Mycoplasma laboratorium, also known as Synthia, is a project to create a synthetic version of a bacterium. The project has changed since it began. At first, scientists aimed to find the smallest number of genes needed to keep an organism alive by studying the genome of Mycoplasma genitalium. They then rebuilt these genes to make a new organism. Mycoplasma genitalium was chosen because it had the fewest genes of any studied organism at the time. Later, the focus shifted to Mycoplasma mycoides, and scientists used a more trial-and-error method.
To find the minimal genes required for life, scientists tested each of the 482 genes in M. genitalium by removing them one by one and checking if the bacteria could still survive. This process identified a group of 382 genes that might be the smallest set needed for life. In 2008, scientists built the full set of M. genitalium genes in the lab, adding special markers to show they were synthetic. However, M. genitalium grows very slowly, so scientists switched to M. mycoides to speed up experiments.
In 2010, scientists successfully created the complete genome of M. mycoides subsp. capri GM12 from a computer record and moved it into a cell of Mycoplasma capricolum that had its own DNA removed. This synthetic genome cost about $40 million and took 200 years of work by scientists. The new bacterium, named JCVI-syn1.0 or Synthia, was able to grow. After further experiments to find a smaller set of genes that could still create a working organism, scientists made JCVI-syn3.0, which contains 473 genes. Of these, 149 have unknown functions. Because the genome of JCVI-syn3.0 is completely new, it is considered the first truly synthetic organism.
Minimal genome project
Synthia is a project in synthetic biology conducted by the J. Craig Venter Institute. A team of about 20 scientists, led by Nobel laureate Hamilton Smith and including DNA researcher Craig Venter and microbiologist Clyde A. Hutchison III, worked on this project. The goal was to identify the basic components needed to create a new living organism from scratch. The project initially focused on M. genitalium, a type of bacteria that must live inside other cells. Its genome contains 482 genes and 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 could be grown outside of a host cell. Scientists used a method called transposon mutagenesis to determine which genes were not necessary for the organism’s survival. This process led to the identification of a minimal set of 382 essential genes. This work was part of the Minimal Genome Project.
Choice of organism
Mycoplasma is a group of bacteria in the class Mollicutes and the division Mycoplasmatota (previously called Tenericutes). These bacteria do not have a cell wall, which makes them Gram negative. They live inside or on other organisms, either as parasites or commensals. In molecular biology, Mycoplasma is important because it is hard to remove from cell cultures and is not affected by certain antibiotics like beta-lactams. It is also studied as a model organism because it has a small genome.
In 1996, scientists Arcady Mushegian and Eugene Koonin compared Mycoplasma genitalium with Haemophilus influenzae, another small bacterium. They suggested that all living organisms might need a common set of 256 genes to survive.
The name "Mycoplasma laboratorium" was chosen for the Synthia project in 2000 by Karl Reich.
By 2005, Pelagibacter ubique, a type of α-proteobacterium in the Rickettsiales order, was found to have the smallest genome of any free-living organism. Its genome is 1,308,759 base pairs long. It may be the most common bacterium in the world, with as many as 10 individual cells. Pelagibacter and other members of the SAR11 group are estimated to make up a quarter to half of all bacterial or archaeal cells in the ocean. Scientists identified it in 2002 using rRNA sequences and fully sequenced it in 2005. It is very difficult to grow in laboratory cultures because it does not reach high population numbers.
Some newly discovered bacteria have fewer genes than Mycoplasma genitalium, but they cannot live independently. For example, Hodgkinia cicadicola, Sulcia muelleri, Baumannia cicadellinicola, and Carsonella ruddi are symbionts of cicadas or other insects. These bacteria may rely on their host organisms to provide some genes they lack. The organism with the smallest known genome as of 2013 is Nasuia deltocephalinicola, an obligate symbiont that lives inside another organism. It has only 137 genes and a genome size of 112 kilobases.
Techniques
For the project, scientists had to create or change several laboratory methods because it involved making and changing very large DNA pieces.
In 2007, Venter's team shared results showing they successfully moved the chromosome from the species Mycoplasma mycoides into Mycoplasma capricolum.
The term "transformation" describes inserting a vector into a bacterial cell using methods like electroporation or heat shock. In this case, the process is called "transplantation," which is similar to nuclear transplantation.
In 2008, Venter's group explained how they created a synthetic genome, a copy of the M. genitalium G37 sequence L43967, using a step-by-step strategy.
The genome from this 2008 project, called M. genitalium JCVI-1.0, is listed on GenBank with the ID CP001621.1. This should not be confused with later synthetic organisms named JCVI-syn, which are based on M. mycoides.
Synthetic genome
In 2010, Venter and his team created a synthetic genome for Mycoplasma mycoides strain JCVI-syn1.0. The first version of the synthetic genome did not work, so the team made several partial synthetic versions to find the problem. This delay caused the project to take 3 months longer than expected. The issue was a single frameshift mutation in DnaA, a protein that helps start DNA replication.
The goal of creating a cell with a synthetic genome was to test the method for making modified genomes in the future. Using a natural genome as a model helped reduce possible mistakes. Scientists made 8 pieces of the genome in yeast and then joined them together. Compared to the original genome, JCVI-syn1.0 has some differences, including a transposon from E. coli (a type of mobile DNA element), an 85-base pair duplication, and parts needed for growth in yeast and leftover DNA from restriction sites.
Some people debate whether JCVI-syn1.0 is a truly synthetic organism. Although the genome was made in pieces using chemical methods, it was designed to closely match the parent genome and then placed into the cytoplasm of a natural cell. DNA alone cannot form a living cell. Proteins and RNA are needed to read DNA instructions, and cell membranes are required to separate DNA from the rest of the cell. In JCVI-syn1.0, the donor and recipient cells are from the same genus, which reduces problems that might happen if the proteins in the host cell did not match the new genome. Paul Keim, a molecular geneticist at Northern Arizona University, said, "There are many challenges ahead before genetic engineers can fully design an organism’s genome from scratch."
A well-known feature of JCVI-syn1.0 is the inclusion of watermark sequences. These are four coded messages written into the DNA, with lengths of 1246, 1081, 1109, and 1222 base pairs. These messages used a special code, not the standard genetic code (which uses groups of three DNA bases to represent amino acids), and readers were challenged to solve them.
Later evolutions
In 2016, the Venter Institute used genes from JCVI-syn1.0 to create a smaller genome called JCVI-syn3.0. This genome contains 531,560 base pairs and 473 genes. Of these genes, 149 have unknown functions as of 2016. Scientists used a strain called JCVI-syn2.0 to test which genes could be safely removed during this process.
JCVI-syn3.0 cells were fragile and hard to work with. They also had irregular shapes. To improve this, scientists created a new strain called JCVI-syn3A in 2017. This strain added 19 genes from JCVI-syn1.0 and removed two others. The added genes included FtsZ and SepF, which help with cell division, and some genes with unknown functions. The resulting bacterium had 493 genes (452 proteins and 38 RNAs) on a 543,000 base pair genome. In 2019, a complete computer model of all metabolic pathways in JCVI-syn3A was published. This model, called an "in silico" model, covered 155 genes involved in 338 chemical reactions. Future models will include processes related to ribosome and tRNA formation.
As of 2019, 91 genes in JCVI-syn3A had unknown functions. Eight of these genes had similar genes (orthologs) in a database called KEGG, which provided some clues about their possible roles.
In 2021 (published in 2023), JCVI-syn3B was first described in a study about evolution in this strain. Its genome was published in 2024 as part of research showing how it interacts with mammalian cells. Unlike JCVI-syn1.0, JCVI-syn3B could not attach to or live inside mammalian cells. Eight genes in JCVI-syn1.0 were found to be necessary for this behavior. When these genes were added to JCVI-syn3B, the modified strain could attach to cells but could not survive being engulfed by them. The genome of JCVI-syn3B (CP146056) is very similar to JCVI-syn3A, except for a few small changes and the addition of a 1,186 base pair segment from yeast, which was also present in JCVI-syn3.0.
Readers are encouraged to review the Introduction section in Bianchi et al. (2022) for details about efforts to identify the functions of unknown genes in JCVI strains. Both laboratory experiments and computer-based methods have been used to study these genes. The work by Bianchi et al. focused on finding similar proteins in other organisms using bioinformatics.
Concerns and controversy
On October 6, 2007, Craig Venter told reporters from The Guardian newspaper in the UK that his team had created a changed version of the single chromosome of Mycoplasma genitalium using chemicals. The new genome had not yet been placed into a living cell. The next day, a Canadian group called ETC Group, through their representative Pat Mooney, said that Venter’s work could be used to make important things like new medicines or dangerous things like biological weapons. Venter said, “We are working on big ideas. We are trying to create a new way of thinking about life. When dealing with such large ideas, not everyone will agree.”
On May 21, 2010, the journal Science reported that Venter’s team had made the genome of Mycoplasma mycoides using information from a computer. They then placed this genome into a Mycoplasma capricolum cell that had had its own DNA removed. The new bacterium was alive and could reproduce. Venter called it “the first species… to have its parents be a computer.”
In March 2016, Science announced the creation of a new synthetic bacterium called JCVI-3.0. It has 473 genes. Venter called it “the first designer organism in history” and said that because 149 of the genes have unknown functions, “the entire field of biology has been missing a third of what is essential to life.”
The project received a lot of media attention because of Venter’s public speaking style. Jay Keasling, a scientist who helped start synthetic biology, joked that “The only regulation we need is of my colleague’s mouth.”
Venter has said that synthetic bacteria could help create organisms that make hydrogen and biofuels or remove carbon dioxide and other greenhouse gases. George M. Church, another scientist, argued that making a fully synthetic genome is not needed because E. coli grows more efficiently than M. genitalium, even with extra DNA. He added that scientists have already used synthetic genes in E. coli to do some of these tasks.
In 2006, the J. Craig Venter Institute applied for patents on the genome of Mycoplasma laboratorium (a “minimal bacterial genome”) in the United States and other countries. ETC Group, the Canadian bioethics group, objected, saying the patent covered too many things.
Similar projects
From 2002 to 2010, scientists at the Hungarian Academy of Science developed a type of Escherichia coli called MDS42. This strain is now sold by Scarab Genomics in Madison, WI, as "Clean Genome. E. coli." Scientists removed 15% of the DNA from the original strain, E. coli K-12 MG1655, to help make experiments easier. They removed parts of the DNA called IS elements, pseudogenes, and phages. These changes helped keep harmful genes from being turned off by jumping genes. The parts of the bacteria that help with chemical reactions and DNA copying were not changed.