over the years, researchers have proposed several theories to explain the general trend of bacterial genome decay and the relatively small size of bacterial genomes. Compelling evidence indicates that the apparent degradation of bacterial genomes is owed to a deletional bias.

<span class="mw-page-title-main">History of genetic engineering</span>

Genetic engineering is the science of manipulating genetic material of an organism. The concept of genetic engineering was first proposed by Nikolay Timofeev-Ressovsky in 1934. The first artificial genetic modification accomplished using biotechnology was transgenesis, the process of transferring genes from one organism to another, first accomplished by Herbert Boyer and Stanley Cohen in 1973. It was the result of a series of advancements in techniques that allowed the direct modification of the genome. Important advances included the discovery of restriction enzymes and DNA ligases, the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Transformation of the DNA into a host organism was accomplished with the invention of biolistics, Agrobacterium-mediated recombination and microinjection. The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch. In 1976, the technology was commercialised, with the advent of genetically modified bacteria that produced somatostatin, followed by insulin in 1978. In 1983, an antibiotic resistant gene was inserted into tobacco, leading to the first genetically engineered plant. Advances followed that allowed scientists to manipulate and add genes to a variety of different organisms and induce a range of different effects. Plants were first commercialized with virus resistant tobacco released in China in 1992. The first genetically modified food was the Flavr Savr tomato marketed in 1994. By 2010, 29 countries had planted commercialized biotech crops. In 2000 a paper published in Science introduced golden rice, the first food developed with increased nutrient value.

<span class="mw-page-title-main">Minimal genome</span> Concept in genetics

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 may 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.

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.

Essential genes are indispensable genes for organisms to grow and reproduce offspring under certain environment. However, being essential is highly dependent on the circumstances in which an organism lives. For instance, a gene required to digest starch is only essential if starch is the only source of energy. Recently, systematic attempts have been made to identify those genes that are absolutely required to maintain life, provided that all nutrients are available. Such experiments have led to the conclusion that the absolutely required number of genes for bacteria is on the order of about 250–300. Essential genes of single-celled organisms encode proteins for three basic functions including genetic information processing, cell envelopes and energy production. Those gene functions are used to maintain a central metabolism, replicate DNA, translate genes into proteins, maintain a basic cellular structure, and mediate transport processes into and out of the cell. Compared with single-celled organisms, multicellular organisms have more essential genes related to communication and development. Most of the essential genes in viruses are related to the processing and maintenance of genetic information. In contrast to most single-celled organisms, viruses lack many essential genes for metabolism, which forces them to hijack the host's metabolism. Most genes are not essential but convey selective advantages and increased fitness. Hence, the vast majority of genes are not essential and many can be deleted without consequences, at least under most circumstances.

Synthetic virology is a branch of virology engaged in the study and engineering of synthetic man-made viruses. It is a multidisciplinary research field at the intersection of virology, synthetic biology, computational biology, and DNA nanotechnology, from which it borrows and integrates its concepts and methodologies. There is a wide range of applications for synthetic viral technology such as medical treatments, investigative tools, and reviving organisms.

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.

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