Mycoplasma laboratorium
This article is missing information about division morphology issues in JCVI-syn3.0 and the fix in JCVI-syn3A.(March 2021) |
Mycoplasma laboratorium | |
---|---|
Scientific classification | |
Domain: | Bacteria |
Phylum: | Mycoplasmatota |
Class: | Mollicutes |
Order: | Mycoplasmatales |
Family: | Mycoplasmataceae |
Genus: | Mycoplasma |
Species: | |
Subspecies: | M. m. JCVI-syn1.0
|
Trinomial name | |
Mycoplasma mycoides JCVI-syn1.0 Gibson et al., 2010[a 1]
| |
Synonyms[a 2] | |
Mycoplasma laboratorium Reich, 2000 |
Mycoplasma laboratorium or Synthia
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
Choice of organism
Mycoplasma
Mycoplasma is a genus of
lifestyle. InOther organisms with small genomes
As of 2005,
species name | number of genes | size (Mbp) |
---|---|---|
Candidatus Hodgkinia cicadicola Dsem [1] | 169 | 0.14 |
Candidatus Carsonella ruddii PV [2]
|
182 | 0.16 |
Candidatus Sulcia muelleri GWSS [3]
|
227 | 0.25 |
Candidatus Sulcia muelleri SMDSEM [4]
|
242 | 0.28 |
Buchnera aphidicola str. Cinara cedri [5] | 357 | 0.4261 |
Mycoplasma genitalium G37 [6] | 475 | 0.58 |
Candidatus Phytoplasma mali [7] | 479 | 0.6 |
Buchnera aphidicola str. Baizongia pistaciae [8] | 504 | 0.6224 |
Nanoarchaeum equitans Kin4-M [9] | 540 | 0.49 |
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:
- 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 electrophoresisand 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-mediated transformation of the circular chromosome to the DNA-free cells followed by selection.[a 15]
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]
- Synthesis → 1kbp: The genome sequence was synthesized by 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 spheroplasts without a vector sequence (present in assembly 811-900).
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
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[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:
- 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: "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.[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
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
Utility
Venter has argued that synthetic bacteria are a step towards creating organisms to manufacture
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
References
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- ^ Ken Shirriff (2010-06-10). "Using Arc to decode Venter's secret DNA watermark". Ken Shirriff's blog. Retrieved 2010-10-29.
- ^ First Minimal Synthetic Bacterial Cell. Astrobiology Web. March 24, 2016.
- ^ a b Yong, Ed (March 24, 2016). "The Mysterious Thing About a Marvelous New Synthetic Cell".
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