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''Mycoplasma laboratorium'' or Synthia refers to a
synthetic Synthetic things are composed of multiple parts, often with the implication that they are artificial. In particular, 'synthetic' may refer to: Science * Synthetic chemical or compound, produced by the process of chemical synthesis * Synthetic ...
strain of
bacterium Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
. 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 ''Mycoplasma genitalium'' (''MG'', commonly known as Mgen) 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 20 ...
'', 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 ''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 i ...
'' and took a more trial-and-error approach. 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. In 2008 the full set of ''M. genitalium'' genes was constructed in the laboratory with watermarks added to identify the genes as synthetic. 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. In 2010, the complete genome of ''M. mycoides'' was successfully synthesized from a computer record and transplanted into an existing cell of ''
Mycoplasma capricolum ''Mycoplasma capricolum'' is a species of Mycoplasma bacteria. It is primarily a pathogen of goat The goat or domestic goat (''Capra hircus'') is a domesticated species of goat-antelope typically kept as livestock. It was domesticated ...
'' that had had its DNA removed. It is estimated that the synthetic genome used for this project cost US$40 million and 200 man-years to produce. 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. 149 of these genes are of unknown function. 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 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 G ...
by a team of approximately 20 scientists headed by Nobel laureate Hamilton Smith and including DNA researcher
Craig Venter John Craig Venter (born October 14, 1946) is an American biotechnologist and businessman. He is known for leading one of the first draft sequences of the human genome and assembled the first team to transfect a cell with a synthetic chromosome. ...
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. The initial focus was the bacterium ''M. genitalium'', an
obligate intracellular parasite Intracellular parasites are microparasites that are capable of growing and reproducing inside the cells of a host. Types of parasites There are two main types of intracellular parasites: Facultative and Obligate. Facultative intracellular par ...
whose genome consists of 482 genes comprising 582,970
base pairs 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 ...
, 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. This effort was known as the Minimal Genome Project.


Choice of organism


''Mycoplasma''

''Mycoplasma'' is a genus of bacteria of the class
Mollicutes Mollicutes is a class of bacteria distinguished by the absence of a cell wall. The word "Mollicutes" is derived from the Latin ''mollis'' (meaning "soft" or "pliable"), and ''cutis'' (meaning "skin"). Individuals are very small, typically only 0 ...
in the division
Mycoplasmatota Mycoplasmatota is a phylum of bacteria that contains the class Mollicutes. The phylum was originally named "Tenericutes" (''tener cutis'': soft skin). Notable genera include ''Mycoplasma'', ''Spiroplasma'', ''Ureaplasma'', and ''Candidatus'' Phy ...
(formerly Tenericutes), characterised by the lack of a
cell wall A cell wall is a structural layer surrounding some types of cells, just outside the cell membrane. It can be tough, flexible, and sometimes rigid. It provides the cell with both structural support and protection, and also acts as a filtering me ...
(making it
Gram negative The gram (originally gramme; SI unit symbol g) is a unit of mass in the International System of Units (SI) equal to one one thousandth of a kilogram. Originally defined as of 1795 as "the absolute weight of a volume of pure water equal to th ...
) due to its
parasitic Parasitism is a close relationship between species, where one organism, the parasite, lives on or inside another organism, the host, causing it some harm, and is adapted structurally to this way of life. The entomologist E. O. Wilson has ...
or
commensal Commensalism is a long-term biological interaction (symbiosis) in which members of one species gain benefits while those of the other species neither benefit nor are harmed. This is in contrast with mutualism, in which both organisms benefit fro ...
lifestyle. In molecular biology, the genus has received much attention, both for being a notoriously difficult-to-eradicate contaminant in mammalian
cell culture Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This t ...
s (it is immune to
beta-lactams A beta-lactam (β-lactam) ring is a four-membered lactam. A ''lactam'' is a cyclic amide, and ''beta''-lactams are named so because the nitrogen atom is attached to the β-carbon atom relative to the carbonyl. The simplest β-lactam possible is ...
and other
antibiotics An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention ...
),Young L, Sung J, Stacey G, Masters JR. "Detection of ''Mycoplasma'' in cell cultures". ''Nat Protoc.'' 2010 5(5): 929–34. Epub 2010 Apr 22. and for its potential uses as a model organism due to its small genome size. The choice of genus for the Synthia project dates to 2000, when Karl Reich coined the phrase ''Mycoplasma laboratorium''.


Other organisms with small genomes

As of 2005, ''
Pelagibacter ubique "''Candidatus'' Pelagibacter", with the single species "''Ca.'' P. communis", was isolated in 2002 and given a specific name, although it has not yet been described as required by the bacteriological code. It is an abundant member of the SAR11 ...
'' (an α-proteobacterium of the order
Rickettsiales The Rickettsiales, informally called rickettsias, are an order of small Alphaproteobacteria. They are obligate intracellular parasites, and some are notable pathogens, including ''Rickettsia'', which causes a variety of diseases in humans, and ' ...
) 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 The Pelagibacterales are an order in the Alphaproteobacteria composed of free-living marine bacteria that make up roughly one in three cells at the ocean's surface. Overall, members of the ''Pelagibacterales'' are estimated to make up between a q ...
, are estimated to make up between a quarter and a half of all bacterial or archaeal cells in the ocean. It was identified in 2002 by rRNA sequences and was fully sequenced in 2005. It is extremely hard to cultivate a species which does not reach a high growth density in lab culture. 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 "''Candidatus'' Karelsulcia muelleri" is an aerobic, gram-negative, bacillus bacterium that is a part of the phylum Bacteroidota. "''Ca.'' K. muelleri" is an obligate and mutualistic symbiotic microbe commonly found occupying specialized cell ...
'', '' Baumannia cicadellinicola'' (symbionts of
cicada The cicadas () are a superfamily, the Cicadoidea, of insects in the order Hemiptera (true bugs). They are in the suborder Auchenorrhyncha, along with smaller jumping bugs such as leafhoppers and froghoppers. The superfamily is divided into two ...
s) and '' Carsonella ruddi'' (symbiote of hackberry petiole gall psyllid, ''
Pachypsylla venusta ''Pachypsylla'' is a genus of psyllids. Species of the genus ''Pachypsylla'' lay eggs on the leaves of the ''Celtis occidentalis'' tree. Upon hatching, the young psyllids become encased in a gall which the young leaf parts grow in response to the ...
'') may be encoded in the host nucleus. The organism with the smallest known set of genes as of 2013 is ''
Nasuia deltocephalinicola ''Nasuia deltocephalinicola'' was reported in 2013 to have the smallest genome of all bacteria, with 112,091 nucleotides. For comparison, the human genome has 3.2 billion nucleotides. The second smallest genome, from bacteria '' Tremblaya prince ...
'', an obligate symbiont. It has only 137 genes and a genome size of 112 kb.


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: * isolating the genome of ''M. mycoides'': gentle lysis of cells trapped in agar—molten agar mixed with cells and left to form a gel—followed by
pulse field gel electrophoresis Pulsed field gel electrophoresis is a technique used for the separation of large DNA molecules by applying to a gel matrix an electric field that periodically changes direction. Historical background Standard gel electrophoresis techniques for s ...
and the band of the correct size (circular 1.25Mbp) being isolated; * making the recipient cells of ''M. capricolum'' competent: growth in rich media followed starvation in poor media where the nucleotide starvation results in inhibition of DNA replication and change of morphology; and *
polyethylene glycol Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular we ...
-mediated transformation of the circular chromosome to the DNA-free cells followed by selection. The term
transformation Transformation may refer to: Science and mathematics In biology and medicine * Metamorphosis, the biological process of changing physical form after birth or hatching * Malignant transformation, the process of cells becoming cancerous * Trans ...
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 sequenc
L43967
by means of a hierarchical strategy: *Synthesis → 1kbp: The genome sequence was synthesized b
Blue Heron
in 1,078 1080bp cassettes with 80bp overlap and NotI restriction sites (inefficient but infrequent cutter). *Ligation → 10kbp: 109 groups of a series of 10 consecutive cassettes were ligated and cloned in ''E. coli'' on a plasmid and the correct permutation checked by sequencing. *Multiplex PCR → 100kbp: 11 Groups of a series of 10 consecutive 10kbp assemblies (grown in yeast) were joined by multiplex PCR, using a primer pair for each 10kbp assembly. *Isolation and recombination → secondary assemblies were isolated, joined and transformed into yeast
spheroplast A spheroplast (or sphaeroplast in British usage) is a microbial cell from which the cell wall has been almost completely removed, as by the action of penicillin or lysozyme. According to some definitions, the term is used to describe Gram-negative ...
s without a vector sequence (present in assembly 811-900). The genome of this 2008 result, ''M. genitalium'' JCVI-1.0, is published on GenBank a
CP001621.1
It is not to be confused with the later synthetic organisms, labelled JCVI-syn, based on ''M. mycoides''.


Synthetic genome

In 2010 Venter and colleagues created ''Mycoplasma mycoides'' strain JCVI-syn1.0 with a synthetic genome. Initially the synthetic construct did not work, so to pinpoint the error—which caused a delay of 3 months in the whole project—a series of semi-synthetic constructs were created. The cause of the failure was a single frameshift mutation in
DnaA Introduction Based on the Replicon Model, a positively active initiator molecule contacts with a particular spot on a circular chromosome called the replicator to start DNA replication. DnaA is a protein that activates initiation of DNA replica ...
, a replication initiation factor. 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. 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 The lipid bilayer (or phospholipid bilayer) is a thin polar membrane made of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around all cells. The cell membranes of almost all organisms and many vir ...
are required to compartmentalize the DNA and
cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. Th ...
. 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.
Paul Keim Paul may refer to: * Paul (given name), a given name (includes a list of people with that name) *Paul (surname), a list of people People Christianity *Paul the Apostle (AD c.5–c.64/65), also known as Saul of Tarsus or Saint Paul, early Chri ...
(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".


Watermarks

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) 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. The content of the watermarks is as follows: # Watermark 1: an HTML script which reads to a browser as text congratulating the decoder, and instructions on how to email the authors to prove the decoding. # 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". # Watermark 3: more authors and a quote from Robert Oppenheimer (uncredited): "See things not as they are, but as they might be". # Watermark 4: more authors and a quote from
Richard Feynman Richard Phillips Feynman (; May 11, 1918 – February 15, 1988) was an American theoretical physicist, known for his work in the path integral formulation of quantum mechanics, the theory of quantum electrodynamics, the physics of the superfl ...
: "What I cannot build, I cannot understand".


JCVI-syn3.0

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.First Minimal Synthetic Bacterial Cell
''Astrobiology Web''. March 24, 2016.
In 1996, after comparing ''M. genitalium'' with another small bacterium ''Haemophilus influenza'', 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. In this new organism, the number of genes can only be pared down to 473, 149 of which have functions that are completely unknown. As of 2022 the unknown set has been narrowed to about 100. 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.


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 ''Mycoplasma genitalium'' (''MG'', commonly known as Mgen) 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 20 ...
'' chemically. The synthesized genome had not yet been transplanted into a working cell. The next day the Canadian
bioethics Bioethics is both a field of study and professional practice, interested in ethical issues related to health (primarily focused on the human, but also increasingly includes animal ethics), including those emerging from advances in biology, med ...
group, ETC Group issued a statement through their representative,
Pat Mooney Pat Roy Mooney, for more than thirty years, has worked with civil society organizations on international trade and development issues related to agriculture, biodiversity and emerging technologies. He was born and lived on the Canadian prairies f ...
, 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." 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. Venter described it as "the first species.... to have its parents be a computer". 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".


Press coverage

The project received a large amount of coverage from the press due to Venter's showmanship, to the degree that
Jay Keasling Jay D. Keasling is a professor of chemical engineering and bioengineering at the University of California, Berkeley. He is also associate laboratory director for biosciences at the Lawrence Berkeley National Laboratory and chief executive officer ...
, a pioneering synthetic biologist and founder of Amyris commented that "The only regulation we need is of my colleague's mouth".Andrew Pollack, His Corporate Strategy: The Scientific Method, NYTimes, September 4, 2010


Utility

Venter has argued that synthetic bacteria are a step towards creating organisms to manufacture hydrogen and
biofuel Biofuel is a fuel that is produced over a short time span from biomass, rather than by the very slow natural processes involved in the formation of fossil fuels, such as oil. According to the United States Energy Information Administration (EIA ...
s, and also to absorb carbon dioxide and other
greenhouse gas A greenhouse gas (GHG or GhG) is a gas that absorbs and emits radiant energy within the thermal infrared range, causing the greenhouse effect. The primary greenhouse gases in Earth's atmosphere are water vapor (), carbon dioxide (), methane ...
es.
George M. Church George McDonald Church (born August 28, 1954) is an American geneticist, molecular engineer, chemist, and a serial entrepreneur who is widely regarded as the "Founding Father of Genomics", and a pioneer in personal genomics and synthetic b ...
, 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.Longest Piece of Synthetic DNA Yet
''Scientific American News'', 24 January 2008


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.Artificial life: Patent pending
, ''The Economist'', June 14, 2007. Retrieved October 7, 2007.
Roger Highfield,
Man-made microbe 'to create endless biofuel'
, ''Telegraph'', June 8, 2007. Retrieved October 7, 2007.
US Patent Application

/ref> The ETC group, a Canadian bioethics group, protested on the grounds that the patent was too broad in scope.


Similar projects

From 2002 to 2010, a team at the Hungarian Academy of Science created a strain of ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Esc ...
'' called MDS42, which is now sold by Scarab Genomics of Madison, WI under the name of "Clean Genome. E.coli", 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 Insertion element (also known as an IS, an insertion sequence element, or an IS element) is a short DNA sequence that acts as a simple transposable element. Insertion sequences have two major characteristics: they are small relative to other transp ...
, pseudogenes and phages, resulting in better maintenance of plasmid-encoded toxic genes, which are often inactivated by transposons. Biochemistry and replication machinery were not altered.


References


Primary sources


Popular press


External links


J. Craig Venter Institute: Research Groups
{{Bacteria classification, state=collapsed Artificial life Synthetic biology Mycoplasma